EP3302539A1 - Manufacturing multi-dose injection ready dendritic cell vaccines - Google Patents

Manufacturing multi-dose injection ready dendritic cell vaccines

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Publication number
EP3302539A1
EP3302539A1 EP16800434.9A EP16800434A EP3302539A1 EP 3302539 A1 EP3302539 A1 EP 3302539A1 EP 16800434 A EP16800434 A EP 16800434A EP 3302539 A1 EP3302539 A1 EP 3302539A1
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EP
European Patent Office
Prior art keywords
cancer
cells
antigen
dendritic cell
her2
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
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EP16800434.9A
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German (de)
French (fr)
Other versions
EP3302539A4 (en
Inventor
Brian J. Czerniecki
Gary K. Koski
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Individual
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Individual
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Priority claimed from PCT/US2015/041022 external-priority patent/WO2016011422A2/en
Application filed by Individual filed Critical Individual
Publication of EP3302539A1 publication Critical patent/EP3302539A1/en
Publication of EP3302539A4 publication Critical patent/EP3302539A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464406—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/49—Breast

Definitions

  • Dendritic cells are white blood cells that acquire protein antigens from microbes or even cancerous cells and show, or "present” these antigens to T cells. The T cells, thus activated by the DCs, then initiate systemic immune responses to challenge the threat.
  • Traditional vaccines against microbes contain: additives known as "adjuvants" that by a number of possible means enhance DC activity with m the vaccinated individual and am lify ' vaccine-induced immune responses.
  • the requirements of vaccines against cancer present a number of unique problems. For example, traditional adjuvants do not provide the proper signals to DCs that allow them to initiate optimal immunity against cancer. Also, the tumors themselves produce an environment thai can affect the proper activation of DCs.
  • a popular solution to this problem is to extract DCs from cancer patients, load them wit tumor antigens . /?* vitro, and then suppl unique activation signals to the cells before re-administering them to the body. This ensures proper DC activation removed from the influence of the tumor environment When returned to the body, the DCs can then interact with T cells and initiate powerful anti-tumor iawmmity.
  • extra-corpoxealized ' DCs has solved many efficacy issues., it has historically come at the price of practical limitations. For example, since the DC vacc ines ate comprised of living cells, a special cell processing and vaccine production facility has been required at the physical location of the medical center administering the therapy. This is an expensive and inefficient, way to deliver the therapy because every institution administering such treatment would have to bui ld and maintain their own special-use facility.
  • Herceptin was developed as a targeted therapy for HER2 ErbB2 positive breast cancer cells, often used in conjunction with other therapies, including the mitotic inhibitor paciitaxe! (sold under the trademark Taxol).
  • Herceptin as a monotherapy is estimated to be less than 30%; combinatorial treatment with microtubule stabilizing drugs such as paclitaxel increases efficacy to approximately 60% (Burns et a!., 2000, Semin. Oncol 27; 19-23). Treatment with Herceptin results in accumulation of the Cdk inhibitor p27 and subse uent GT/S cell cycle arrest, and paciitaxe! stalls the entry of mitosis which can lead to cell death. In spite of great promise, however, high doses of Herceptin or paclitaxel result in undesirable side effects. Further, the cancer often develops resistance to
  • Herceptin and/or paclitaxel Herceptin and/or paclitaxel.
  • Figure 1 is a chart showing the viability and yield of post-thawed cryopreserved DO . Recovery of cells was on a verage 89% and viabi lit was 95% when cells were directly thawed and counted.
  • figure.5 is a chart showing production of lFM- ⁇ from cryopreserved and non ⁇ cryopreserved DCs.
  • Figures 6A-6D show Th 1 cytokines TNF-a and lFN ' - ⁇ synergize to induc senescence in breast cancer cells and the doses required are ' in an inverse correlation with the HE 2 expression.
  • Figure 6 A shows results of studies in which SK-BR.-3 breast cancer cells were incubated with 10 ng/ral TNF-a and 100 U/ml IF - ⁇ for 5 days, cultured for 2 more passages in absence of cytokines and then stained for SA-j3- galactosidase (SA-p-gal) expression (senescence marker) and compared to untreated control ceils. Only paired cytokines induced senescence.
  • Top panel shows photographs of representative data from 1 of 3 independent experiments.
  • FIG. 6B shows photographs of western blot analysis in which cell lysaies of the cells described in Figure 6A were analyzed for piSI Kb and l6!N 4a expression. Vinculra was used as loading control.
  • Figure 6C shows results of studies in which T-47D breast cancer cells were untreated (1 ) or incubated with the following concentrations of TNF-a and INF-y: 10 ng/ml and 100 U/n l (2), SO ng/ml and 500 U/rnl .(3) honor 75 ng/nil and 750 U/ml (4), .and 100 ng/ml and 1000 U/ml (5) for 5 days and cultured for 2 more passages in absence of cytokines . The cells were then stained for SA-p-gal and compared to control untreated cells or those treated with 8 ⁇ etoposide as a positive control (6). Top panel shows photographs of representative data from 1 of 3 independent experiments.
  • Bottom panel shows a histogram of densitometric analysis of the 6 studies. Data are presented as % of SA- ⁇ - gal-positive cells and presented as mean ⁇ S.D. (n ⁇ 3). P-va ' lues were calculated using a paired Student's t-test. Statistical significance was determined at *P ⁇ 0.05, **P ⁇ 0.01 , ***P ⁇ 0.001.
  • Figures 7 ⁇ -7 ⁇ show HE 2 induces senescence and apopiosis in MDA- MB-231 breast cancer cells.
  • Figure 7 A left panel, is a histogram showing results of densitometric analysis in which SA-f3-gal staining was performed i MDA-MB-231 cells transfected with wt HER2 (pcDNAHBR.2) or with empty vector (pcDNA3) which were treated with the l isted concentrations of TNF-a and IFN- ⁇ for 5 days and cultured for 2 more passages in absence of cytokines.
  • Figures 8A-SB show combined HE 2 and HER3 blockage expression enhances Th! cytokines TNF-a and JFN-y senescence induction and apoptosis in. $K ⁇ BR-3 breast cancer cells.
  • Figure 8 A shows results of studies in which S A-P ⁇ gal staining was performed in SK-BR-3 cells transfecied with non-target siRNA (siRNA NT), HER2 siRNA, HER3 siRNA or a combination of HER2 and H.ER3 siRNA, and then treated with the concentrations listed of TNF-a and lFN- ⁇ for 5 days and cultured for 2 more passages in absence of cytokines.
  • Left panel shows a histogra of densitonieroc analysis.
  • Figures 9A-9C show combined HER2 inhibition and HER2-F1ER dimerization inhibition enhances Tii! cytokines TNF-a and IFN-y senescence induction and apoptosis in SK-BR-3 breast cancer cells.
  • Figure 9 A shows results of SA-p-gai staining performed in SK-BR-3 cells which were untreated (I) or treated with 10 ng ml TNF- ⁇ and 100 U/mi IFN- ⁇ (2), or with 10 ug/ml of trastuzutnab (Tzm), pertuzumab (Per)(3), or with the combination of both treatments (4) for 5 days and cultured for 2 more passages in absence of the antibodies and the cytokines. Left pane!
  • FIG. 9B shows photographs of western blot analysis in which cell lysaies of the cells described in Figure 9 A were analyzed for plSlNKb or cleaved caspase-3 expression. Vinculin was used as loading control. Similar results were observed in 3 independent experiments.
  • Fi gure 9C shows resul ts of studies of induction of apoptosis of SK-BR-3 c ells untreated or treated as described above performed by staining for annexin V and PI and analyzed by flow cytometry. Top panel shows plots of representative data from 1 of 3 independent experiments. Bottom panel, shows a histogram of densitoraetric analysis. Data are presented as average £ SE. of annexin V* ⁇ cells from 3 independent experiments. P- vahies were calculated using a paired Student's Mest. Statistical significance was determined at **P ⁇ 0.0.1.
  • Figures I OA- 10B show combined treatment with trastimanab and pertiizumab enhance CD4 'r Thl -mediated Senescence and apoptosis of HER2- ovexpressing human breast cancer cells.
  • Figure I OA shows results of studies in which, using a transwell system, 0,5x10 s SK-BR-3 cells were co-cultured with 5xl0 5 CD4 : T- cells alone (CD4 f only), CD4* T-celis + 0.5x10 s each of HER2 Class It peptide (DC H)- or irrelevant Class II BRAF or survivin peptides (DC B or DC S)-pulsed type 1 polarized mature DCs, and CD4 " * T-celis + HER2 (iDC H)-pul$ed immature DCs ( IDC H), with or without 10 tig/ml of trastuziimab (Tzm) and pertuzumab (Per) for 5 days.
  • DC H Class It peptid
  • Top panel is a histogram of densitometric analysis. Data are presented as % of $A ⁇ P ⁇ gal ⁇ positive cells and presented as .mean ⁇ S.D. (n ⁇ 3). P-values were calculated using a paired Student's t-test. Statistical significance was determined at ***P ⁇ 0.0001. Bottom panel shows photographs of representative data from 1 of 3 independent experiments.
  • Figure 10B shows photographs of western blot analysis of cell lysates of SK-BR-3 cells co-cultured as indicated and analyzed for pl 51 ' NK4b and cleaved caspaee- 3 expression.
  • Increased pi 5INK4b and cleaved caspase-3 expression is seen that suggests induced senescence and apoptosis of SK-BR-3 ceils, respectively when co- cultured wit the DC H/CD4 r T-celis in presence of trastuzuraab and pertuzurnab, but not from DC B, DC S and IDC H groups, Vioculin was used as loading control.
  • Results are representative of 3 independent experiments, photographs of western blot analysis in which cell lysates of the cells described m Figure A were analyzed for pISINKh or cleaved caspase-3 expression.
  • Figures 11 A ⁇ l IB show. Th .1 cytokines- TNF-a. and IFN- ⁇ sensitize trastuzumab and pertuzumab resistant breast cancer cells to senescence and apoptosis induction.
  • Figure i 1 A shows results of SA-fi-gai staining performed in HOC- ⁇ 419 and JiMT-I cells, respectively untreated (!) or treated with 50 »g ml TNF- ⁇ and 500 U/m IFN- ⁇ (2), or treated with 10 ag/ml of trastuzumab (Tzm), pertuzumab (Per) (3), or treated with the combination of the same concentrations of trastuzumab, pertoznmab and TNF-a, IFN-y (4) for 5 days and cultured for 2 more passages in absence of the
  • Top panel is a histogram of densiiometric analysis. Data are presented as % of SA-P-gal-positive ceils and presented as mean j; S.D. (n-3). P- values were calculated using a paired Student's t-test. Statistical significance was determined at **P ⁇ 0.01. Bottom panel, top layer, shows photographs of representative data from 1 of 3 independent experiments in HC-14I9 ceils. Bottom panel, bottom layer, shows photographs of representative data from 1 of 3 independent experiments in JIMT-I cells.
  • Figure 1 I B shows results of cell lysates of the cells described in Figure 1 1 A which were analyzed by western blotting for pi SINKb or cleaved caspase-3 expression in HC-141 (left pane! and JIMT-l ⁇ right panel). Vmculin was used as loading control. Similar results were observed, in 3 independent experiments.
  • FIG.1.2 shows IFN-yRa nd TNF-R.1 ate- expressed in similar levels in breast cell lines independently from their HER2 level.
  • Figures 13A-13B sho combined HER2 and HER3 blockage expression enhances Thl cytokines TNF-a and IFN-y senescence and apoptosis induction in MCF-7 breast cancer cells.
  • Figure ⁇ 3 A shows results of studies in which S A- ⁇ -gal staining was performed in MCF-7 cells transfected with non-target siRN (siRNA NT), HER2 siRNA, HER3 siRNA or a combination of HER2 and BER3 siRNA, and then treated with the concentrations listed of TNF-a and IFN-y for 5 days and cultured for 2 more passages in absence of cytokines.
  • Left panel shows a histogram of densitometric analysis. Data are presented as % of SA-p-gal-positive cells and presented as mean ⁇ S.D. (n ⁇ 3). P-vahies were calculated using a paired Student's t-test.
  • Statistical analysis of S A- ⁇ -gal staining was performed in M
  • Inset shows photographs of western blot analysis of SK-BR-3 cells transfected with NT, HER2, or HER3 siRNA probed with HER and HER3 specific antibodies. Vineulk was used as loading control. Similar results were observed k 3 independent experiments.
  • Figures I4A-14B show the effect of Till -elaborated cytokines on S -BR- 3 senescence and apoptosis.
  • Figure 14A shows he results of studies using a transwell system, whereby O.S l O 5 SK-BR-3 cells were co-cultwed with 5x10 5 CD4 ' T-cells alone (CD4 ⁇ only), CD4 ; T-cells + O.SxlO 5 each of HER2 Class ⁇ peptide (DC H) ⁇ or
  • Figure 15 shows the effect of trastuzumab and pertuzumab on AKT activation by heregulin in breast cancer ceil lines.
  • Serum-starved T-47D, HCC-I41 and JIMT-I cells were treated with rrastoizumab (Tzm) and pertuzumab (Per 10 ug/mh 90 min) and then stimulated with (HRG, 20 ng ml, 5 ruin).
  • Top panel sliows representative data from of 3 independent experiments. Data are expressed as % of the HRG response in the absence of trastuzumab and pertuzumab and presented as mean S.D. (n-3).
  • Figure 16 shows vaccination procedure. Patients with biopsy diagnosed FfER2 $>l s DQI$ W ere enrolled in the trial. Patient's monocytes were collected by
  • Figures 17A-17B shows results of SKBR3 and MCF7 breast cancer cell lines treated with Thl cytokines (IFNy and TNFa), a tamoxifen metabolite (4-F!ydroxy ⁇ Tamoxifen, "4HT"), or both.
  • Thl cytokines IFNy and TNFa
  • a tamoxifen metabolite 4-F!ydroxy ⁇ Tamoxifen, "4HT”
  • SKBR3 (ER ae3 ⁇ 4 ) Figure 17 A
  • CF7 ER !X,i
  • Figure 17B did not increase anti-tumor activity in response to either Th 1 cytokine treatment or anti-estrogen treatment, but the combination -.resulted in an. increase in metabolic activity.
  • Figure 18 shows patient distribution for the combination anti-estrogen ("AE") therapy and anti ⁇ HER2 DC! vaccination study.
  • HER-2 positivity was defined as >53 ⁇ 4 of cells expression 2+ or 3+ intensity of the HER-2 protein on immunohistocbemistry.
  • AE therapy (Tamoxifen., Letrozole, or Anastrozole) was .given concurrently with DC vaccination.
  • Figure 19 is a histogram showing pathologic complete response rate comparing patients by ER status and AE treatment (ER iit3 ⁇ 4 ; £W* w/o AE; ER pos w AE).
  • Figures 20A-20B shows subsequent breast events of the study pallets comparing patients by pathologic complete response (“pCR”) ( Figure 20A) and ER status and AE treatment. ( Figure 20B).
  • pCR pathologic complete response
  • Figures 21 A-21C show CD4 - systemic immune response measured in the peripheral blood.
  • Thl response respoiisivity
  • Figure 2 IB response repertoire
  • Figure 21C cumulative response
  • Figures 22A-22C show CD4 i local regional immune response measured in patient sentinel lymph nodes. By each metric (responsivity (Figure 22 A); response repertoire (Figure 22B) or cumulative response (Figure 22C)) the post-vaccination immune responses were higher in the ER*** atients who received AE compared to the EW patients who did not receive AE.
  • Figure 23 shows CDS ' systemic immune response measured i the peripheral blood. Responsivity of patients with E * ® status and those with ER 51 "* status with and without anti-estrogen treatment (EE 550 * w/o AE; ER wAE) are shown.
  • Figure 24 shows BRAF M3 ⁇ 4)i; ⁇ DCl vaccines overcome vemurafenib resistance in BRAF-mutant murine melanoma.
  • the present embodiments provide compositions and methods for producing an FDA-approved injectable multi-dose antigen pulsed dendritic cell vaccine for the personalized treatment and prevention of cancer or other disorders.
  • the embodiments ' provide compositions and methods for producing an FDA-approved injectable muiti-dose antigen pulsed type ⁇ polarized dendritic ceil vaccine (DC 1).
  • DC 1 polarized dendritic ceil vaccine
  • a method to efficiently produce injectable multi-dose antigen pulsed dendritic cell vaccine preferably injectable multi- dose antigen pulsed type 1 polarized dendritic ceil vaccine that exhibit maximal efficacy.
  • an FDA-approved, injectable multi-dose antigen pulsed dendritic cell vaccine is produced by collecting DCs in a single patient
  • the leukapheresis and production of the dendritic cell vaccine is performed at a first location whereby the first location can be a centralized vaccine production facility where the DCs are manipulated to create an activated, antigen-loaded DC vaccine comprised of an initial immunizing dose and multiple "booster" doses thereof.
  • An advantage of the present embodiments is that all FDA mandated quality control/quality assurance steps would be performed at the central facility, and after completion and release, all vaccine doses are cryopreserved and shipped to remote medical centers for serial administration to the patient.
  • the FDA- approved injectable multi-dose antigen pulsed dendritic cell vaccine of the embodiments does not -requirement any mandated quality control/quality assurance steps at the adminislxation site.
  • an effective therapy to treat cancer includes changing the immune response in. the tumor so that the immune cells in the tumor site are more effective in. attacking the tumor cells.
  • the effective therapy inclodes improving the migratio and activity of immune cells in the tumor site.
  • the embodiments provide compositions and methods of using a dendritic cell vaccine in combination with a composition that inhibits one or more of HER-2 and IiER-3 (e.g., trastuzutnab, pertrrzumab, and the like) as a treatment regimen to treat cancer.
  • the treatment regimen comprises the use of dendritic cell vaccines, an inhibitor of HER-2, and a chemok ne modulator.
  • compositions and methods of using a dendritic cell vaccine in combination with blockage of HER-2 and HER-3 with the addition of TNF- ⁇ and IF -y in another embodiment, there is provided compositions and methods of blocking one or more of HER-2 and HER- 3 with the addition ofTNF- and IFN-y as a treatment regimen to treat cancer.
  • the treatment re gimen of the embodiments comprise a combination therapy of inducing an anti-oncodriv er Thl immune response (e.g., TNF ⁇ a and lFN- ⁇ ) and oncodri ver blockade for one or more of HER-2 and HER-3
  • Thl immune response e.g., TNF ⁇ a and lFN- ⁇
  • oncodri ver blockade for one or more of HER-2 and HER-3
  • the treatment regimen of the embodiments can be used to treat cancer and therefore can be considered as a type of anti-cancer therapy, in another embodiment, the treatment, regime of the embodimen ts can be used in
  • anti-cancer therap including but is not limited to surgery, chemotherapy, radiatio therapy (e.g. X ray), gene therapy, immunotherapy, honuone therapy, viral therapy, DMA therapy, RNA therapy, protein, therapy, cellular therapy, nanotherapy, and the like.
  • the treatment regimen of the embodiemems is used prior to receiving the other anti-cancer therapy.
  • the treatment regimen of the embodiments is used concurrently with receiving the other anti-cancer therapy .
  • the treatment regimen of the embodiments is used after recei ving the other anti-cancer therapy.
  • concurrent neoadjuvant anti-estrogen therapy and anti-HER2 DC1 vaccination increases the immune response in the local sentinel lymph nodes and the rate of pathological complete response in HER2 po VER iH>* DOS patients.
  • Standard techniques are used for nucleic acid and peptide synthesis.
  • the techniques and procedures are generally performed, according to conventional methods in the art and various general references (e.g., Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY, and Ausubel et al, 2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout this document.
  • abnormal when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day , etc.) from those organisms, tissues, cells or components thereof that display the "normal” (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.
  • ⁇ antigen or "ag* as used herei n, is defined as a molecule that provokes an immune response. This immune respons may involve either antibody production, or the activation of specific immiinologicaily-competent cells, or both.
  • any niacroraolecuie can serve as an antigen.
  • antigens can be derived from recombinant or genomic DNA.
  • any DNA which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein.
  • an antigen need not be encoded solely by a full length nucleotide sequence of a gene, it is readi!y apparent that the present embodiments include, but are not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
  • APC Antigen presenting cell
  • T cells includes, but is not limited to, monocytes/macrophages, B cells and dendritic ceils (DCs).
  • Antigen-loaded APC or an.
  • antigen-pulsed APC includes an APC, which has been exposed to an antigen and activated by the antigen. For example, an ARC may become Ag ⁇ loaded in vitro, e.g. , during culture in the presence of an antigen. The APC may also be loaded in vivo by exposure to an antigen.
  • An "'antigen- loaded APC” is traditionally prepared in one of two ways: (1) small peptide fragments, known as
  • antigenic peptides are "pulsed” directly onto the outside of the APCs; or (2) the APC is incubated with whole proteins or protei particles whic are then ingested by the APC. These proteins are digested into smallpepti.de fragments by the APC and are eventually transported to and presented on the APC surface.
  • the antigen-loaded APC can also he generated by introducing a polynucleotide encoding an antigen into the cell.
  • Anti-.HER2 response is the immune response specifically against HER2 protein.
  • Apoptosis- 5 is the process of programmed eel! death.
  • Caspase-3 is a frequently activated death protease.
  • the term ''autqtminune disease as used herein is defined as a disorder that, results from an autoimmune response.
  • An autoimmune disease i s the result of an inappropriate and excessive response to a self-antigen.
  • Examples of autoimmune diseases include bin are not limited to, Addision's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes- (Type I)*
  • dystrophic epidermolysis bullosa epididymitis, glomerulonephritis.
  • Graves' disease Guiilain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, .rayxedema, pernicious anemia, ulcerative colitis, among others .
  • autologous is meant to refer to any material derived from the same individual to which it is later to be re -introduced into the
  • B ceil as used herein is defined as a cell derived from the bone marrow and/or spleen. B cells can develo into plasma ceils whic produce: antibodies.
  • cancer as used herein is defined as a hyperproliferatioii of cells whose unique trait—loss of normal control—esults in unregulated growth, Sack of differenti tion, local tissue invasion, and/or metastasis. Examples include -but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer and lung cancer.
  • CD4 ; Thl cells,” “Thl cells,” “CD4+ T-heiper type 1 cells,” “CD4+ T cells,” and the like are defined as a subtype of T-heiper cells that express the surface protein CD4 and produce high levels of the cytokine IFN-y.
  • DC vaccination “DC immunization/' ' ' $ DC 1 immunization ' and the like refer to a strategy using autologous dendritic ceils to harness the immune system to recognize specific molecules and mount specific responses against them.
  • DC dendritic cell
  • Dendritic ceils and their precursors can be isolated from a variety of lymphoid organs, e.g., spleen, lymph nodes, as well as from bone marrow and peripheral blood.
  • the DC has a characteristic morphology with thin sheets (iarneSlipodia) extending in multiple directions away from the dendritic cell body.
  • dendritic cells express high levels of MHC and costimulatory (e.g., B7-3 and B7-2) molecules. Dendritic cells can induce antigen specific differentiation of T cells in vitro, and are able to initiate primary T cell responses in vitro and in vivo.
  • an " ' activated DC” is a DC that has been exposed to a ' Foil- like receptor agonist
  • the activated DC may or may not be loaded with an antigen.
  • mature DC is defined as a dendritic ceil that expresses molecules, including high levels of MHC class ⁇ , CD80 (B7.1) and CD86 (B7.2). in contrast, immature dendritic cells express low levels of MHC class II, CD80 (B7.1 ) and CD86 ⁇ B7.2) molecules, yet can still take up an antigen.
  • Mature DC also refers to an antige presenting cell existing in vivo, in vitro, ex vivo, or to a host or subject that is DCI-polarized (i.e., fully capable of promoting cell-mediated, immunity).
  • a "disease ' is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is riot ameliorated then the animal's health continues to deteriorate.
  • a “disorder" in an animal is a state of health in which the animal is able to maintain .homeostasis, bat in which the animal's state of health is less favorable than, it would be in the absence of the disorder. Left untreated, a disorder does not necessari ly cause a farther decrease in the animal's state of health.
  • a disease or disorder is "alleviated” i f the severity or frequency of at least one sign or symptom of the disease or disorder experienced by a patient is reduced,
  • Effective amount or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herei effective to achieve a particular biological result. Such results may include, but are not l imited to, the inhibition of virus infection as determined by any means suitable in the art
  • endogenous refers to any material from or produced inside an organism, ceil, tissue or system.
  • exogenous refers to any material introduced, from or produced outside an organism, cell, tissue or system.
  • Estrogen receptor (ER) positive cancer is cancer which tests positive for expression of ER. Conversely, "ER negative” cancer tests negative for such expression. Analysis of ER status can be performed by any method known in the art.
  • freeze drying medium refers to any medium mixed with a. cell sample in preparation for freezing, such that at least some of cells within the cell sample can be recovered and remain viable after thawing.
  • HER2 is a member of the human epidermal growth factor receptor
  • EGFR EGFR family
  • HER2 is overexpressed in approximately 20-25% of human breast cancer and is expressed in many other cancers
  • a "HER receptor” is a receptor protein tyrosine kinase which belongs to, the HER receptor family and includes EGFR (ErbBL HER!), HER2 (ErbB2), HER3 ( E.rbB3) and HER4 (ErbB4) receptors.
  • the HER receptor will generally comprise an extracellular domain, which may bind an HER ligand and/or dimerize with another HER receptor molecule; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-termraal signaling domain harboring several tyrosine residues which can be phosphorylated.
  • the HER receptor may be a "native sequence” HER receptor or an "amino acid sequence variant" thereof.
  • the HER receptor is a native sequence huma HER. receptor.
  • the "HER pathway” refers to the signaling network mediated, by the HER receptor family.
  • HE activation refers to activation,. or phosphorylation, of any one or more HER receptors. Generally, HER activation results i signal transduction (e.g. thai caused by an intracellular" kinase domain of HER receptor phosphorylating tyrosine residues in the HER receptor or a substrate polypeptide), HER activation may be
  • HER ligand binding to a HER dimer comprising the HER receptor of interest
  • HER ligand binding to a HER dimer may activate a kinase domain of one or more of the HER receptors in the dimer and thereby results in phosphorylatio of tyrosine residues in one or more of the HER receptors and/or phosphorylation of tyrosine residues in additional substrate polypeptides(s), such as Akt orMAPK intracellular kinases.
  • HER2 binding peptides refer to MHC Class II peptides derived from or based on the sequence of the i IER2/ «i3 ⁇ 4 protein, a target found on approximately 20-25% of ail human breast cancers and their equivalents.
  • HER2 extracellular domain refers to a domain of HER2 that is outside of a cell, either anchored to a cell membrane, or in circulation, including fragments thereof.
  • HER2 intracellular domain "ICD” refers to a domain of the
  • HER2/ «t3 ⁇ 4 protein within the cytoplasm of a cell HER2 epitopes or otherwise binding peptides comprise 6 HER2 binding peptides which include 3 HER2 ECD peptides and 3 HER2 ICD peptides.
  • Preferred HER2 ECD peptides comprise:
  • Peptide 42-56 BLDMLRHLYQGCQVV (8BQ ID NO: 1);
  • Peptide 9S-1 1 RLRiVRGTQLFEDr ⁇ YAL (SEQ ID NO: 2);
  • Peptide 328-345 TQRCEKCS PCARVCYGL (SEQ ID NO: 3); Preferred HE 2 ICD peptides comprise:
  • Peptide 776-790 GVGSPYVSRLLGICL (SEQ ID NO: 4);
  • Peptide 927-941 PAREIPDLLEKGERL (SEQ ID NO: 5);
  • Peptide 1 166-1 180 TLERPKTLSPG NGV (SEQ ID NO: 6).
  • HC class I peptides or epitopes comprise:
  • Peptide 369-377 lFGSLAFL (SEQ ID NO:7);
  • HER2 P is the classification or molecular subtype of a type of breast cancer as well as numerous other types of cancer. HER2 positivity is currently defined by gene amplification b FISH (fluorescent in situ hybridization) assay and 2 ⁇ or 3+ on intensity of pathological staining.
  • FISH fluorescent in situ hybridization
  • HER ⁇ ' is. defined! by the lack of gene amplificat on by FISH, and can encompass a . range of pathologic staining from 0 to 2+ i most cases.
  • hyperproliferative disease is defined as a disease that results from a hyperproliferation of cells.
  • exemplary hyperproliferative diseases include, but are not limited to, cancer or autoimmune diseases.
  • Other hyperproliferative diseases may include vascular occlusion, restenosis, atherosclerosis, or inflammatory bowel disease, for example.
  • inhibitor means to suppress or block an activity .or unction, for example, about ten percent relative to a control value. Preferably, the. activity is suppressed or blocked by 50% compared to a control value, more preferably by 75%, and even more preferably by 95%. "Inhibit,” as used herein, also means to reduce a molecule, a reaction, an interaction, gene, an mRNA, and or a protein's expression, stability, function or activity b a measurable amount or to prevent entirely.
  • Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., antagonists.
  • an "instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to
  • the instructional material of the kit of the embodiments may, for example, be affixed to a container which contain the nucleic acid, peptide, arid/or composition of the
  • the instnictiona! material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • Thl responses are defined for each subject group analyzed for aoti-HER2 CD * Thl immune response; (a) overall anti ⁇ HER2 responsivtty (expressed as percent of subjects responding to >! reactive peptide); (b) response repertoir (expressed as mean number of reactive peptides (n) recognized by each subject group); and (c) cumulative response (expressed as total sum of reactive spots (spot-forming cells "SFC" per 10* cells from iFN- ⁇ ELiSPOT analysis) from 6 MHC Class II binding peptides from each subject group.
  • SFC spot-forming cells
  • moduleating mediating a detectable increase or decrease in the level of a response in a subject compared with the le vel of a response in the subject in the absence of a treatment or compound, and/or compared wi th the level of a response in an otherwise identical but untreated subject.
  • Neoadjuvant therapy for breast cancer as used herein refers to treatment given before primary therapy (i.e., surgery).
  • adjuvant therapy is treatment given after primary therapy to increase the chance of long-term survival.
  • a “population.” includes reference to an isolated culture comprising a homogenous, a substantially homogenous, or a heterogeneous culture of cells. Generally, a “population” may also be regarded as an "isolated" culture of cells.
  • a "recombinant DC l" is a host cell that comprises a recombinant polynucleotide.
  • esponsivity or "antt-HE 2 responsivity” are used interchangeably herein to mean the percentage of subjects responding to at. least I of 6 binding peptides.
  • Response repertoire is defined as the mean n umber ("n") of reactive peptides recognized by each subject group.
  • sample or “biological sample” as used herein means a biological material from a subject, including but is not limited to organ, tissue, exosome, blood, plasma, saliva, urine and other body fl uid, A sample can be any source of materia! obtained from a subject.
  • Senescence refers to cells no longer capable of dividing but which are stil! alive and metabo!ica!iy active. Hallmarks of senescent cells include an essentially irreversible growth arrest, and expression of SA-p-gal, PI SINK
  • Signal 1 as used herein generally refers to the first biochemical signal passed from an acti vated DC to a T cell.
  • Signal 1 is provided by an. antige expressed at the surface of the DC and is sensed by the. T cell through the T ceil receptor .
  • Signal 2 as used herein generally refers to the second signal provided by DCs to T cells.
  • Signal 2 is provided by "costimulatory" molecules on the activated DC, usually CD80 and/or CD86 (although there are other co-stimulatory molecules known), and is sensed by the T cell through the surface receptor CD28.
  • Signal 3 as used herein generally refers to the signal generated from soluble proteins (usually cytokines) produced by the activated DC. These are sensed through receptors on the T lymphocyte. The 3 rd signal instructs the T cell as to which phenotypical or functional feature they should ac uire to best deal with the current threat.
  • ⁇ specifically binds as used herein, is meant a molecule, such as an antibody, which recognizes and binds to another molecule or feature, but does not substantially recognize or bind other molecules or features i a sample.
  • the patient, subject or individual is a human.
  • T/C is defined as uastuzumab and chemotherapy. This refers to patients that receive both trastazumab and chemotherapy before/after surgery for breast cancer.
  • T cell as used herein is defined as a thymus-derived cell that participates in a variety of cell-mediated immune reactions.
  • T ⁇ helper cells are used herein with reference to cells indicates a sub-group of lymphocytes (a type of white blood ceil or leukocyte) including different cell types identifiable by a skilled person in the art.
  • T-helper ceils are effector T-cells (such as Th 1, Th2 and Thl7) whose primary function is to promote the activaticm and functions of other B and T lymphocytes and/or macrophages.
  • Helper T cells differentiate into two major subtypes of cells known as "Th! or "Type I” and "Th2" or “Type 2" phenotypes. These Th cells secrete cytokines, proteins, or peptides that stimulate or interact with other leukocytes.
  • Th! cell “CDC Th! cell,” “CD4 ! T-helper type! cell,” “CM* T csH w and the like as used herein refer to a mature T-cell that has expressed the surface glycoprotein CD4.
  • CD4 ' T-helper ceils become activated when they are presented with peptide antigens by MHC class H molecules which are expressed on the surface of antigen-presenting peptides ("APCs" ) suc h as dendr itic cells.
  • CM T helper ceil Upon acti vation of a CM T helper ceil by the MHC -antigen complex, it secretes high levels of cytokines such as interferon-y flFN- ⁇ ("cytokines such as interferon-y flFN- ⁇ ”), Such cells are thought to be highly effective against certain disease-causing microbes that Hve inside host cells, and are critical in antitumor response in hum
  • Thi T celF refers to a T cell that produces high levels of the cytokine !F - ⁇ and is thought to be highly effective against certain disease-causing microbes that live inside host cells, and cancer as well.
  • Thl7 T cell refers to a T cell thai produces high levels of the cytokines IL ⁇ 17 and !L-22 and is thought to be highly effective against disease- causing microbes that live on mucousat surfaces.
  • “Therapeutically effective amount” is an amount of a compound of the embodiments, that when administered to a patient, ameliorates a symptom, of the disease.
  • the amount of a compound of the erabodiements which constitutes a “therapeutically effective amount” will vary depending on the compound, the disease state and its severity , the age of the patient to be treated, and the like.
  • the therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.
  • compositions of the present embodiments for example, a subject afflicted a disease or disorder, or a subject who ultimately may acquire such a d sease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorat one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • TLR Toll like receptor
  • Toll like receptor agonists or 4l TLR agonists
  • 4l TLR agonists as used herein is defined as a ligand that binds to the TL to acti ate immune cell response.
  • the term "vaccine” as used herein is defined as a material used to provoke an immune response after administration of the materia! to an animal, preferably a mammal, and more preferably a human. Upon introduction into a subject, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies, cytokines and/or other cellular responses. Ranges; throughout this disclosure., various aspects of the embodiment can be presented in a range format, it should be understood that the description i range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the embodiments.
  • a range should be considered to have spec.ifica.lly disclosed all the possible subranges as well as individual numerical values withi that range .
  • description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges soch as from 1 to 3, from 1 to 4, from 3 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example. 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range .
  • the present embodiments include a preparation of DCs.
  • the DC preparations are greater than 90% pure.
  • the DC preparations are fully activated.
  • the DCs are activated with a cytokine and''or a Toil like receptor Hgaiich a state which is fully maintained by the
  • a benefit of the DC preparation of the embodiments is that the cells are efficientl cryopreserved from a single leukapheresis (patient collection) into an initial vaccine plus multiple "booster" doses (e.g., 10 or more) that can be thawed as needed at remote treatment locations without any specialized cell processing facilities or further required quality control testing.
  • multiple "booster" doses e.g. 10 or more
  • the present embodiments provide a method for generating and cryopreservmg DCs with superior functionality in producing stronger signals to T cells, and thus resulting in a more potent DC -based vaccine.
  • samples can be stored and thawed for later use, thereby reducing the need for repeated pheresis and elutriation processes during vaccine production.
  • Being able to freeze DCs and then thaw them out later is an advantage because it means that a single round of vaccine production ca be divided into small parts, frozen away, and then administered one at a time to a patient over the course of weeks, months, or years to give "booster" vaccinations that strengthen immunity.
  • the present embodiments includes an FDA- approved injectable multi-dose antigen pulsed dendritic cell vaccine produced by coileciing DCs in a single patient leukaplieresis.
  • the FDA- approved injectable multi-dose antigen pulsed dendritic cell vaccine comprises an initial immunizing dose and multiple "booster" doses.
  • the FDA- approved injectable multi-dose antigen pulsed dendritic ceil vaccine are cryopreserved and can be shipped to remote medical centers for serial administration to the patient with no special requirements at the administrati site (e.g., FDA mandated QC QA steps).
  • the present embodiments also relate to the cryopreservatio of these activated DCs in a. manner that retains their potency and functionality in presenting antigen as well as their production of various cytokines and chemokines after thawing, such that the cryopreserved and subsequently thawed activated DCs are as clinically effective as freshly harvested and activated DCs,
  • the present embodiments also relate to inducing tumor senescence and apoptosis in a cell by blocking one or more of HER-2 and HER-3 in combination with activating anti-HER-2 CD4 Till cells. Accordingly, the embodiments include a combination and method for promoting an anti-oncodriver T i immune response with an oncodri ver blockade for HER-2 in order to promote tumor senescent in HER-2
  • promoting an anti-oncodriver Thi immune response comprises TNF ⁇ and IFN- ⁇
  • an oncodri ver blockade for HER-2 includes any composition that blocks HER-2 i cluding but is not limited to trastuxumab and pertuzumab.
  • compositions and methods for the combination of blocking one or more of HER-2 and HER-3 together with, the addition of TNF-a and IFN- ⁇ for inducing senescence of HER-2 expressing breast cancer in one embodiment, the TNF-a and lFN-y is secreted from CD4 Thi cells,
  • HER2 is required in the mechanism of TNF-a and IFN-y inducing senescence and apoptosis in breast cancer cells.
  • TNF-a and IFN- ⁇ restores the sensitivity to trastuzuBiah and pertumtmab to breast cancer resistant cells, in one embodiment, the Thi cytokines, IFN ⁇ y and TNF-a, revert the resistance to the therapeutic agents that, is affecting cancer patients widely.
  • DCs are derived from pluripotent monocytes that serve as antigen- presenting cells (APCs). DCs are ubiquitous in peripheral tissues, where they are prepared to capture antigens. Upon antigen capture, DCs process the antigen into small peptides and move towards secondary lymphoid organs. It is within the lymphoid organs that DCs present antigen peptides to naive T cells, thereby initiating a cascade of signals that polarizes T cell differentiation. Upon exposure, DCs present antigen molecules bound to either MHC class I or class II binding peptides and activate CDS' " or CD4* T cells, respectively (Stehnnan, 1991 , Anna. Rev. Immunol 9:271—296; Banchereaa.
  • DCs are responsible for the induction,, coordination and regulation of the adapti ve immune response and also serve to orchestrate communication, between effectors of the innate arm and the adaptive arm of the immune system. These features have made DCs strong candidates for immunotherapy, DCs have a unique capacity to sample the environment through macropinocytosis and receptor-mediated endocytosis (Gemer et al, 2008, J. Immunol.181:155-164; Sto.itar.ner et al. , 2008, Cancer Immunol Iimmmother 5 ; 1665- 1673; Lanzevecchia A., 1 96, Curr. Opin. Immunol 8:348-354; Delamarre et al, 2005, Science, 307(5715): 1630-1634).
  • DCs also require maturation signals to enhance their antigen-presenting capacity.
  • DCs upregulate the expression of surface molecules, such as CD80 and CD86 (also known as second signal molecules) by providing additional maturation signals, such as TNF-a. CD40L or calcium signaling agents (Czerniec et al, 1997,. J.
  • DCs can also be matured with calcium ionophore prior to being pulsed with antigen,
  • pathogen-recognition receptors such as PK.R and MDA-5
  • DCs also contain a series of receptors, known as Toll-like receptors
  • TLRs that are also capable of sensing danger from pathogens.
  • TLRs that are also capable of sensing danger from pathogens.
  • DCs DCs
  • DCs can activate and extend the various arms of the cell-mediated response, such as natural killer ⁇ T and ⁇ T cells and, once activated, DCs retain their immunizing capacity (Steinman, 19 1 , Anrai. Rev. Immunol 9:271 -296; anchereau et al., 1 98,
  • the present embodiments include mature, antigen loaded DCs activated by Toll-like receptor agonists that induce clinically effecti ve immune responses, preferably when used earlier in the disease process.
  • the DCs of the present embodiments produce desirable levels of cytokines and cheniokmes, and further have the capacity to induce apoptosis of tumor cells.
  • the method comprises rapidly maturing dendritic cells, cryopreservmg the dendritic cells, and thawing the cryopreserved cells wherein the thawed dendritic cells produce an effective amount of at least one cytokine to generate a T cell response.
  • the maturation, of dendritic cells comprise contacting the cells with IF -gamma and LPS.
  • the thawed cells maintain. DO phenotype to drive a
  • the thawed cells maintain the ability to primarily sensitize T cells.
  • an AFC such as a DC
  • an AFC may become Ag-ioaded in vitro, e.g., by culture ex vivo in the presence of an antigen, or in vivo by exposure to an antigen.
  • APC can be "pulsed” in a manner that exposes the APC to an antigen for a time sufficient to promote presentation of that antigen on the surface of the APC.
  • an APC can be exposed to an antigen in the form of small peptide fragments, known as antigenic peptides, which axe "pulsed" directly onto the outside of the APCs (Mehta-Damaiii t al., 1 94); or APCs can be incubated with whole proteins or protein particles which are then ingested by the APCs, These whole proteins are digested into small peptide fragments by the APC and eventually carried to and presented on the APC surface (Cohen et a!,, 1994). Antigen in peptide form may be exposed to the cell by standard "pulsing" techniques described herein.
  • the antigen in the form of a foreign or an autoantigen is processed by the APC of the embodiments in order to retain the immunogenic form of he antigen.
  • the immunogenic form of the antigen implies processing of the antigen through fragmentation to produce a form of the antigen that can be recognized b and stimulate immune cells, for example T cells.
  • a foreign or an autoantigen is a protein which is processed into a peptide b the APC.
  • the relevant peptide which is produced by the APC may be extracted and purified for use as an immunogenic composition.
  • Peptides processed by the APC may also be used to induce tolerance to the proteins processed by the APC.
  • the antigen-loaded APC is produced by exposure of the APC to an. antigen either in vitro or in vi .
  • the APC can be plated on a culture dish and exposed to an antigen in a sufficien t amount and for a s uffic ient period of time to allow the antigen to bind to the APC,
  • the amount and time necessary to achieve binding of the antigen to the APC may be determined by using methods known in. the art or otherwise disclosed herein. Other methods known to those of skill in the art, for example immunoassays or binding assays, may be used to detect the presence of antigen on the APC following exposure to the antigen.
  • the APC may be transfected with a vector which allows for the expression of a specific protein by the APC
  • the protein, which is expressed by the APC may then be processed and presented on the cell surface.
  • the transfected APC may then be used as an immunogenic composition to produce an.
  • vectors may be prepared to include a specific polynucleotide which encodes and expresses a protein to which an immunogenic response is desired.
  • retroviral vectors are used to infect the cells.
  • adenoviral vectors are used to infect the cells,
  • a vecior may be targeted to an APC by modifying the viral vector to encode a protein or portions thereof that is recogni zed by a receptor on the APC, whereb occupation of the APC receptor b the vector will initiate endocytosis of the vector, al lo wing for processing and presentation of the antige encoded by the nucleic ac id of the viral vector .
  • the nucleic acid which is delivered by the virus may be native to the virus, which when expressed on the APC encodes viral proteins which are then processed and presented on the MHC receptor of the A P
  • various methods can be used for transfecting a polynucleotide into a host cell.
  • the methods include, but are not limited to, calcium phosphate precipitation, lipofection, particle bombardment, microinjection,
  • colloidal dispersion systems i.e. macromolecute complexes
  • nanoeapsules microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes).
  • a polynucleotide encoding an antigen in another embodiment can be cloned into an expression vector and the vector can be introduced into an APC to otherwise generate a loaded APC.
  • the expression vector can be transferred into a host cell by physical, chemical or
  • polynucleotide encoding an antigen yields a pulsed cell.
  • the present embodiments include various methods for pulsing APCs including, but not limited to, loading APCs with whole antige in the form of a protein, cDNA or mRNA.
  • the embodiments should not be construed to be limited to the speci fic form of the antigen used for pulsing the APC, R ather, the embodiments encompass other methods known in the art for generating an antigen loaded APC.
  • the APC is tranfected with mRNA encoding a defined antigen, mRNA corresponding to a gene product whose sequence is known can be rapidly generated in vitro using appropriate primers and reverse transcriptase-polymerase chain reaction (RT- PCR) coupled with transcripti on reactions.
  • Transfection of an APC with an mRNA provides an advantage over other antigen-loading techniques for generating a pulsed APC. For example, the ability to amplify RNA from a microscopic amount of tissue, i.e. tumor tissue, extends the use of the APC fo vaccination to large number of patients .
  • an antigenic composition for an antigenic composition to he useful as a vaccine, the antigenic composition must induce an immune response to the antigen in a cell, tissue or mammal (e.g., a human).
  • an "immunological composition” may comprise an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding an antigen (e.g., an antigen expression vector), or a cell expressing or presenting an antigen or cellular component.
  • the antigenic composition comprises or encodes all or part of any antigen described herein, or an immunologically functional equivalent thereo
  • th antigenic composition is in a mixture that comprises m additional immunostimulatory agent or nucleic acids encoding such an agent.
  • Imm.unostimulatory agents include but are not limited to an additional antigen, an immunomodulatory an antigen presenting cell or an adjuvant, in other embodiments, one or more of ' the additional agentfs) is covalently bonded to the antigen or an
  • the antigenic composition is conjugated to or comprises an HLA anchor motif amino acids.
  • a vaccine may vary in its composition of nucleic acid and/or cellular components.
  • a nucleic encoding an antigen might also be formulated with an adjuvant.
  • compositions described herein may further comprise additional components.
  • one or more vaccine components may be comprised in a lipid or liposome, in another non-limiting example, a vaccine may comprise one or more adjuvants.
  • a vaccine of the present embodiments, and its various components may be prepared and/or administered by any method disclosed herein or as would be known to one of ordinary skill in the art, in light of the present disclosure.
  • an antigenic composition of the presen embodiments may be made by a method thai is well known, in the art, including hut not limited to chemical synthesis by solid phase synthesis and purification away from the other products of the chemical reactions by HPLC, or production by the expression of a nucleic acid sequence (e.g., DNA sequence) encoding a peptide or polypeptide comprising an antigen of the present embodiments in an in vitro translation system or in a living cell, in addition, an antigenic composition can comprise a cellular component isolated from a biological sample. The antigenic composition isolated, and extensively dialyzed to remove one o more undesired small molecular weight molecules and/or lyopliilized for more ready formulation into a desired vehicle. It is further understood that additional amino acids, mutations, chemical modification and such like, if any, that are made in a vaccine component will preferably not substantially interfere with the antibody
  • determinants of the present embodiments should generally be at least five or six amino acid residues in length, and may contain up to about 10, about 15, about 20, about 25, about 3.0, about 35, about 40, about 45 or about 50 residues or so.
  • a peptide sequence may be synthesized by methods known to those of ordinary skill in the art, such as, for example, peptide ' synthesis using automated peptide synthesis machines, such as those available from Applied Biosysiems, lac, Foster City. CA (Foster City, CA).
  • a nucleic acid encoding an antigenic composition .and/or a component desc ed herein may be used, for example, to produce an antigenic composition in vitro or in vivo for me various compositions aod methods of the present embodiments.
  • a nucleic acid encoding an antigen is comprised in, for example, a vector in a recombinant cell.
  • the nucleic acid may be expressed to produce a peptide or polypeptide comprising an antigenic sequence.
  • the peptide or polypeptide may be secreted from the cell, or comprised as part of or within the cell.
  • an immune response may be promoted by transfectiftg o inoculating a mamma! with a nucleic acid encoding an antigen.
  • One or more cells comprised within a target mamma! then expresses the sequences encoded by the nucleic acid after administration of the nucleic acid to the mamma!.
  • a vaccine may also be in the form, for example, of a nucleic acid (e.g., a cDN A or an RNA) encoding all or part of the peptide or polypeptide sequence of an antigen.
  • Expression in vivo by the nucleic acid may be, for example, by a plasraid type vector, a viral vector, or a viral/plasmid construct vector.
  • the nucleic acid comprises a coding region that encodes all or part of the sequences encoding an appropriate antigen, or an
  • nucleic acid may comprise and/or encode additional sequences, including but not limited to those comprising one or more immunomodulators or adjuvants.
  • the present embodiments may include use o f any antige suitable for loading into an. APC to elicit an. immune response.
  • any antige suitable for loading into an. APC to elicit an. immune response.
  • tumor antigens may be used. Tumor antigens can be divided into two broad categories: shared tumor antigens; and unique tumor antigens. Shared antigens are expressed by many tumors, while unique tumor antigens can result from mutations induced through physical or chemical carcinogens, and are therefore expressed only by individual tumors. In certain embodiments, shared tumor antigens are loaded into the DCs of the present embodiments. In other embodiments, unique tumor antigens are loaded into the DCs of the present embodiments.
  • inmor antigen refers to antigens that are common to specific hyperproliferative disorders. I certain aspects, the hyperproliferative disorder antigens of the present embodiments are derived from
  • cancers including but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkins lymphoma, leiikeraias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.
  • Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include, but are not limited to, tissue- specific antigens such as MART- 1 , tyrosinase and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer.
  • tissue-specific antigens such as MART- 1 , tyrosinase and GP 100 in melanoma
  • PAP prostatic acid phosphatase
  • PSA prostate-specific antigen
  • Other target molecules belong to the group of transformation-related molecules, such as the oncogene HBR ⁇ 2/ eu/ErbB ⁇ 2.
  • antigens are onco-fetal antigens, such as carcinoembryonic antigen (CEA).
  • the tumor- specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor, B cell differentiation, antigens, such as CD 19, CD20 and CD37, are other candidates for target antigens in B cell lymphoma.
  • B cell differentiation, antigens such as CD 19, CD20 and CD37.
  • Some of these antigens have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.
  • the tumor antigen and the antigenic cancer epitopes thereof may be purified and isolated from natural sources such as from primary ciinicai isolates, ceil lines and the like.
  • the cancer peptides and their antigenic epitopes may also be obtained by chemical synthesis or by recombinant DMA techniques known hi the arts. Techniques for chemical synthesis are described in Steward et at. (1969); Bodansk et al. (!9?6);
  • the present embodiments may include microbial antigens for presentation by the AFCs.
  • microbial antigens may be viral, bacterial, or fungal, in origin.
  • infectious virus include: Retroviridae (e.g. human immunodeficiency viruses, such as HlV-1 (also referred to as HTLV-IH, LAV or HTLV-III/LAV, or HIV- ⁇ ; and other isolates, such as HIV-LP; Pico navMdae (e.g. polio viruses,, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echov ruses); Calciviridae (e.g.
  • Togaviridae e.g. equine encephalitis viruses, rubella viruses
  • Flaviridae e.g. dengue viruses, encephalitis viruses, yellow fever viruses
  • Coronaviridae e.g. corona viruses
  • Rhabdoviridae e.g. vesicular stomatitis viruses, rabies viruses
  • FiSoviridae e.g. ebola viruses
  • Para yxoviridae e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus
  • Orthoniyxoviridae e.g. influenza viruses
  • Bungaviridae e.g.
  • Arena viridae hemorrhagic fever viruses: Reoviridae (e.g. reoviruses, orbiviruses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);
  • Papova iridae papilloma viruses, polyoma viruses
  • Adenoviridae most adeiiovimses
  • i lerpesviridae herpes simplex virus (BSY) 1 and 2, varicella zoster virus
  • CMV cytomegalovirus
  • Poxviridae variola viruses, vaccima viruses, pox viruses
  • Iridoviridae e.g. African, swine fever virus
  • unclassified viruses e.g. the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis
  • infectious bacteria examples include'.. Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophila, Mycobacteria, sps (e.g. M. tuberculosis, M. avium, M. intraceHulare, M. kansasii, M. gordonae). Staphylococcus aureus, Neisseria gonorrhoeae. Neisseria meningitidis. Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
  • Streptococcus (viridans group). Streptococcus faecalis, Streptococcus bovis,
  • Streptococcus (anaerobic sps.), Streptococcus pneumoniae., pathogenic Campylobacter sp. s Enterococcus sp., Haemophilus influenzae, Bacillus antbracis, corynebacteriura diphtheriae, corynebacterium sp,, Erysipelothrix rhusiopathiae, Clostridium perfrin.ge.ns, Clostridium tetaai, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bactero des sp., Fusobacterium nucleatum, StxeptobaciUus moniliformis, Treponema Treponema permur, Leptospira, and Actinomyces israelii.
  • infectious fungi examples include: Cryptococeus neoformans,
  • DC-based vaccines that have previously dominated clinical trials
  • a cytokine cocktail mixture including combinations of TNF, IL-6, PGE2 and IL- ⁇ ⁇ , which ultimately stimulate aseptic inflammation
  • the present embodiments instead utilize TLR agonists to mature the DCs and stimulate production of signal.
  • DCs with a combination of TLR !igands leads to the production of increased amounts of IL-12. Further, activation of DCs with a combination of TLR agonists can yield a more pronounced CD4 and CDS T ⁇ eell response (Warger et al., 2006, Blood .108:544-550). Thus, the DCs of the present embodiments can secrete Th l driving cytokines, such as IL- 2, by exposure to these !igands that trigger TLRs.
  • a TLR3 agonist to IL-.1 ⁇ , TNF- ⁇ , and IFN- ⁇ can generate a potent type-1 polarized DC, characterized by robust levels of IL-12 produc tion (Heifler et al. , 1996, Em. J. Immunol.. 26: 59-668).
  • antigen can be loaded into the DC prior to TLR agonist exposure. In other embodiments, antigen can he loaded into the DC subsequent to TLR agonist exposure.
  • the injectable multi- dose antigen pulsed dendritic cell vaccine is produced by collecting DCs in a single patient leukapheresis whereby the cells are activated with biomolecules mat simulate bacterial infection (e.g., LPS).
  • biomolecules mat simulate bacterial infection (e.g., LPS).
  • This unique activation method endows the DCs with qualities not found in DCs that are matured with a cytokine cocktail of TNF, IL-6, PGE2 and IL-lp (the "traditional maturation"), which also, simulates aseptic inflammation (Lombardi et aL, 2009, J. Immunol. 182:3372-3379).
  • the DCs of the present embodiments can be acti vated wit the combination of the TLR4 agonist, bacteria! !ipopo!ysaccharide (LPS), the TLR7/8 agonist, resiniiquod (R848) and/or IFN-y (Amati et aL 2006, Curr. Pbarra, Des 12:4247-4254),
  • acti vating DCs with a TLR4 agonist and bacterial LPS DCs are generated that are at least virtually identical (in phenotype) to DCls generated via traditional maturation methods.
  • These DCs have a high expression of surface molecules, including CD83, CD80, CD86 and HLA-DR.
  • TLR2 agonists such as lipotechoic acid (LTA), TLR3 agonists, such as polyCLC), and/or other TLR4 agonists, siich as MPL, may be used.
  • LTA lipotechoic acid
  • TLR3 agonists such as polyCLC
  • TLR4 agonists siich as MPL
  • any TLR agonist, or combination of TLR agonists can be used to active DCs, provided such ligands stimulate tire production of cytokine and chemokiae signals by the activated DCs.
  • Many other TLR agonists are known in the art and can be found in the published literature for use with the present embodiments.
  • the ceils ar harvested and the vaccine is cryopreserved.
  • peripheral blood monocytes are obtained by leukapheresis.
  • the cells are cultured in se um fee medium with GM-CSF and I L ⁇ 4 for a period, of time followed by pulsing the cells with a desired antigen.
  • the antigen pulsed dendritic ceils are incubated with 1 FN- ⁇ followed by a TLR agonist (e.g., LPS),
  • the activated antigen pulsed dendritic cell is harvested and eryopreserved in a freezing medium and stored in liquid nitrogen.
  • the freezing medi um comprises 55% plasmalyte, 40% human serum
  • the eryopreservation aspect of the embodiments allow for the generation of an FDA- approved injectable multi-dose antigen pulsed dendritic cell vaccine.
  • An advantage of the embodiments is that the multi-dose antigen pulsed dendritic ceils retain their ability to produce signals critical to T cell function after thawing.
  • the present embodiments include a variety of eryopreservation techniques and cr omedia, as would be understood by those skilled in the art.
  • the freezing medium comprises 55% plasmalyte, 40% human serum albumin, and 5% DMSO.
  • the embodiments provide the ability to produce the multi-dose antigen pulsed dendritic cell vaccine of the embodiments at a centralized area comprising of an initial immunizing dose and multiple "booster" doses. Therefore the multi-dose antigen pulsed dendritic cell vaccine can be shipped to remote medical cemers for serial administration to the patient with no special FDA qualit control/quality assurance requirements at the administration site.
  • the dendritic cell vaccine of the embodiments is eryopreserved in a!iquots for multiple doses.
  • the cells are eryopreserved at a concentration of 30x10 6 cells/mL.
  • a bag of freezing medi um containing a volume equal to the cell volume is prepared. Working rapidly, the freezing medium is added to the cell bag and the cells are transferred to labeled cryovials..
  • the vials are frozen using a rate controlled freezer.
  • cryovials are frozen using an automated rate controlled freezer at C/min and stored in vapor phase nitrogen.
  • the vials are frozen using a rate controlled freezer.
  • the vials are placed in a freezing chamber and liquid nitrogen enters the chamber through an electronic solenoid valve. Since vaporization is almost instantaneous, controlling the rat at which liquid nitrogen enters the chamber directly controls the rate at which heat is absorbed and removed from the freezing chamber and its contents.
  • the present embodiments include a variety of eryopreservation techniques and freezing medium, as would be understood by those skilled in the art.
  • the freezing medium for cultured cells can include about 55% pJasraal te, about 40% human serum albumin, and about 5% DMSO.
  • the cryomedia can be serum-free,
  • controlled rate freezing may be used, while other embodiments can include use of insulated containers in which vials of cells mixed with freezing medium are placed in the freezer, such as at temperatures ranging from about -70*C to -80 & C, The present
  • embodiments provide a method to preserve acti vated DCs in such a manner so as to further facilitate clinical application of such cells., and to reduce the need for extensive and repeated pherisis and . etutriation steps.
  • cryopreservatton techniques may be used for both small-scale and large-scale batches.
  • cryopreserved activated DC When considering the extensive utility for activated DC, the ability to provide a steady supply of cryopreserved activated DC represents a significant advantage that can facilitate various therapeutic uses of such cells. For example, a large-scale culture of activated DC may be cryopreserved in aliquots of the appropriate size
  • activated DCs can be cryopreserved for 2-24 weeks at temperatures of approximately -70°C or lower. At lower temperatures, such as at about -120°C or lower, acti vated DCs can be
  • the DCs axe suspended in human serum and approximately 5% DMSO (v v).
  • serum types such as fetal calf serum
  • the suspended cells can be aliqitoted into smaller samples, such as in 1.8 ml vials, and stored at approximately -70°C or lower.
  • the freezing medium may include about 20% serum and about 10% DMSO, and suspended cells can be stored at about. - I SO ⁇ C.
  • Stii! further embodiments may include medium containing about 55% piasrnalyte, and about 5% DMSO.
  • Other exemplary freezing media may include about 12% DMSO and about 25-40% serum.
  • present embodiments as described herein may include specific concentrations of serum, it should be understood by those skilled in the art that the exact amount of serum in the freezing medium may vary, and in some embodiments ma be entirely absent, but will generally be within the range of about 1% to 30%.
  • any concentration of serum that results in a cell viability of around 50% and/or a cell recovery of around 50% may be used in any D composition of the present embodiments., as well as with any cryopreservation method as described herein.
  • cell viability and recovery of at least 60%, more preferably at least about 70%, or even 80% is desired when recovering cryopreserved ceils in the selected freezing medium.
  • DM SO may be entirely absent in some embodiments, while in other embodiments,
  • concentrations from about 5% to as high as about 20% may be used in the freezing medium and included within the cryopreservation. methods described herein. Generally, Sower concentrations of DMSO are preferred, such as between about 5% to about 10%. However, any concentration of DMSO that results, after thawing, in ceil viability of at least 50% and a cell recovery of at least 50% . and preferably a cell, viabil ity and recovery of at least 60%, more preferably about 70%, more preferably about 80% and even more preferably about 90% and higher, may be used.
  • cryopreservation media as described herein may either include serum or may be serum free.
  • serum free media can include XV1VO 10, XVIVO 15, XV1VO 20, StemP.ro, as well as any commercially available serum free media.
  • the cryopreservation methods of the present embodiments are generall free of infectious agents, antibodies and foreign proteins, which may be antigenic, and any other foreign molecule that may typically be found in serum-based freezing medium.
  • Cryopreservation ' .of antigen loaded, active DCs can occur at an point after activation of the cells with a TLR agonist, in one embodiment, the activated DCs are cryopreserved approximately 6-8 hr after exposure to the TLR agonist. Preferably, the time point chosen to cryopreserve the activated cells should be based on the
  • the present embodiments provide compositions and methods for producing large scale dendritic cell vaccines.
  • the large scale dendritic cell vaccines In one embodiment, the large scale
  • dendritic cell vaccines allows for the production of an FDA-approved injectable multi-dose antigen pulsed dendritic ceil vaccine for the personalized treatment and prevention of cancer or other disorders.
  • die embodiments provide a method to cryopreserve dendritic ceils that are in an antigen-loaded, pre-activated state in a large scale that is "syringe-ready", i.e. suitable for immediate injection into the patient without the
  • die embodiments provide a method to efficiently produce in a large scale injectable multi-dose antigen pulsed dendritic cell vaccine, preferably injectable multi-dose antigen pulsed type I polarized dendritic cell vaccine that exhibit maximal efficacy.
  • Suitable containers for compositions of the embodiments include vials, syringes (e.g. disposable syringes), etc. These containers should be sterile.
  • the vial is preferably made of a glass or plastic material.
  • the via! is preferably sterilized before the
  • vials are preferably sealed with a latex-free stopper, and the absence of latex in all packaging material, is preferred.
  • the vial may include a single dose of vaccine, or it may inc lude more than one dose (a "raultidose" vial) e.g. 10 doses.
  • Preferred vials are made of colorless glass.
  • a vial can have a cap (e.g. a Liter lock) adapted such that a pre- filled syringe can be inserted into the cap, the contents of the syringe can he expelled into the vial, and the contents of the vial can he removed back into the syri nge.
  • a needle can then be attached and the composition can be administered to a patient.
  • the cap is preferably located inside a seal or cover, such that the seal or cover has to be removed before the cap can be accessed.
  • a vial may have a cap that permits aseptic removal of its contents, particularly for multidose vials.
  • the syringe may have a needle attached to it. If a needle is not attached, a separate needle may be supplied with the syringe for assembly and use. Such a needle may be sheathed. Safety needles are preferred. 1-inch 23-gauge, 1-inch 25-gauge and 5/8-inch 25-gauge needles are typical. Syringes may be provided with peel-off labels o which the lot number, influenza season and expiration date of the contents may be printed, to facilitate record keeping.
  • the plunger in the syringe preferably has a stopper to prevent the plunger from being accidentally removed during aspiration.
  • the syringes may have a latex rubber cap and/or plunger.
  • Disposable syringes contain a single dose of vaccine.
  • the syringe will generally have a tip cap to seal the tip prior to attachment of a needle, and the tip cap is preferably made of a butyl rubber. If the syringe and needle are packaged separately then the needle is preferably fitted with a butyl rubber shield,
  • Containers may be marked to show half-dose volume e.g. to facilitate delivery to children. For instance, a syringe containing a 0,5 ml dose ma have a mark showing a 0.25 ml volume.
  • a glass container e.g. a syringe or a vial
  • a container made from a borosiiicate glass rather than from a soda lime glass.
  • a kit or composition may be packaged (e.g. in the. same box) with a leaflet including details of the vaccine e.g. instructions for administration, details of the antigens within the vaccine, etc.
  • the instructions may also contain warnings e.g, to keep a solution of adrenaline readily available in case of anaphylactic reaction following vaccination, etc.
  • Methods for Treating a Disease also encompass methods of treatment and/or prevention of a disease caused by pathogenic microorganisms, autoimmune disorder and/or a hyperproliferative disease.
  • inventions include diseases caused by viruses, bacteria, yeast, parasites, protozoa, cancer cells and the like.
  • the pharmaceutical composition of the present embodi ments may be used as a generalized immune enhancer (DC activating composition, or system) and as such has utility in treating diseases.
  • Exemplary diseases that can. be txeaied and/or prevented utilizing the pharmaceutical compositio of the present embodiments include, but are not limited to infections of viral etiology such as HIV, influenza.
  • Preneoplastic or hyperplastic states that may be treated or prevented using the pharmaceutical composition of the present embodiments (transduced DCs, expressio vector, expression construct; etc. ) of the present embodiments include but are not limited to preneoplastic or hyperplastic states such as colo polyps, Crohn's disease, ulcerative colitis, breast lesions and the like.
  • Cancers that may be treated using the composition of the present embodiments include, but are not limited to primary or metastatic melanoma,
  • adenocarcinoma adenocarcinoma, squamous ceil carcinoma, adenosquamous cell carcinoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leuke.rn.ias, uterine cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, NPC, bladder cancer, cervical cancer and the like.
  • hyperproliferative diseases that may be treated using DC activation system of me present embodiments include, but are not limited to rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas.
  • hemangiomas hemangiomas, fibronras, vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions (such as adenomatous hyperplasia and prostatic intraepithelial neoplasia), carcinoma in situ, oral hair ⁇ ' leukoplakia, or psoriasis.
  • Autoimmune disorders that ma be treated using the composition of the present embodiments include, but are not limited to, AIDS.
  • Graves' disease hypereosinophiiia, irritable bowel syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
  • osteoarthritis osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, and autoimmune thyroiditis; complications of cancer, hemodialysis, and extracorporeal circulation; viral, bacterial, fungal, parasitic, protozoal, and helminthic infections; and trauma.
  • the administration of the composition of the embodiments may be for either "prophylactic" or '"therapeutic" purpose.
  • the composition of the present embodiments is provided in advance of any symptom, although in particular embodiments the vaccine is provided following the onset of one or more symptoms to prevent farther symptoms from developing or to prevent present symptoms from becoming worse.
  • the prophylactic administration of composition serves to prevent or ameliorate any subsequent infection or disease.
  • the pharmaceutical composition is provided at or after the onset of a symptom of infection or disease.
  • the present embodiments may be provided either prior to the anticipated exposure to a disease-causing agent or disease state or after the initiation of the infection or disease.
  • an effective amount of the composition would be the amount that achieves this selected result of enhancing the immune response, and such an amount could be determined as a matter of routine by a person skilled in the art.
  • an effective amount of for treating an immune system defici ncy against cancer or pathogen could be that amount necessary to cause activation of the immune system, resulting in th development of an antigen specific immune response upon exposure to antigen.
  • the term is also synonymous with "sufficient amount.”
  • the effective amount for any particular application cm. vary depending on such factors as the disease or condition being treated, the particular composition being administered, the size of the subject, and/or the severity of the disease or condition.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular composition of the present embodiments without necessitating undue experimentation.
  • the present embodiments include the generation of an antigen loaded, activated APC that produces significant levels of cytokines and chernokin.es when thawed from ci opreservation, where the antigen loaded and activated APC is used in
  • the response to aa antigen presented by an APC may be measured by monitoring the induction of a cytolytic T-cell response, helper T-cell response, and/or antibody response to the antigen using method well known in the art.
  • the present embodiments include a method of enhancing the immune response in a mammal comprising the steps of; generating immature DCs from monocytes obtained from a mamma! (e.g., a patient); pulsing the immature DCs with a composition comprising an antigenic composition; activating the antigen loaded DCs with at least one TL agonist; cryopreserving the activated, antigen loaded DCs; thawing the activated, antigen loaded DCs and then administering the activated, antigen loaded DCs t a mammal in need thereof.
  • the composition includes ' at least an antigen, and may further be a vaccine for ex vivo immunization and/or in vivo therapy in a mammal.
  • the mammal is a human.
  • cells are isolated from a mammal (preferably a human).
  • the cells can be administered to a mammalian recipient to provide a therapeutic benefit.
  • the mammalian recipient may be a human and die cells can be autologous with respect to the recipient.
  • the cells can be allogeneic, syngeneic or xenogeneic with, respect to the recipient
  • peripheral blood monocytes are obtained from, a patient by combined leukapheresis and ehitriation.
  • the monocytes can be cultured in SFM with GM-CSF and IL-4 overnight.
  • the next clay, immature DCs can. he pulsed with antigen, followeded by contacting the DCs with IFN- ⁇ and LPS.
  • the activated DCs can then be suspended in a freezing medium and frozen until ready for use in
  • Cryo preserved D s can be : .cultured ex vivo under conditions effective to generate the percent recovery and percent viabi lity of the cells as compared freshly activated DCs.
  • DCs generated from crvopreserved samples ca show similar stability as compared to freshly prepared DCs.
  • comparisons of cryopreserved mature DCs with those of freshly prepared DCs can show virtually identical phenoiypes as well as signal secretion profiles.
  • DCs can be preserved at both small and large scale for approximately 2 to 24 weeks, in the various freezing media described herein, at temperatures of approximately ⁇ 70*C to -80 .
  • the duration of storage can be extended indefinite ly or at least beyond 24 weeks without impacting cell recovery, viability, and functionality of the DCs.
  • the activated ceils can be preserved for at least one year and still retain their ability to produce signal after thawing.
  • the present embodiments provide fo effective recovery and viabilit profiles upon thawing the cells, and furthermore the cryopreservation conditions described herei do not affect the ability of DCs to retain their signal profiles as explained herein throughout.
  • the mixture can then be a!iquoted in 1.8 ml via!s and frozen at about -80°C in a cryochamber overnight. Vials can then be transferred to liquid nitrogen tanks the following day.
  • the frozen DCs ca be thawed and examined for their recovery and viability. Recovery of such DCs can be greater than or equal to about 70% with a viability of greater than or equal to about 70%.
  • DCs or even monocytes can be cryopreserved prior to cell activation.
  • a variety of cell selection techniques are known for identifying and separating ceils from a population of cells.
  • monoclonal antibodies or other specific cell binding proteins
  • markers or cell surface antigens are known in the art..
  • the presen embodiments further include vaccine formulations suitable for use in immunotherapy.
  • vaccine formulations are used for the prevention and/or treatment of a disease, such as cancer and infectious diseases.
  • the administration to a patient of a vaccine in accordance with, the present embodiments for the prevention and/or treatment of cancer can take place before or after a surgical procedure to remove the cancer, before or after a che otherapeutic procedure for the treatment of cancer, and before or after radiation therapy for the treatment of cancer and any combination thereof.
  • the vaccine formulations may be administrated to a patient in conjunction or combination with another composition or pharmaceutical product. It should be appreciated that the present embodiments can also be used to prevent cancer in individual ' s without cancer, but who might be at risk of developing cancer.
  • a cancer vaccine prepared in accordance with the present embodi men ts is broadly applicable to the prevention or treatment of cancer, determined in part b the selection of antigens forming part of the cancer vaccine.
  • Cancers that can be suitably treated in accordance with the practices of the present embodiments include, without limitation, cancers of the lung, breast, ovary, cervix, colon, head and neck, pancreas, prostate, stomach, bladder, kidney, bone, liver, esophagus, • gastroesophageal., brain, testicle., uterus and the various leuke ias and lymphomas,
  • vaccines can be derived from the tumor -or cancer cells to be treated.
  • the lung cancer cells would be treated as described hereinabove to produce a lung cancer vaccine.
  • breast cancer vaccine, colon cancer vaccine, pancreas cancer vaccine, stomach cancer vaccine, bladder cancer vaccine, kidney cancer vaccine and the like would be produced and employed as im unotherapeutc agents in accordance with the practices tor the prevention and/or treatment of the tumor or cancer cell from which the vaccine was produced.
  • vaccines could, as stated, also be prepared to treat various infectious diseases which affect mammals, by collecting the relevant antigens shed into a culture medium by the pathogen.
  • polyvalent vaccines can be prepared by preparing the vaccine from a pool of organisms expressing the different antigens o importance.
  • the vaccine can be administered by intranodal injection into groin nodes.
  • the vaccine can he i tradermally or snbcu!aneously administered to the extremities, arms and legs, of the patients being treated.
  • this approach is generally satisfactory for melanoma and other cancers, including the prevention or treatment of infectious diseases, other routes of administration, such as intramuscularly or into the blood stream may also be used.
  • the vaccme can be given together with adjuvants and/or immune-modulators to boost the activity of the vaccine and t he patient's response.
  • adjuvants and/or mununo-modulators are understood by those skilled in the art, and are readily described in available published literature.
  • the production of vaccine can, if desired, be scaled up by culturing cell s in bioreactors or fermentors or other such vessels or devices suitable for the growing of cells in bulk.
  • the culture medium would be collected regularly, frequently or continuously to recover therefrom any materials or antigens before such materials or antigens are degraded in. the culture medium.
  • devices or compositions containing the vaccine or antigens produced and recovered, in accordance with the present embodiments, and suitable for sustained or intermitteBt release could be, in effect, implanted in the body or topically applied thereto for a relatively slow or timed release of such materials into the body.
  • the present embodiments provide an effective therapy to treat cancer wherein the therapy includes changing the immune response in the tumor so that the immune cells in the tumor site are more effective in attacking the tumor cells, m some instances, the effective therapy includes improving the migration and activity of immune cells in the tumor site.
  • the treatment regimen comprises the use of a dendritic cell vaccine, an inhibitor of one or more of H0ER2 and HE 3, and a chemokine .modulator.
  • the chemokine modulator is a chemokine-activating agent.
  • An example of a chemokine-activating agent is a TLR8 agonist.
  • compositions and methods of using a dendritic cell vaccine in combination with blockage of HER-2 and HER-3 as a treatment regimen to treat cancer in another embodiment, there are provided
  • compositions and methods of using a dendritic cell vaccine in combination with blockage of HER-2 and HER-3 with TNF ⁇ a and I F -v are provided.
  • compositions and methods of blocking both of HER-2 and HER-3 with the addition of TNF ⁇ a and IFN- ⁇ as a treatment regimen to treat cancer are provided.
  • a treatment regimen can be used to treat cancer and therefore can be considered as a type of anti-cancer therapy
  • a treatment regimen can be used m the context of a combination therapy with another anticancer or anti-tumor therapy including but not limited to surgery, chemotherapy.
  • radiation therapy e.g. X ray
  • geae therapy e.g., immunotherapy
  • hormone therapy e.g., viral therapy
  • DMA therapy e.g., DMA therapy
  • R A therapy e.g., protein therapy, cellular therapy, and nanotherapy.
  • a treatment regimen in combination with another cancer medicament for the treatment or prevention of cancer in subjects is provided.
  • the other cancer med cament ' is administered in synergistic amounts or in various dosages or at various time schedules with the treatment regimen of the embodiments.
  • the embodiments also relate to kits and compositions concerning the combination of treatment regi mens of the embodiments alone or in combination with a desired cancer medicament.
  • a treatment regimen is used prior to receiving another anti-cancer therapy. In another embodiment, a treatment regimen is used concurrently with receiving another anti-cancer therapy. In another embodiment, a treatment regimen is used after receiving another anti-cancer therapy.
  • the present embodiments provide a method of treating breast cancer that is negative for ER in a subject. In some embodiments, there is a method of treating breast cancer that, is negative for ER and positive for HER2 in a subject. In some embodiments, the breast cancer is a metastatic breast cancer, in some embodiments, the breast cancer is at stage ⁇ , stage II, or stage III
  • the treatment regimen may be used in combination with existing therapeutic agents used to treat cancer.
  • existing therapeutic agents used to treat cancer In order to evaluate potential the rapeutic efficacy of the treatment regimen of the embodiments in
  • a treatment regimen may be used in combination with a therapeutic agent such as an anti-tumor agent including but is not. limited to a chemotherapeutic agent, an anti-cell proliferation agent or any combination thereof.
  • a therapeutic agent such as an anti-tumor agent including but is not. limited to a chemotherapeutic agent, an anti-cell proliferation agent or any combination thereof.
  • any chemotherapeutic agent can be used with the treatment regimen of the embodiments.
  • any conventional chemotherapeutic agents of the following non- limiting exemplary classes are included in the embodiments: alkylating agents; nitrosoureas; antimetabolites; antitumor antibiotics; plant alkyloids; iaxaoes; hormonal agents; and miscellaneous agents.
  • Alkylating agents are so named because of their ability to add aikyl groups to many electronegative groups under conditions present in cells, thereby interfering with DNA. replication to prevent cancer cells from reproducing. Most alkylating agents are cell cycle non-specific, In specific aspects, they stop tomor growth by cross-linking guanine bases i DMA double-helix strands.
  • Non-limiting examples include busul&n, earboplatin, chlorambucil, eisplatin, cyclophosphamide, dacarbazroe, ifosfamide, mechlorethamine hydrochloride, melphaian, procarbazine, thiotepa, and uracil mustard.
  • Anti-metabolites prevent incorporation of bases into DNA during the synthesis (S) phase of the cell cycle, prohibiting normal development and division.
  • antimetabolites include drugs such as 5-fliiorou.racil, 6- mercaptopurine, capecitab e, cytosine arabinoside, iloxuridine, tludarabine,
  • gemcitabine methotrexate, and thioguanine.
  • antitumor antibiotics that generally prevent cell division by interfering with enzymes needed for cell division or by altering the membranes that surround cells. Included in this class are the anthracyc lines, such as doxorubicin, which act to prevent cell division by disrupting the structure of the DNA and terminate its function. These agents are cell cycle non-specific, Non-limiting examples of antitumor antibiotics include daetiftomycin, daunorubicin, doxorubicin, idarubicin, mitomyein ⁇ C, and mitoxamraiie.
  • Plant alkaloids inhibit or stop mitosis or inhibit enzymes that prevent cells from making proteins needed for cell growth. Frequently used plant alkaloids include vinblastine, vincristine, vindesine, and vinorelbine. However, the embodiments should not be construed as being limited solely to these plant alkaloids.
  • taxanes affect cell structures called microtubules that are important in cellular functions, in normal cell growth, microtubules are formed when a cell starts dividing, but once the cell stops dividing, the .microtubules ' are disassembled or destroyed. Taxanes prohibit the microtubules from breaking clown such that the cancer cells become so clogged with microtubules that they cannot grow and divide.
  • Non- limiting exemplary taxanes include pacHtaxel and docetaxel.
  • Homionai agents and hormone- like drugs are - utilized for certain types of cancer, including, for example, leukemia, lymphoma, and multiple myeloma. They are often employed with other types of chemotherapy drags to enhance their effectiveness.
  • Sex hormones are used to alter the action or production of female or male hormones and are used to slo w the growth of breast, prostate, and endometrial cancers. Inhibiting the production (aromatase inhibitors) or action (tamoxifen) of these hormones c an often be used as an adjunct to therapy. Some other tumors are also hormone dependent.
  • Tamoxifen is a non-limiting example of a homionai agent that interferes with the activity of estrogen, which promotes the growth of breast cancer ceils.
  • Miscellaneous agents include chemotherapeuttcs such as bleomycin, hydroxyurea, L-asparaginase, and procarbazine that are also useful in the embodiments.
  • An anti-cell proliferation agent can further he defined as an apoptosis- induciiig agent or a cytotoxic agent
  • the apoptosis-inducing agent may be a granzyme, a Be 1-2 family member, cytochrome C, a caspase, or a combination thereof.
  • Exemplary granzymes include granzyme A, granzyme B, granzyme C, granzyme D, granzyme E, granzyme F, granzyme G, granzyme H
  • granzyme I granzyme J, granzyme , granzyme L, granzyme , granzyme N s or a combination thereof.
  • the Bcl- 2 family member is, for example, Bax, Bak, Bcl-Xs, Bad, Bid, Bik, Hrk, Bok, or a combination thereo
  • the caspase is caspase- 1 ,. caspase-2, easpase-3, easpase-4, easpase ⁇ 5, caspase-6, caspase-7, easpase ⁇ 8, easpase-9, caspase- 1.0, caspase- .1 1 , caspase- 12, caspase- 13, caspase- 14, or a combination thereof.
  • the cytotoxic agent is TNF- , gelonin, Prodigiosan, a rihosome-inhihiting protein (RIP), Pseudomonas exotoxin.
  • Clostridium difficile Toxin B Helicobacter pylori VacA, Yersinia enterocoiitica YopT, Vlolacein, diemylenetriaminepentaacetic acid, irofulven, Diptheria Toxin, mitogillin, ricin, botulinum toxin, cholera toxin, saponin 6, or a combination thereo
  • a treatment regimen is used in combination with an anti-tumor agent wherein the anti-tumor agent is an antitumor .alkylating agent, antitumor antimetabolite, antitumor antibiotics, plant-derived antitumor agent, antitumor platinum complex, antitumor campthotecin derivative, antitumor tyrosine kinase inhibitor, monoclonal antibody, interferon, biological response -modifier, hormonal anti-tumor agent, anti-tumor vital agent, angiogenesis inhibitor, differentiating agent,
  • the anti-tumor agent is an antitumor .alkylating agent, antitumor antimetabolite, antitumor antibiotics, plant-derived antitumor agent, antitumor platinum complex, antitumor campthotecin derivative, antitumor tyrosine kinase inhibitor, monoclonal antibody, interferon, biological response -modifier, hormonal anti-tumor agent, anti-tumor vital
  • the anti-tumor agent is citabme, capecitabine, valopicitabrae or gemcitahine. in some embodiments, the anti-tumor agent is selected from the group consisting of Avasttn, Sutent, Nexavar, Recenttn, ABT-869, Axidnib, Irinotecan.
  • topotecan pac!itaxel, docetaxel, lapatinib, Berceptin. lapatinib, tamoxifen, a steroidal aromatase inhibitor, a non-steroidal aroraatase inhibitor, Fulvestrant, an inhibitor of epidermal growth factor receptor (EGFR), Cetuximab, Panituraimab, an inhibitor of insulin-like growtli factor I receptor (IGF ), and CP-751871 ,
  • the anti-tumor agent is a chernotherapeutic agent
  • a chemotherapeutie agent as used herein is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN); alkyl sulfonates such as bnsu!fan, iraprosulfan and piposuifan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethyleneme!amine, trietylenephosphoramide, triethiy!enef hipphosphoratnide and trimethylolomelamine; aeetogenins (especially huOatacin and bullatacinone); delta ⁇ 9 ⁇ tetrahydrocafmabinol (dronabinol,
  • teniposide teniposide
  • cryptophycms particularly cryptophycin 1 and etyptophycin 8
  • dolastatin duocan.nycin (including the synthetic analogues, KW-2189 and CBl -TMl); eleiithero n; pancrati statin; a sareodictyin; spongistatin; nitrogen mustards such as- chlorambucil, chlornaphazine, cholopfaospharnide, estramustine, ifosfamide, rnecbloretharnine, raecMoretharaine oxide hydrochloride, tnelphalan, noverabichin, phenesterine. precinimustine, trofosfamide, uracil mustard; iikrosureas such as camiusiine,
  • antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especi ally ealieheamiein gamma Li and ealkheamkin omegall (see, e.g., Agnew, Chem Intl. Ed. Engl, 33: 183-186 (1994) ⁇ ; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chxomoprotein enediyne antiobioiic chromophores),
  • antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especi ally ealieheamiein gamma Li and ealkheamkin omegall (see, e.g., Agnew, Chem Intl. Ed. Engl, 33: 183-186 (1994) ⁇ ; dynemicin, including
  • doxorubicin (including ADRIAMYCI , morpholino-doxorubicin, eyanomo ⁇ holino-doxorabicin, 2-pyrroHno-doxorubicin, doxorubicin HC! liposome injection (DOXIL), liposomal doxorubicin TLC D-99
  • YOCET peglylated liposomal doxorubicin
  • CAELYX deoxydoxorubicin
  • deoxydoxorubicin 5 epirubkin esorubkin, idarubicm, marceliomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalanrycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamychi, rodorubicin, streptonigrin, streptozocin, tubercidin, iibenimex, zmostatin, zorubicm
  • anti-metabolites such as methotrexate, gemciiabine (GEMZAR), tegafur ( IJFTORAL), capecitabiae (XELODA), an epothilone, and S-fluorouracil (5-FU); folic acid analogues such as denopterra, methotrexate, pieropterin, trimetrex
  • mitotane, trilostane; folic acid reple isher such as frolinic acid; aceglatone;
  • aidophosphamide glycoside aminolevulinic acid; eniluracii: amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziqtione; elforaithine; elliptinimn acetate; etoglucid; gallium nitrate; hydroxyurea; lentiiian; kmidainine; maytansraoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;
  • procarbazine PS polysaccharide complex (JHS ' Natural Products, Eugene, Qreg. ⁇ ; razoxane rhizoxin; sizofiran; spirogermamura; te uazonk acid triaziquone; 2 i 2',2" ⁇ trichiorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); tirethan; dacarbazme; raai oraustme; mitobroniiol; mitolactol; pipobxoman; gacytosine; arabinoside ("Ara-C"); ioiepa; taxoM, e.g., paclitaxel (TAXOL), albumin engineered nanoparticle formulation of paclitaxel (ABRAX A E), an d doceta el (TAXOTE E); chioranbucil; 6-thio
  • vincas which prevent tubulin polymerization .from forming microtubules, including vinblastine (VELBA ), vincristine (ONCOVIN), vindesine (ELDISIME, FILDESIN), and. vinorelbme ( AVELBINE); etoposide (VP- 16); ifosfamide; mitoxantrone; leucovovin; novantrone; edatrexate;
  • daunomycin aninopterin
  • ibandronate topoisomerase inhibitor RFS 2000
  • DMFO difluorometlhyloraifhtee
  • retinoids such as reiinoic acid, including hexarotene (TARGRETIN)
  • bisphosphonates such as clodronate (for example, BONEEOSO or
  • OSTAC etidronate
  • DIDROCAL etidronate
  • ZOMETA alendronate
  • a .EDIA alendronate
  • SKELID tihidronate
  • ACTONEL risedronate
  • troxacitabine a 1 ,3-dioxolane nucleoside cytosine analog
  • antisense oligonucleotides particularly those that inhibit expression of genes in signaling pathways implicated m aberrant cell proliferation, such as, tor example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF ' -R)
  • vaccines such as THERATOPE.RTM vaccine and gene therapy vaccines, for example, ALLOVECTIN vaccine, LEUVECTIN vaccine, and VAXID vaccine
  • topoisomerase 1 inhibitor e.g., LURTOTECAN
  • niiRH e.g., ABARELIX
  • topoisomerase 1 inhibitor e.
  • bottezoraib VELCADE
  • GCI-779 tipf&mib (Rl 1577); orafeftib, ABT5I0
  • Be! ⁇ 2 inhibitor such as oblimersen sodium (GENASENSE); pixantrone; EGFR inhibitors; tyrosine kinase inhibitors; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFQX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN) combined with 5-FU and leucovovin.
  • ELOXATIN oxaliplatin
  • combinations.of ' immnno.therapie;S are used to treat estrogen receptor -positive/HER -positive (ER 3 ⁇ 4>0S HER i>os ) DCIS breast cancer patients.
  • Anti-estrogen therapy such as, for example, tamoxifen is combined with amti-HER2 dendritic eel! vaccination to improve pathologic complete response.
  • Example I A cryopreserved, pre-aetivated, multi-dose dendritic cell vaccine
  • DO vaccines pulsed with tumor antigen whereby the fully activated DO vaccines are cryopreserved as multi-dose syringe ready 6 pack DO vaccines.
  • the DO are cryopreserved activated as DO as described elsewhere herein. For example, they are cryopreserved in 55% Plasmalyte medium with 40% Human serum albumin and 5% DM SO. These vaccines have been generated and extensively tested in the laboratory and consistently meet quality standards set by the FDA for administration to ⁇ patients.
  • Dendritic cells were matured with TLR agonist LPS (TLR .4, lOng/nii) or R848 (TLRS, 1 ⁇ «1 ⁇ 21). The maturation time was at least about 6hr. After that, the TLR agonist-activated DCs were ready for cryopreservation or immediate use.
  • the DCs are activated with combinations of the cytokine IFN.-y, or the TLR agonists bacterial LPS and/or R848. This should induced T ceils that produce IF - ⁇ .
  • the DCs can be activated with combinations of ATP, bacterial LTA, LPS and prostaglandin E2
  • DCs were harvested by gentle scraping. All medium and the cells were kepi at wet ice at all times. Cells were gently washed, by centrifogation at about 80GRPM for 10 min. Cells (e.g., 10 x l O 6 cells were cryopreserved in freezing medium of piasmalyte 55%, human serum a lbumin 40% wit h 5% DM SO and stored in liquid nitrogen. The results of the experiments presented herein are now described.
  • peripheral ' blood monocytes were obtained by combined
  • IL-12 and Thl chemokines were produced for 36 hours post thaw from multi-dose DCl vaccines. Thawed cells produced high levels of IL-12 from about 6 hoors post thaw through 36 hours. These levels of IL-12 are comparable to prepared DCl vaccines made from cryopreserved monocytes.
  • the multi-dose syringe ready pack DCl vaccines can be used in HER-2 non-expressing breast cancer.
  • HER-2 is expressed on approximately 25% of ail breast cancers.
  • Breast cancers that, do not produce detectable levels of HER-2 may not be susceptible to vaccination.
  • additional target proteins can be added to the vaccine.
  • many breast cancers that do not produce high levels of HER-2 instead produce other, related proteins including HER-i and HER-3. Without wishing to be bound by any particular theory, it is believed, that adding these other proteins to the vaccine would allow the targeting of these other breast cancer phenotypes.
  • the multi-dose syringe read pack DCl vaccines can be used in other cancer types, besides- breast cancer. Anticipated target proteins such as HER-i , HER-2 and HER-3 can also be present on otiier types of cancer including ovarian, prostate, pancreatic, colorectal, gastric, head and neck and non-small cell lung carcinoma, as well as other common cancers.
  • the multi-dose syringe ready pack DO vaccines can be vised to treat chronic infectious diseases, including but not limited to, chronic infections like HIV or hepatitis virus C.
  • proteins specific for these viruses would replace the HE -2 or other cancer proteins to mobilize the patient's immune response against these persistent infections. It is possible that the enhanced immunity would greatly reduce viral load and attendant disease symptoms and progression, or could possibly help clear the infection entirely.
  • the multi-dose syr inge ready pack DC I vaccines can. be used to treat autoimmune diseases. Diseases like rheumatoid arthritis- and Lupus occu when the immune system mistakenly attacks the body's own normal tissues.
  • the current vaccine inimunotherap formulation is designed to initiate and strengthen immune responses, without wishing to be bound by any particular theory, it is believed that in vitxo signals can be provided to the DCs during vaccine production that induce these cells to switch off pathological immune responses.
  • Dendritic cell-based vaccine therapy is a promising directed therapy against a variety of cancers. While a variety of strategies have been employed to mature DCs to a phenotype that optimizes sensitization of CD4+ and CD8+ T cells to recognize tumor epitopes and elicit antitumor immunity, experiments were designed to utilize a method that employs the rapid maturation of monocytes in serum free media (SFM) using interferon gamma (lFN- ⁇ ) and lipopol saccharide (LPS), a toll-like receptor (TLR) 4 agonist, resulting in mature DCs capable of polarizing the immune response to a Thl- type response and eliciting sensitization: via an IL-12 dependent mechanism. Results demonstrate the potential for this vaccine strategy to be used as an adjunct therapy in early breast cancer. Cryopreservation of dendritic cells (DCs) in a matured state permits easier production of and accessibility to personalised, therapy.
  • SFM serum free media
  • LPS lipopol saccharide
  • the resul ts presented herein demonstrate that rapid maturation .method of DO ca be cryopreserved functionally matured and. maintains, pfaenotype and function, thus can be used to manufacture syringe ready DC 1 for use world-wide in cancer therapy.
  • the results presented herein demonstrate that the cryoDCs maintained the ability to primarily sensitize T cells. This also likely relates to the maturation strategy, as the DCis matured with IFN-gamma and LPS exhibit an enhanced ability to primarily sensitize CD 4+ T cells compared to cytokine matured DCs.
  • Example 3 Cytokines from CD4 T cells and Hereeptm make high HE -2-expressi.ng breast cells susceptibl to killing by CDS T cells
  • CDS T cells allow them to be killed by these immune cells. It has been shown that the cytokines interferon-gamma (IFN-y) and tumor necrosis factor-alpha (TMF-a) produced by CD4 ceils, when combined with Herceptin, cause the intermediate and high
  • HER-2 -expressing breast cancer cells to increase their Class I molecule expression.
  • the CDS T ceils are able to better see the breast cancer cells and kill them or to produce cytokines to kill them.
  • a phase I DCIS vaccine trial combining Herceptin with DC 1 vaccines was designed.
  • a Phase I trial was designed for patients with high HER-2- expressing DCIS to receive a DO vaccine combined with 2 doses of Herceptin at week 1 and week 4.
  • this combination will increase the complete response rate from 30% to greater than 50% in patients with HE -2-expressmg DCIS,
  • a Phase III DCIS vaccine trial was designed.
  • a vacci ne trial was de vel oped to prevent recurrence of breast cancer i patients with estrogen independent ⁇ ER *a*3 ⁇ 4 ⁇ l,ive ) > l iER-2 "t,osa! 3 ⁇ 4 DCIS.
  • those patients who have complete responses to the treatment can avoid radiation after surgery. It is also believed that this trial serves demonstrate the prevention of recurrence in patients with DOS using a vaccine.
  • phase 1 neoadjuvant DC I vaccine in combination with
  • Herceptin in patients with early invasive HER-2 ⁇ posmvi breast cancer was designed.
  • a Phase I trial was designed to test whether the combination of Herceptin and vaccines along with a chemokine modulator (e.g., a cheniokme-activating agent) can eliminate small HER-2-expressing invasive breast cancers prior to surgery and avoid the need for chemotherapy.
  • a chemokine modulator e.g., a cheniokme-activating agent
  • this neoadjuvant (before surgery) stxategy with the possibility of adding an immune antibody that takes the brakes off the immune response, may eliminate the need for toxi c chemotherapies for the treatment of breast cancer and therefore make imm une therapy the standard of care for this disease.
  • the treatment regimen disclosed herein provides a step forward in the quest to eradicate breast cancer using the natural immune response, which can be restored with vaccines regimens of the embodiments.
  • the regiments discussed herein can drive immune cells into the tumor by changing the immune response in the tumor and enable the immune cells to work longer by taking the brakes off the cells. It is believed that combining DC 1 vaccine with Herceptin and also adding the chemokine modulator improves the migration and activity of the immune cells within the tumor in the breast.
  • an. effective therapy to treat cancer would include agents that rapidly change the immune response in the tumor prior to surgery to improve outcomes for patients with breast cancer and other types of cancer.
  • This strateg can be applied to a variety of cancers including but is not limited to colon cancer, melanoma. Sung, brain, pancreas, prostate, esophagus, and the like.
  • HCC- 1.419 and MDA-MB-231 were obtained from the American Type Culture Collection (Manassas, VA) and grown in RPMI-1640 (Life technologies. Grand island, NY) supplemented with .10% FBS (Cellgro, -Herndon, VA), IiMT- 1 cells were a kind gift from Dr.
  • DMEM Dulbecco's modified Eagle's medium
  • Normal immortalized MCF-10 cells were obtained from the armanos Cancer Institute (Detroit, MI) and. grown in DMEM/F 2 (Invitrogen) supplemented with 10 mM HEPES, 10 pg/ml insulin, 20 ng/m! EGF, .100 ng/ml cholera toxin, 30 mM sodium bicarbonate, 0.5 pg/rnl hydrocortisone, and 5% fetal horse serum. All cells were grown at 37°C in a humidified 5% C02 incubator.
  • HERCEPTiNTM pertuzumab
  • PERJETATM pertuzumab
  • 0.5x10 5 cells were incubated in the presence of DC/CD4 1 T-eell co-culture su ematants tor 5 day at 37°C In both approaches, cells were then .cultured for 2 more passages in absence of cytokines and subjected to senescence studies (SA-p-gal activity at pH 6 and pl51NK4b and pl6!N 4a western blot) or apoptosis studies (cleaved caspase-3 western blot).
  • MDA-MB-231 cells were transiently transieeted for 48 fa with 2 ⁇ 3 ⁇ 4 of the wt HER2 expression vector (pcDNAHER2).
  • pcDNAHER2 the wt HER2 expression vector
  • Both vectors were kindly provided by Dr. Mark Greene (University of Pennsylvania, Philadelphia, PA).
  • the cells were transfeeted in complete .med um ' ithout antibiotics with Tmbofect (13 ⁇ 4emio Scientific, Waltham., MA).
  • Ttansfection efficiency was evaluated by western blot 48 h after transfectton. Fort eight hours later, transfeeted ceils were transferred to complete culture medium containing 0.4 mg ml G418 (Life Technologies). After 15 days of culture, colonies resistant to G418 were selected by limiting dilution. Transfection efficiency was evaluated by westera blot.
  • siRNA Small interfering RNA
  • HERS siRNA HERS siRNA
  • SMART Pool ON-TARGETplus Non-targeting Pool were purchased from GE Dharmacon-iLafayette, CO). The following target sequences were used: BER2: UGGAAGAGAUCACAGGUUA (SEQ ID NO: 9%
  • UGGUUUACAUGUUUUCCUA SEQ I D NO: 20
  • siRN A sequences 25 nM
  • R Ai Max Lipofeetamine life Technologies
  • the percentage of SA-$-gal- positive (blue) cells in each sample was determined after scoring 300 cells using a bright- field microscope (Eyes Corexr,, Bothel, WA 40X/ 2048 x 1536, 3,2 mu pixel; 3,1 MP COLOR/ Captured images: Color TIFF, PNG, JPG or B P-2048 x 1536 pixels) Western blot analysis
  • Lysates were prepared from MCF-lOA, SK-BR-3, and CF-7, T-47 D or MDA-MB-231 cells. Cells were lysed in a buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 m.M EDTA, 1 mM EGTA, .10% glycerol, 70% Tergitol, 0.1% SDS, 1 mM gsCl and protease inhibitor cocktail Sigma-Aldrieli (St. Louis, MO). Lysates were centrifuged at .12,OO ' 0 x g for 15 mln i4 C.
  • Proteins were soluhiiized in sample buffer (Life Technologies) and subjected to SDS-PAGE, Proteins were eleetrohiotted onto PVDF.
  • Membranes were immunob lotted with the following antibodies: pl.51NK4b (K- 18), pl6INK4a (50.1 ), IFN-yRa (C-20), HER3 (C- 17) all from Santa Cruz Biotechnology (Santa Cruz, CA); VincuHn (V913 I) from Sigma-Aidrich; HER2 (2908), cleaved caspase-3 (Aspl 75) and TNP-R1 (C25C1) and phospho-Akt (Ser473) from Ceil
  • SK-BR-3 cells were untreated, treated with IFN-y (.1 0 U/ml.) and T F-a (l Ong/ml), treated with trastuzumah (l Opg/ml) and pettoznmab (1 Opg/mi), or treated with a ' ⁇ combination of IFN-y, T F- , trastuzumah, and pertuzumab, for 24 hours.
  • apoptosis induction was determined using FITC-Annexin ' V apoptosis detection kit (BD biosciences) according to manufacturer's instructions. Briefly,
  • SK-BR-3 (2 > 10 i! cells/mouse in 200 ⁇ PBS) were injected into the flanks of six-week old female oathymic (nude) mice (F ' axmw, Ffarlam Laboratories, 5 mice/group).
  • the animals were treated s.c. with trastuzumab and pertuzumab (30 ug/kg) and then injected .v. c. twice a week with hrTNF-a and hrEFN-y ( 10 ng/kg). Tumor formation was monitored by palpation and tumor volume in was determined with a caliper twice a. week: width2 x !ength/2.
  • Ail animal experiments were carried out in compliance with the institutions guidelines.
  • S -BR-3 cells were incubated with human recombinant tumor necrosis factor alpha (T F- ) and interferon gamma (If -y) alone or combined for 5 days at 37°C to study if the elaborated cytokines produced by the immune system cells could induce a specific senescence response in tumor cells. The cells were then cultured for 2 more passages and subjected to senescence studies.
  • T F- tumor necrosis factor alpha
  • If -y interferon gamma
  • T-47D cells were treated with several concentrations of TNF-a, from 10 to 100 ng/rni, and lFN- ⁇ , from 100 to 1000 ' ⁇ ⁇ ⁇ , or in combination, it was observed that the induction of the senescence phenotype was dose dependent (Figure 6C). Similar results were found in SK-BR-3. BT-474 and MCF-7 cells. inverse correlation between the HER2 expression level in breast cancer cells and the Thl cytokines TNF- and IFN-y doses requited to induce senescence
  • HE 2 is eq uired lor Thl cytokines T F-a a d lFN- ⁇ tnediated senescence and apoptosis in MDA-MB-231 breast cancer cells
  • Cytokine receptors are expressed in sirni jar le els i breast eel 1 1 ines
  • the receptor TNFR1 expression mediates TNF ⁇ a ⁇ induced proliferation of mammary epithelial cells, and TNFR2 activation induces casein accumulation (Varela et aL 1.996 Endocrinology .137; 4915-4924).
  • the active form of IFN-y interacts with its receptor expressed on the surface of almost all normal cells (Ealick et aL, 1991 Science 252: 698-702; Farrar et al., 1993 Annu. Rev. Immunol. 1 1 :571-611).
  • Combined HER2 and HE 3 blockage expressio enhances Th I cytokines TNF-a and IFN- ⁇ senescence induction in breast cancer ceils.
  • Trastuzumab and pertuzuraab are antibodies that have bee widely used in the clinic to treat HER2- ositive breast cancer.
  • Thl cytokines-induced senescence and apoptosis were studied in a translational approach, experiments were designed to pretreat SK-BR-3 cells with trastuzumab and pertuztimab and then, the ceils were additionally treated with TNF-a. and IFN-y for 5 days at37° € followed by 2 more passages without the cytokines and antibodies.
  • TNF-o and IFN-y induce senescence and apoptosis in breast cancer cells in a dose dependent manner. Also revealed is an inverse correlation between the HER2 expression level in breast cancer cells and the doses of TNF-o: and IFN- ⁇ required to induce senescence i those cells. It has also been shown thai cytokine receptors are expressed in similar levels in all the breast cell lines tested, implicating that this is not the cause of the differential response.
  • HER2 signaling is required to induce senescence and apoptosis by till cytokines, because in cells that lack HER2 or express very low levels is not possible to induce senescence or apoptosis even with high doses of cytokines.
  • cells that express high or intermediate levels of HBRZ knocking down the gene induces
  • DCs dendritic cells
  • the antigen-presenting capacity of dendritic cells has led to enthusiasm for their use in anti-tumor vaccination.
  • the present group designed a HER2 peptide-pulsed autologous DC vaccine uniquely engineered to promote anti-HER2 T l sensitization and attraction. See, United States Ser. No. 14/658,095, filed March 13, 2015; United States Ser. No. 14/985,303, filed. December 30, 2015, the disclosures of which are incorporated by reference herein in their entireties; Datta, 1, et al.,
  • Vaccine preparation and delivery have been described in detail previously. See, for example, Sharma, et al, Czeraieeki, BJ,, et al. Cancer Res. 67(4): 1842-52 (2007) (Czemiecki, et al.), and Koski, G.K., et al, J. Jmmunother. 35(1) 54-56 (2012).
  • the vaccination procedure is shown in Figure 16. Briefly, monocytic dendritic cell precursors were obtained f om patients via tandem ieukapheresis/coiraierctarent centrifugal eiutriation. Monocytes were cultured at 37°C in serum free medium.
  • TQR.CE CSKPCAR.VCYGL (SEQ ID NO: 3» and three intracellular domain (ICO) peptides (peptide 776-790: GVGSPYVSRLLGICL (SEQ ID NO: 4);. peptide 927-941 : PAREIPDLLEt GERL (SEQ ID NO: 5); and peptide 1 166-1 180
  • TLERPK FLSPGKNGV (SEQ ID NO: 6)
  • IFN-gamma Intermune, Brisbane, CA
  • M1H National Institute of Health
  • Pathologic response was examined at the time of surgical resection ⁇ lumpectomy (n - 48 ⁇ or mastectomy (n - 33).
  • a pathologic complete response to immunization was defined as no residual DOS or invasive breast cancer at the time of surgical resection.
  • Patients were monitored after surgical resection for the development of subsequent breas events.
  • a subsequent breast event was defined as a lesion - DCIS or invasive breast cancer, identified in either the ipsilateral or contralateral breast.
  • Anti-Estrogen Therapy BR positive patients were treated with ao.ti ⁇ estrogen therapy concurrent with four to six weekly a «ii-BBR2 DCl vaccinations.
  • a physician investigator decided which of the following ami-estrogen therapies was best suited for each patient: Tamoxifen (4- hydroxytamoxifea (“4HT") (NOLVADEXTM)); Letrozole (FEMARA ' TM); Anastrozole (ARMIDEXTM); Exemestane (AROMASiNTM); Raloxifene (EVISTATM); or any other suitable anti-estrogen agent that blocks or modifies the ac tions of estrogen.
  • Tamoxifen (4- hydroxytamoxifea
  • FEMARA ' TM Letrozole
  • ARMIDEXTM Anastrozole
  • Exemestane AROMASiNTM
  • Raloxifene EVISTATM
  • any other suitable anti-estrogen agent that blocks or modifies the ac tions
  • PB C peripheral blood mononuclear cells
  • MHC major histocompatibility complex
  • HER2 CD4+ T -cell responses were measured in the locoregional sentinel lymph nodes (SEN) in 40 patients who underwent SEN biopsy. IFN- ⁇ production was quantified via enzyme- 1 inked immunosorbent spot (EL!SPOT) assays as previously described in detail. (FracoL M Chandler et al. ⁇ Am. Surg. Oncol.
  • PVDF membrane plates (Mabfcech, Cincinnati, OH) were coated overnight with aati-IFN- ⁇ capture antibody (IDI K), The following day, alter the plates were washed with PBS (Mediatech, Manassas, VA) and blocked with 10% human serum DMEM, 2x 10 s PBMCs on SEN cells were plated in each well either unstimulated, ulsed with HER2-derived Class I peptides (4p.g) (42-56, 98-1 1 , 328-345, 776-790, 927-94.1 , 1166-1 1 SO), or pulsed with anti-human CD3 and CD28 antibodies (0.5pg/mL) (positive control, BD Pharmingen, San Diego, CA), and incubated at 37°C+ 5%C02 for 24-36 horn s.
  • IDI K aati-IFN- ⁇ capture antibody
  • a positive response to an individual HER2 Class II peptide was defined as minimum of 20 SFC/2. J O 5 cells after subtracting the unstimulated background and at least a two-fold increase over the unstimulated background. Three metrics were used to quantify the CD4+ Thl response (I) overall response rate (the proportion of patients responding to 1 peptide), (2) response repertoire (the number of peptides to which a patient responds), and (3 ) cumulative response (the sum of the SFCs across all 6 peptides).
  • Systemic anti-HER2 CD8+ T-cell responses were measured m twenty two HLA- A2 pos (HLA.2.1) patients .
  • Anti-HER.2 CD8+ T ⁇ ceil responses were generated by in vitto sensitization assays as previously described in detail by Czemieeki, et ai.. Briefly, CD8+ T-cells were selected from the cryopreserved 120-140 lymphocyte cell fractions via negative selection (StemCell Technologies., Vancouver, BC).
  • ELISA A positive response to the BER2 Glass I peptide wa defined as a two-fold increase in CD8+ T-cell IFNy production compared to the irrelevant peptide controls.
  • the aati ⁇ HER2 Thl immune response was significantly higher in the patients with ER pf,s DOS who were treated with combination anti ⁇ HER2 vaccination and anti-estrogen therapy compared with the patients with ER pos DOS who were treated with anti-HER2 vaccination alone.
  • the S BR3 breast cancer cell line (ER riS3 ⁇ 4 ) increased anti-tumor activity in response to Th l cytokine treatment, but not in response to anti-estrogen treatment.
  • the MCF7 breast cancer cell One did not increase anti-tumor activity in response to either Th l cytokine treatment or anti -estrogen treatment; however, the combination of Th l cytokine treatment and anti-estrogen treatment together resulted in an increase in metabolic activity as show in Figure 1 B.
  • Vaccination was administered into groin lymph nodes in 47 patients (58%), into the breast in 18 patients (22.2%), and into both the groin lymph nodes and the breast in 16 patients (20%).
  • Surgical resection was completed via lumpectomy in 48 patients (59.3%) and via mastectomy in 33 patients (40.7%). Of those patients who underwent lumpectomy, 37.5% received post-operative radiation therapy.
  • the vaccine was well tolerated with only grade 1-2 adverse events.
  • Figure 18 shows that in the overall cohort, 35 patients (43.2%) had E ueg disease and 46 patients (56.8%) had ER poif disease. Of those patients with ER*** disease, 25 patients (54.3%) received the DC! vaccine alone and 21 patients (45.7%) received the DO vaccine and concurrent AE therapy.
  • Demographic and. clinical characteristics of these treatment groups are summarized in Table 1 below, and did not show any significant difference between the groups. Table 1 ; Demographic and clinical characteristics comparing patients by Ell status and
  • Lumpectomy 19 (54.3 ⁇ 13 (52.0) 16 (76.2) 0.18
  • ER estrogen receptor
  • AE anti-estrogen
  • 1QR ⁇ interquartile range
  • HER2 human epidermal growth factor receptor 2.
  • Pathologic coi.npl.ete response .cor relates with a decreased risk of recurrence See, for example, Tanioka, M. 5 ei al', Br, J, Cancer 103(30:297-302 (2010) Subsequent breast lesions, defined as either DC S or invasive breast cancer identified in either breast, occur red in 6 vaccinated patients (7.4%) . All of the patients who experienced subsequent breast even ts had residua! disease identified at the time of surgical resection, ⁇ pCR ( Figure 20 A). Two of the patients had ER ne DCIS and four of the patients had ERT !,% DCIS but did not receive AE therapy.
  • Responsiviiy Following vaccination, responsivii increased significantly in each group (ER !J ⁇ 3 ⁇ 4 58.3% to 87.5%, p ⁇ 0,01; ER iJ ⁇ )A without AE treatment 50.0% to 75.0%, p ⁇ 0,01 ; ER with AE treatment 57.1 to 90.5%, p ⁇ 0.01). Pre-vaceination responsivity rates were similar across all three groups (ER ii3 ⁇ 4i 58.3%, EE 5 TM* without AE treatment 50.0%, EW* with AE treatment 57.3%; p . ⁇ 0.9).
  • Response Repertoire Following vaccination, response repertoire increased in eac group (ER** 8 1 to 3, p » 0,05; ER* 08 without AE treatmeni 0 to! .5, - 0.1; ER pi!S with AE treatment 1 to 3. p - 0.03 ).
  • Pre-vaceination median response repertoire was similar across ail three groups (ER Beg 3 (iQR 0-2), ER t, S without AE treatment 0 (IQR 0- 1.5), ER r " ,s with AE treatment 1 (iQ 0-2); p - 0,5).
  • Cturtulati ye Response Following vaccination cumulative response increased in each group (ER i,£3 ⁇ 4 56.3 to 149.7, p ⁇ 0.01; ER pl s without AE treatment 4 ⁇ .1 to 178.7, p ⁇ 0.01 ; ER pftS with AE treatment 58.6 to 100.9, p ⁇ 0.0 ⁇ ).
  • Pre- vaccination median ER i,£3 ⁇ 4 56.3 to 149.7, p ⁇ 0.01; ER pl s without AE treatment 4 ⁇ .1 to 178.7, p ⁇ 0.01 ; ER pftS with AE treatment 58.6 to 100.9, p ⁇ 0.0 ⁇ ).
  • Example 6 A Novel Dendritic Cell Vaccine Targeting Mutated BRAF Overcomes Vemurafemb Resistance and Synergi stically Improves Survival in BR AF-Mutant M urine Melanoma
  • BRAF inhibitor vemurafemb improves survival in B AF-mutant
  • BRAF 6im melanoma, but resistance is common.
  • BRAF V ⁇ 3 ⁇ 4H3 ⁇ 4; ⁇ DC 1 induces antigen-specific CDS * T-cells that impact murine BRAP ⁇ >!t>E melanoma.
  • BRAF 3 ⁇ 4ft " E PTEN ' ⁇ melanoma model was developed in the C57BS/6 background.
  • DC I were generated from bone marrow precursors using FH3, IL ⁇ 6, GM-CSF, lL-4, CpG and LPS, and pulsed with class I BRAF VS aE peptide.
  • BRAF vtitffi: -DCi (2x weekly injections) and PLX were administered alone or in designated combinations to tumor-bearing mice
  • TME tumor micrqenvitonments
  • Figure 24 shows mice receiving B AF 6u0h -DCRPLX combinations, either initiated concurrently or after B AF 3 ⁇ 4&00fe -DCI induction, demonstrated dramatically delayed tumor growth (P ⁇ 0.0Q1) and improved median survival (86d and 733d,
  • BRAF 6,,0i - DC1 +PLX, compared with individual, treatments induced synergisticaily improved systemic CD8 * T-ceil recognition of BRAF t,, - -pulsed antigen-presenting cells and BRAF V600E tumor cells (p ⁇ 0.001 ) measured by IFN-y release in vitro.
  • I TME I TME
  • BRAF v ⁇ -DCi+PLX generated highsr niRNA levels of Thl (IFN ⁇ f ⁇ - ⁇ ) and T-eeil homing (CXCL CCL5) cytokines, while attenuating PD-Ll expression: CDS f TIL trafficking was augmented by BRAF V3 ⁇ 400£ -DC1 +PLX.
  • BRAF WM3 ⁇ 4 ⁇ DC I vaccines overcome vemurafenib resistance in

Abstract

The present embodiments relate to an FDA- approved injectable multi-dose antigen pulsed dendritic cell (DC) vaccine. In one embodiment, the activated antigen-loaded DC vaccine comprises an initial immunizing dose and multiple "booster"doses. Also provided is a method of blocking both HER-2 and HER-3 as a treatment in causing permanent tumor senescence in HER-2 expressing breast cancers. Also provided is combination anti-estrogen therapy and anti-HER2 dendritic call vaccination for ERpos/HER2pos DCIS breast cancer patients.

Description

MANUFACTURING MULTI-DOSE INJECTION READY DENDRITIC CELL VACCINES
CROSS-REFERENCE TO RELATED APPLICATIONS:
This application is a continuation-in-part application of Serial N .
PCT/US 15/41022 filed July 17, 2015 which in turn claims priority and benefit from U.S. Provisional Application Serial No. 62/025,673, filed July 17, 2014, U.S. Provisional Application Serial No. 62/165.445, filed May 22, 2015, U.S. Provisional Application Serial No. 62/025,685, filed July 17, 2014, and U.S. Provisional Application Serial No. 62/028,774, filed July 24, 2014, the contents of each of which are incorporated by reference herein in their entireties.
ACKNOWLEDGMENT
The present invention was developed in part with government support under grant number R0I CA0 6 ? awarded by the National Institutes of Health. The government has certain rights in this invention.
BACKGROUND
Dendritic cells (DCs) are white blood cells that acquire protein antigens from microbes or even cancerous cells and show, or "present" these antigens to T cells. The T cells, thus activated by the DCs, then initiate systemic immune responses to challenge the threat. Traditional vaccines against microbes contain: additives known as "adjuvants" that by a number of possible means enhance DC activity with m the vaccinated individual and am lify' vaccine-induced immune responses. The requirements of vaccines against cancer, however, present a number of unique problems. For example, traditional adjuvants do not provide the proper signals to DCs that allow them to initiate optimal immunity against cancer. Also, the tumors themselves produce an environment thai can affect the proper activation of DCs.
A popular solution to this problem is to extract DCs from cancer patients, load them wit tumor antigens./?* vitro, and then suppl unique activation signals to the cells before re-administering them to the body. This ensures proper DC activation removed from the influence of the tumor environment When returned to the body, the DCs can then interact with T cells and initiate powerful anti-tumor iawmmity. Whereas the use of extra-corpoxealized 'DCs has solved many efficacy issues., it has historically come at the price of practical limitations. For example, since the DC vacc ines ate comprised of living cells, a special cell processing and vaccine production facility has been required at the physical location of the medical center administering the therapy. This is an expensive and inefficient, way to deliver the therapy because every institution administering such treatment would have to bui ld and maintain their own special-use facility.
Management of breast cancer currently relies on a combination of early diagnosis and aggressive treatment, which can include one or more treatments such as surgery, radiation therapy, chemotherapy, and. hormone therapy. Herceptin (trastuzumab) was developed as a targeted therapy for HER2 ErbB2 positive breast cancer cells, often used in conjunction with other therapies, including the mitotic inhibitor paciitaxe! (sold under the trademark Taxol).
The efficacy of Herceptin as a monotherapy is estimated to be less than 30%; combinatorial treatment with microtubule stabilizing drugs such as paclitaxel increases efficacy to approximately 60% (Burns et a!., 2000, Semin. Oncol 27; 19-23). Treatment with Herceptin results in accumulation of the Cdk inhibitor p27 and subse uent GT/S cell cycle arrest, and paciitaxe! stalls the entry of mitosis which can lead to cell death. In spite of great promise, however, high doses of Herceptin or paclitaxel result in undesirable side effects. Further, the cancer often develops resistance to
Herceptin and/or paclitaxel.
Therefore, there remains an unmet need for compositions and efficient methods for producing maxima! therapeutic DC vaccines and for new methods of treating cancer using Herceptin, Accordingly, there is a need in the art to have additional immunotherapeutic approaches for treating or preventing breast cancer and other malignancies. The present embodiments fulfil! this need.
BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description of preferred embodiments will be better understood when read in conjunction with the appended drawings, for the purpose of illustrating the embodiments, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that die embodiments are not limited to the preci se arrangements and instrumentalities of the embodiments shown in the drawings.
Figure 1 is a chart showing the viability and yield of post-thawed cryopreserved DO . Recovery of cells was on a verage 89% and viabi lit was 95% when cells were directly thawed and counted.
figure 2 is a chart showing that here was no significant difference in the viability (p= 4807X and recovery (p™ 1220) of the ceils.
Figure 3 is a chart showing that both populations had similar initial (7 hours post LPS addition) EL-12 p70 secretion (p=.076S). The populations continued to exhibit comparabie secretion levels of EL- 12 p70 over a 30 hour observation period with no significant differences between the populations.
Figure 4 is a chart showing that there was no significant difference between populations and production of IL-Ι β (p-Q..7690}:, lL-ία (p=s:0.0841}! Rantes (p-0. 02), MDC (p-0.1514), IL-8 (p-0.7844); ΜίΡ-Ια (p- 2673), IP-10 (p=0.7366), 1L- 6 (p-0.24), TNF-a (p-0.8972); IL-5 (p-0.0735), IL~15(p-0.887¾ IL-K) (p=, 1 37), iP-lp (p-Q.9217)?
figure.5 is a chart showing production of lFM-γ from cryopreserved and non~cryopreserved DCs.
Figures 6A-6D show Th 1 cytokines TNF-a and lFN'-γ synergize to induc senescence in breast cancer cells and the doses required are' in an inverse correlation with the HE 2 expression. Figure 6 A. shows results of studies in which SK-BR.-3 breast cancer cells were incubated with 10 ng/ral TNF-a and 100 U/ml IF -γ for 5 days, cultured for 2 more passages in absence of cytokines and then stained for SA-j3- galactosidase (SA-p-gal) expression (senescence marker) and compared to untreated control ceils. Only paired cytokines induced senescence. Top panel shows photographs of representative data from 1 of 3 independent experiments. Bottom panel, shows a histogram of densitometric analysis. Dat are presented as % of SA-p-gal-positive cells and presented as mean ± S.D. (n~3). ). P-vaiues were calculated using a paired Student's Mest. Statistical significance was determined at *P < 0.05. Figure 6B shows photographs of western blot analysis in which cell lysaies of the cells described in Figure 6A were analyzed for piSI Kb and l6!N 4a expression. Vinculra was used as loading control. Figure 6C shows results of studies in which T-47D breast cancer cells were untreated (1 ) or incubated with the following concentrations of TNF-a and INF-y: 10 ng/ml and 100 U/n l (2), SO ng/ml and 500 U/rnl .(3)„ 75 ng/nil and 750 U/ml (4), .and 100 ng/ml and 1000 U/ml (5) for 5 days and cultured for 2 more passages in absence of cytokines . The cells were then stained for SA-p-gal and compared to control untreated cells or those treated with 8 μΜ etoposide as a positive control (6). Top panel shows photographs of representative data from 1 of 3 independent experiments. Bottom panel shows a histogram of densitometric analysis of the 6 studies. Data are presented as % of SA-β- gal-positive cells and presented as mean ± S.D. (n~3). P-va'lues were calculated using a paired Student's t-test. Statistical significance was determined at *P < 0.05, **P < 0.01 , ***P < 0.001. Figure 6D is a histogram showing results of studies in which combination treatment with Thl cytokines IFN-y and NF-α resulted in greater senescence in S -8R- 3 (10 ng/ml TNF-a + 100 U/ml IFN-γ) and T-47D (100 ng/ml TNF-a + 1000 U/ml IF - v) cells, compared with untreated controls; MDA-MB-231 cells (200 ng/ml TNF-a + 2000 U/ml JFN-γ) remained largely unaffected by dual IFN-γ + TNF-a treatment. Data are presented as % of S Α-β-gal-positive cells and presented as mean ± S.D. (n=3). P- values were calculated using a paired Student's t-test. Statistical significance was determined at **P < 0.01, ***P < 0.001.
Figures 7Ά-7Β show HE 2 induces senescence and apopiosis in MDA- MB-231 breast cancer cells. Figure 7 A, left panel, is a histogram showing results of densitometric analysis in which SA-f3-gal staining was performed i MDA-MB-231 cells transfected with wt HER2 (pcDNAHBR.2) or with empty vector (pcDNA3) which were treated with the l isted concentrations of TNF-a and IFN-γ for 5 days and cultured for 2 more passages in absence of cytokines. Inset above the histogram are photographs of western blot analysis in which MDA-MB-231 cells transfected with pcD AHER2 or p.cDNA3 and probed with HER2 specific antibod were analysed for confirmation of HER2 expressio or lack thereof. Vmcolm was used as loading control. Data are presented as % of SA~$-gal-posMve cells and presented as mean ± SIX (iH5). P-values were calculated using a paired Student's t-test. Siatisticai significance was determined at ***P < 0.001. Figure 7 A, right panel, shows photographs of representative data from 1 of 3 independent experiments. Figure 7B shows photographs of western blot analysis of the cell lysates of the cells described in Figure 7 A for expression of p lSI Kb and cleaved caspase-3. Vmculin was used as loading control
Figures 8A-SB show combined HE 2 and HER3 blockage expression enhances Th! cytokines TNF-a and JFN-y senescence induction and apoptosis in. $K~ BR-3 breast cancer cells. Figure 8 A shows results of studies in which S A-P~gal staining was performed in SK-BR-3 cells transfecied with non-target siRNA (siRNA NT), HER2 siRNA, HER3 siRNA or a combination of HER2 and H.ER3 siRNA, and then treated with the concentrations listed of TNF-a and lFN-γ for 5 days and cultured for 2 more passages in absence of cytokines. Left panel shows a histogra of densitonieroc analysis. Data are presented as % of SA-p-gal-positive cells and presented as mean + SIX (n=3). P-vakes were calculated using a paired Student's ΐ-test. Statistical significance was determined at. ***P < 0,001 , Inset shows photographs of western blot analysis of SK-BR-3 cells transacted with NT, HER2, or HER3 siRNA probed with HER and HER3 specific antibodies. Yincidin was used as loading control Similar results were observed in 3 independent experiments. Right panel shows photographs of representative data from 1 of 3 independent experiments. Figure 8B shows results of studies in which lysates of the cells described in Figure 8 A were analyzed by western, blotting for l'5INKb and cleaved caspase-3 expression
Figures 9A-9C show combined HER2 inhibition and HER2-F1ER dimerization inhibition enhances Tii! cytokines TNF-a and IFN-y senescence induction and apoptosis in SK-BR-3 breast cancer cells. Figure 9 A shows results of SA-p-gai staining performed in SK-BR-3 cells which were untreated (I) or treated with 10 ng ml TNF-α and 100 U/mi IFN-γ (2), or with 10 ug/ml of trastuzutnab (Tzm), pertuzumab (Per)(3), or with the combination of both treatments (4) for 5 days and cultured for 2 more passages in absence of the antibodies and the cytokines. Left pane! is a histogram of densitometric analysis. Data are presented as % of SA-f5~gal-positive ceils and presented as mean ± S.D. (n-3). ), P-values were calculated using a paired Student's t- test Statistical significance was determined at ***P < 0.001. Right panel are
photographs showing representative data from 1 of 3 independent experiments. Figure 9B shows photographs of western blot analysis in which cell lysaies of the cells described in Figure 9 A were analyzed for plSlNKb or cleaved caspase-3 expression. Vinculin was used as loading control. Similar results were observed in 3 independent experiments. Fi gure 9C shows resul ts of studies of induction of apoptosis of SK-BR-3 c ells untreated or treated as described above performed by staining for annexin V and PI and analyzed by flow cytometry. Top panel shows plots of representative data from 1 of 3 independent experiments. Bottom panel, shows a histogram of densitoraetric analysis. Data are presented as average £ SE. of annexin V* ΡΓ cells from 3 independent experiments. P- vahies were calculated using a paired Student's Mest. Statistical significance was determined at **P < 0.0.1.
Figures I OA- 10B show combined treatment with trastimanab and pertiizumab enhance CD4'r Thl -mediated Senescence and apoptosis of HER2- ovexpressing human breast cancer cells. Figure I OA shows results of studies in which, using a transwell system, 0,5x10s SK-BR-3 cells were co-cultured with 5xl05 CD4 : T- cells alone (CD4f only), CD4* T-celis + 0.5x10s each of HER2 Class It peptide (DC H)- or irrelevant Class II BRAF or survivin peptides (DC B or DC S)-pulsed type 1 polarized mature DCs, and CD4"* T-celis + HER2 (iDC H)-pul$ed immature DCs ( IDC H), with or without 10 tig/ml of trastuziimab (Tzm) and pertuzumab (Per) for 5 days. The cells were then .cultured for 2 more passages in absence of the blocking antibodies and the immune system ceils and then stained for SA-P-gal expression and compared to untreated control ceils. Top panel is a histogram of densitometric analysis. Data are presented as % of $A~P~gal~positive cells and presented as .mean ± S.D. (n~3). P-values were calculated using a paired Student's t-test. Statistical significance was determined at ***P < 0.0001. Bottom panel shows photographs of representative data from 1 of 3 independent experiments. Figure 10B shows photographs of western blot analysis of cell lysates of SK-BR-3 cells co-cultured as indicated and analyzed for pl 51'NK4b and cleaved caspaee- 3 expression. Increased pi 5INK4b and cleaved caspase-3 expression is seen that suggests induced senescence and apoptosis of SK-BR-3 ceils, respectively when co- cultured wit the DC H/CD4r T-celis in presence of trastuzuraab and pertuzurnab, but not from DC B, DC S and IDC H groups, Vioculin was used as loading control. Results are representative of 3 independent experiments, photographs of western blot analysis in which cell lysates of the cells described m Figure A were analyzed for pISINKh or cleaved caspase-3 expression.
Figures 11 A~l IB show. Th .1 cytokines- TNF-a. and IFN-γ sensitize trastuzumab and pertuzumab resistant breast cancer cells to senescence and apoptosis induction. Figure i 1 A shows results of SA-fi-gai staining performed in HOC- Ϊ 419 and JiMT-I cells, respectively untreated (!) or treated with 50 »g ml TNF-α and 500 U/m IFN-γ (2), or treated with 10 ag/ml of trastuzumab (Tzm), pertuzumab (Per) (3), or treated with the combination of the same concentrations of trastuzumab, pertoznmab and TNF-a, IFN-y (4) for 5 days and cultured for 2 more passages in absence of the
antibodies and the cytokines. Top panel is a histogram of densiiometric analysis. Data are presented as % of SA-P-gal-positive ceils and presented as mean j; S.D. (n-3). P- values were calculated using a paired Student's t-test. Statistical significance was determined at **P < 0.01. Bottom panel, top layer, shows photographs of representative data from 1 of 3 independent experiments in HC-14I9 ceils. Bottom panel, bottom layer, shows photographs of representative data from 1 of 3 independent experiments in JIMT-I cells. Figure 1 I B shows results of cell lysates of the cells described in Figure 1 1 A which were analyzed by western blotting for pi SINKb or cleaved caspase-3 expression in HC-141 (left pane!) and JIMT-l {right panel). Vmculin was used as loading control. Similar results were observed, in 3 independent experiments.
Figure .1.2 shows IFN-yRa nd TNF-R.1 ate- expressed in similar levels in breast cell lines independently from their HER2 level. IFN-yRo, TNF-Rl and HER2 expression in immortalized MCF-IOA mammary epithelial cells and breast cancer cell lines (SK.-BR-3, BT-474, MCF-7, T-47D and MDA-MB-231 ) as determined by Western blot, Vinculin was used as loading control. Similar results were observed in 3 i ndependent experi ents.
Figures 13A-13B sho combined HER2 and HER3 blockage expression enhances Thl cytokines TNF-a and IFN-y senescence and apoptosis induction in MCF-7 breast cancer cells. Figure Ϊ 3 A shows results of studies in which S A- β-gal staining was performed in MCF-7 cells transfected with non-target siRN (siRNA NT), HER2 siRNA, HER3 siRNA or a combination of HER2 and BER3 siRNA, and then treated with the concentrations listed of TNF-a and IFN-y for 5 days and cultured for 2 more passages in absence of cytokines. Left panel shows a histogram of densitometric analysis. Data are presented as % of SA-p-gal-positive cells and presented as mean ± S.D. (n~3). P-vahies were calculated using a paired Student's t-test. Statistical
significance was determined at ***P < 0.001. Inset shows photographs of western blot analysis of MCF-7 cells transfeeted with NT, HER2 or HEK3. or a combination of HER2 and HER3 siRKA probed with HER2 and HERS specific antibodies. Vineulk was used as loading control. Similar results were observed in 3 independent experiments Right panel shows photographs of representative data from 1 of 3 independent experiments. Figure I3B shows results of studies in which cell lysates of the cells described in Figure 13A were analyzed by western blotting for p! SlNKb or cleaved caspase-3 expression. ***P < 0.001. Inset shows photographs of western blot analysis of SK-BR-3 cells transfected with NT, HER2, or HER3 siRNA probed with HER and HER3 specific antibodies. Vineulk was used as loading control. Similar results were observed k 3 independent experiments.
Figures I4A-14B show the effect of Till -elaborated cytokines on S -BR- 3 senescence and apoptosis. Figure 14Ashows he results of studies using a transwell system, whereby O.S l O5 SK-BR-3 cells were co-cultwed with 5x105 CD4' T-cells alone (CD4÷ only), CD4; T-cells + O.SxlO5 each of HER2 Class ΪΪ peptide (DC H)~ or
irrelevant Class II BRAF peptide (DC B)-pulsed type I .polarized mature DCs, and CD * T-ceils + HER2 (iDC H)- or BRAF (iDC Bj-puised immature DCs for 5 days. The cells were then cultured for 2 more passages in absence of immune -system ceils and then stained for SA-f¾-gaI expression and compared to untreated control ceils. Compared with IgG isotype control, senescence induced in SK-BR-3 treated with CD47DC H is partially rescued by neutralizing !FN-γ and TNF-a with specific antibodies (75.27 % rescue). Top panel is a histogram of densitometric analysis. Data are presented as % of SA-p-gal- positive ceils and presented as mean ± S.D. (n-3). ). P- values were calculated using a paired Student's t-test. Bottom panel shows corresponding photographs of representati ve data from I of 3 independent experiments. Figure MB shows photographs of western blots showing increased pi 5IN 4b and cleaved oaspase 3 expressions which suggest senescence and apoptosis induction of SK-B -3 ceils when co-cultured with DC H/CD4 ' T-ceils compared with DC B, iDC II and sDC B groups. Compared with IgG isotype control, senescence and apoptosis induced in SK-BR-3 treated with CD47DC H were partially rescued by neutralizing IFN-γ and T F-a specific antibodies, Vinculin was used as loading control. Results are representative of 3 experiments.
Figure 15 shows the effect of trastuzumab and pertuzumab on AKT activation by heregulin in breast cancer ceil lines. Serum-starved T-47D, HCC-I41 and JIMT-I cells were treated with rrastoizumab (Tzm) and pertuzumab (Per 10 ug/mh 90 min) and then stimulated with (HRG, 20 ng ml, 5 ruin). Top panel, sliows representative data from of 3 independent experiments. Data are expressed as % of the HRG response in the absence of trastuzumab and pertuzumab and presented as mean S.D. (n-3).
Figure 16 shows vaccination procedure. Patients with biopsy diagnosed FfER2$>l s DQI$ Were enrolled in the trial. Patient's monocytes were collected by
leukapheresis and elutriaiion. The monocytes were rapidly matured into type 1 DCs, and the pre- vaccination anti-HE 2 CD4 Th! immune response was measured. Patients underwent 4-6 weekly vaccinations (+ - anti-estrogen therapy). Patient's monocytes were collected again by a second leukapheresis and eiutriation, and the post-vaccination anti~HBR2 CD4 Thl immune response was measured. Following vaccination, patients underwent surgical resection to cure them of residual disease. The clini cal response was measured in the surgical specimen and the immune response was measured in the sentinel lymph nodesfSLNs") when available.
Figures 17A-17B shows results of SKBR3 and MCF7 breast cancer cell lines treated with Thl cytokines (IFNy and TNFa), a tamoxifen metabolite (4-F!ydroxy~ Tamoxifen, "4HT"), or both. SKBR3 (ERae¾) (Figure 17 A) increased anti-tumor activity in response to Thl cytokine treatment, but not to response to anti-estrogen treatment or the combination treatment. CF7 (ER!X,i) (Figure 17B) did not increase anti-tumor activity in response to either Th 1 cytokine treatment or anti-estrogen treatment, but the combination -.resulted in an. increase in metabolic activity.
Figure 18 shows patient distribution for the combination anti-estrogen ("AE") therapy and anti~HER2 DC! vaccination study. HER-2 positivity was defined as >5¾ of cells expression 2+ or 3+ intensity of the HER-2 protein on immunohistocbemistry. AE therapy (Tamoxifen., Letrozole, or Anastrozole) was .given concurrently with DC vaccination.
Figure 19 is a histogram showing pathologic complete response rate comparing patients by ER status and AE treatment (ERiit¾; £W* w/o AE; ERpos w AE).
Figures 20A-20B shows subsequent breast events of the study pallets comparing patients by pathologic complete response ("pCR") (Figure 20A) and ER status and AE treatment. (Figure 20B).
Figures 21 A-21C show CD4 - systemic immune response measured in the peripheral blood. By each metric of Thl response (respoiisivity (Figure 21 A); response repertoire (Figure 2 IB) and cumulative response (Figure 21C)), there was a significant increase in the immune response following anti-HER2 DC1 vaccination. However, the pre- and post-vaccination immune responses were similar across all three groups.
Figures 22A-22C show CD4i local regional immune response measured in patient sentinel lymph nodes. By each metric (responsivity (Figure 22 A); response repertoire (Figure 22B) or cumulative response (Figure 22C)) the post-vaccination immune responses were higher in the ER*** atients who received AE compared to the EW patients who did not receive AE.
Figure 23 shows CDS' systemic immune response measured i the peripheral blood. Responsivity of patients with E *® status and those with ER51"* status with and without anti-estrogen treatment (EE550* w/o AE; ER wAE) are shown.
Figure 24 shows BRAF M¾)i;~DCl vaccines overcome vemurafenib resistance in BRAF-mutant murine melanoma.
DETAILED DESCRIPTION
The present embodiments provide compositions and methods for producing an FDA-approved injectable multi-dose antigen pulsed dendritic cell vaccine for the personalized treatment and prevention of cancer or other disorders. In one embodiment, the embodiments' provide compositions and methods for producing an FDA-approved injectable muiti-dose antigen pulsed type ί polarized dendritic ceil vaccine (DC 1). In one embodiment, there is provided a method to cryopreserve dendritic cells in multiple-dose aliquots that are in an antigen-loaded, pre-activated state that is "syringe-ready", i.e. suitable for immediate injection into the patient without the necessity of any further cell processing that would require (e.g., by FDA mandate) additional facilities and quality control/assurance steps.
In one embodiment, there is provided a method to efficiently produce injectable multi-dose antigen pulsed dendritic cell vaccine, preferably injectable multi- dose antigen pulsed type 1 polarized dendritic ceil vaccine that exhibit maximal efficacy.
In one embodiment, an FDA-approved, injectable multi-dose antigen pulsed dendritic cell vaccine is produced by collecting DCs in a single patient
leukapheresis. Preferably, the leukapheresis and production of the dendritic cell vaccine is performed at a first location whereby the first location can be a centralized vaccine production facility where the DCs are manipulated to create an activated, antigen-loaded DC vaccine comprised of an initial immunizing dose and multiple "booster" doses thereof. An advantage of the present embodiments is that all FDA mandated quality control/quality assurance steps would be performed at the central facility, and after completion and release, all vaccine doses are cryopreserved and shipped to remote medical centers for serial administration to the patient. In one embodiment, the FDA- approved injectable multi-dose antigen pulsed dendritic cell vaccine of the embodiments does not -requirement any mandated quality control/quality assurance steps at the adminislxation site.
In another aspect, th present embodiments are based on the discovery that an effective therapy to treat cancer includes changing the immune response in. the tumor so that the immune cells in the tumor site are more effective in. attacking the tumor cells. In some instances, the effective therapy inclodes improving the migratio and activity of immune cells in the tumor site. Accordingly, the embodiments provide compositions and methods of using a dendritic cell vaccine in combination with a composition that inhibits one or more of HER-2 and IiER-3 (e.g., trastuzutnab, pertrrzumab, and the like) as a treatment regimen to treat cancer. In one embodiment, the treatment regimen comprises the use of dendritic cell vaccines, an inhibitor of HER-2, and a chemok ne modulator. In one embodiment, there is provided compositions and methods for using a dendritic cell vaccine in combination with blockage of one or more of HEE-2 and
HER-3 as a treatment regimen to treat cancer. In another embodiment, there is provided compositions and methods of using a dendritic cell vaccine in combination with blockage of HER-2 and HER-3 with the addition of TNF-α and IF -y. in another embodiment, there is provided compositions and methods of blocking one or more of HER-2 and HER- 3 with the addition ofTNF- and IFN-y as a treatment regimen to treat cancer.
In one embodiment, the treatment re gimen of the embodiments comprise a combination therapy of inducing an anti-oncodriv er Thl immune response (e.g., TNF~a and lFN-γ) and oncodri ver blockade for one or more of HER-2 and HER-3
in one embodiment, the treatment regimen of the embodiments can be used to treat cancer and therefore can be considered as a type of anti-cancer therapy, in another embodiment, the treatment, regime of the embodimen ts can be used in
combination with another anti-cancer therap including but is not limited to surgery, chemotherapy, radiatio therapy (e.g. X ray), gene therapy, immunotherapy, honuone therapy, viral therapy, DMA therapy, RNA therapy, protein, therapy, cellular therapy, nanotherapy, and the like.
in one embodiment, the treatment regimen of the embodiemems is used prior to receiving the other anti-cancer therapy. In another embodiment, the treatment regimen of the embodiments is used concurrently with receiving the other anti-cancer therapy . I n another embodiment, the treatment regimen of the embodiments is used after recei ving the other anti-cancer therapy.
In another embodiment, concurrent neoadjuvant anti-estrogen therapy and anti-HER2 DC1 vaccination increases the immune response in the local sentinel lymph nodes and the rate of pathological complete response in HER2poVERiH>* DOS patients.
Dejjpitiorts
Unless defined otherwise, all technical and scientific terms used herein ha ve the same meaning as commonly understood by one of ordinary skill, in the art to which these embodiments belong. Although any methods and materials- similar or equivalent to those descri bed herein caa be used in the practice or testing of the present embodiments, the preferred methods and materials are described.
Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization, are those well-known and commonly employed in the art.
Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed, according to conventional methods in the art and various general references (e.g., Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY, and Ausubel et al, 2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout this document.
The nomenclature used herei and the laboratory procedures used in analytical chemistry and organic syntheses described below are those well-known and commonly -employed in the art. Standard techniq ues or modifications thereof are used for chemical syntheses and chemical analyses.
As used herein, each of the following terms has the meaning associated with it in this section.
The articles "a" and "an" are used: herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the arti cle . By way o f example, "an element" means one element or more than one element.
"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, or ±10%, or .±5%, or ±]%, or i .Wo from the specified value, as such variations are appropriate to perform the disclosed methods.
The term "abnormal" when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day , etc.) from those organisms, tissues, cells or components thereof that display the "normal" (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type. The term ^antigen" or "ag* as used herei n, is defined as a molecule that provokes an immune response. This immune respons may involve either antibody production, or the activation of specific immiinologicaily-competent cells, or both. The skilled artisan will understand that any niacroraolecuie, including virtually ail proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand mat any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein.
Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene, it is readi!y apparent that the present embodiments include, but are not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
"An antigen presenting cell" (APC) is a cell that are capable of activating T cells, and includes, but is not limited to, monocytes/macrophages, B cells and dendritic ceils (DCs).
"Antigen-loaded APC" or an. "antigen-pulsed APC" includes an APC, which has been exposed to an antigen and activated by the antigen. For example, an ARC may become Ag~loaded in vitro, e.g. , during culture in the presence of an antigen. The APC may also be loaded in vivo by exposure to an antigen. An "'antigen- loaded APC" is traditionally prepared in one of two ways: (1) small peptide fragments, known as
antigenic peptides, are "pulsed" directly onto the outside of the APCs; or (2) the APC is incubated with whole proteins or protei particles whic are then ingested by the APC. These proteins are digested into smallpepti.de fragments by the APC and are eventually transported to and presented on the APC surface. In addition, the antigen-loaded APC can also he generated by introducing a polynucleotide encoding an antigen into the cell. "Anti-.HER2 response" is the immune response specifically against HER2 protein.
"Apoptosis-5 is the process of programmed eel! death. Caspase-3 is a frequently activated death protease. The term ''autqtminune disease" as used herein is defined as a disorder that, results from an autoimmune response. An autoimmune disease i s the result of an inappropriate and excessive response to a self-antigen. Examples of autoimmune diseases include bin are not limited to, Addision's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes- (Type I)*
dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis. Graves' disease, Guiilain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, .rayxedema, pernicious anemia, ulcerative colitis, among others .
As- used herein, the term "autologous" is meant to refer to any material derived from the same individual to which it is later to be re -introduced into the
individual.
The terra "B ceil" as used herein is defined as a cell derived from the bone marrow and/or spleen. B cells can develo into plasma ceils whic produce: antibodies.
The term "cancer" as used herein is defined as a hyperproliferatioii of cells whose unique trait—loss of normal control—esults in unregulated growth, Sack of differenti tion, local tissue invasion, and/or metastasis. Examples include -but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer and lung cancer.
"CD4; Thl cells," "Thl cells," "CD4+ T-heiper type 1 cells," "CD4+ T cells," and the like are defined as a subtype of T-heiper cells that express the surface protein CD4 and produce high levels of the cytokine IFN-y. See also, "T- eiper cells." "Cumulative response" me ns the combined immune response of a patient group expressed as tke total sum of reactive spots (spot-forming cells "SFC" per 106 cells from IF -γ ELISPOT analysis) from all 6 MHC class II binding peptides from a given patient group- The term "cryopreserved" or "cryopreservation" as used herein refers to cells that have been resuspended in a freezing medium and frozen at a temperature of around --70°C or lower,
"DC vaccination," "DC immunization/'' '$DC 1 immunization ' and the like refer to a strategy using autologous dendritic ceils to harness the immune system to recognize specific molecules and mount specific responses against them.
The term "dendritic cell" (DC) is an antigen presenting cell existing in vivo, in vitro, e vivo, or in host or subject, or which can be derived from a
hematopoietic stem ceil or a monocyte. Dendritic ceils and their precursors can be isolated from a variety of lymphoid organs, e.g., spleen, lymph nodes, as well as from bone marrow and peripheral blood. The DC has a characteristic morphology with thin sheets (iarneSlipodia) extending in multiple directions away from the dendritic cell body. Typically, dendritic cells express high levels of MHC and costimulatory (e.g., B7-3 and B7-2) molecules. Dendritic cells can induce antigen specific differentiation of T cells in vitro, and are able to initiate primary T cell responses in vitro and in vivo.
As used herein, an "'activated DC" is a DC that has been exposed to a 'Foil- like receptor agonist The activated DC may or may not be loaded with an antigen.
The term "mature DC" as used herein, is defined as a dendritic ceil that expresses molecules, including high levels of MHC class Π, CD80 (B7.1) and CD86 (B7.2). in contrast, immature dendritic cells express low levels of MHC class II, CD80 (B7.1 ) and CD86 { B7.2) molecules, yet can still take up an antigen. "Mature DC" also refers to an antige presenting cell existing in vivo, in vitro, ex vivo, or to a host or subject that is DCI-polarized (i.e., fully capable of promoting cell-mediated, immunity). A "disease ' is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is riot ameliorated then the animal's health continues to deteriorate.
A "disorder" in an animal is a state of health in which the animal is able to maintain .homeostasis, bat in which the animal's state of health is less favorable than, it would be in the absence of the disorder. Left untreated, a disorder does not necessari ly cause a farther decrease in the animal's state of health.
A disease or disorder is "alleviated" i f the severity or frequency of at least one sign or symptom of the disease or disorder experienced by a patient is reduced,
"Effective amount" or "therapeutically effective amount" are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herei effective to achieve a particular biological result. Such results may include, but are not l imited to, the inhibition of virus infection as determined by any means suitable in the art
As used herein "endogenous" refers to any material from or produced inside an organism, ceil, tissue or system.
As used herein, the term "exogenous" refers to any material introduced, from or produced outside an organism, cell, tissue or system.
"Estrogen receptor (ER) positive" cancer is cancer which tests positive for expression of ER. Conversely, "ER negative" cancer tests negative for such expression. Analysis of ER status can be performed by any method known in the art.
The term "freezing medium" as used herein refers to any medium mixed with a. cell sample in preparation for freezing, such that at least some of cells within the cell sample can be recovered and remain viable after thawing.
"HER2" is a member of the human epidermal growth factor receptor
("EGFR") family, HER2 is overexpressed in approximately 20-25% of human breast cancer and is expressed in many other cancers
A "HER receptor" is a receptor protein tyrosine kinase which belongs to, the HER receptor family and includes EGFR (ErbBL HER!), HER2 (ErbB2), HER3 ( E.rbB3) and HER4 (ErbB4) receptors. The HER receptor will generally comprise an extracellular domain, which may bind an HER ligand and/or dimerize with another HER receptor molecule; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-termraal signaling domain harboring several tyrosine residues which can be phosphorylated. The HER receptor may be a "native sequence" HER receptor or an "amino acid sequence variant" thereof. Preferably the HER receptor is a native sequence huma HER. receptor.
The "HER pathway" refers to the signaling network mediated, by the HER receptor family.
"HE activation" refers to activation,. or phosphorylation, of any one or more HER receptors. Generally, HER activation results i signal transduction (e.g. thai caused by an intracellular" kinase domain of HER receptor phosphorylating tyrosine residues in the HER receptor or a substrate polypeptide), HER activation may be
mediated by HER. ligand binding to a HER dimer comprising the HER receptor of interest, HER ligand binding to a HER dimer may activate a kinase domain of one or more of the HER receptors in the dimer and thereby results in phosphorylatio of tyrosine residues in one or more of the HER receptors and/or phosphorylation of tyrosine residues in additional substrate polypeptides(s), such as Akt orMAPK intracellular kinases.
"HER2 binding peptides," "HER2 MHC class Π binding peptides ' "binding peptides," "HER2 peptides," "immunogenic MHC class II binding peptides," "antigen binding peptides," "HER2 epitopes," "reactive peptides," and the like as used herein refer to MHC Class II peptides derived from or based on the sequence of the i IER2/«i¾ protein, a target found on approximately 20-25% of ail human breast cancers and their equivalents. HER2 extracellular domain "BCD" refers to a domain of HER2 that is outside of a cell, either anchored to a cell membrane, or in circulation, including fragments thereof. HER2 intracellular domain "ICD" refers to a domain of the
HER2/«t¾ protein within the cytoplasm of a cell. According to a preferred embodiment HER2 epitopes or otherwise binding peptides comprise 6 HER2 binding peptides which include 3 HER2 ECD peptides and 3 HER2 ICD peptides.
Preferred HER2 ECD peptides comprise:
Peptide 42-56: BLDMLRHLYQGCQVV (8BQ ID NO: 1);
Peptide 9S-1 1 : RLRiVRGTQLFEDr^YAL (SEQ ID NO: 2); and
Peptide 328-345: TQRCEKCS PCARVCYGL (SEQ ID NO: 3); Preferred HE 2 ICD peptides comprise:
Peptide 776-790: GVGSPYVSRLLGICL (SEQ ID NO: 4);
Peptide 927-941 : PAREIPDLLEKGERL (SEQ ID NO: 5); and
Peptide 1 166-1 180: TLERPKTLSPG NGV (SEQ ID NO: 6).
In embodiments where patients are HLA~A2po7have A2.1 blood type HC class I peptides or epitopes comprise:
Peptide 369-377: lFGSLAFL (SEQ ID NO:7); and
Peptide 689-69? iRLLQETELV (SEQ ID NO:8)
"HER2P"-" is the classification or molecular subtype of a type of breast cancer as well as numerous other types of cancer. HER2 positivity is currently defined by gene amplification b FISH (fluorescent in situ hybridization) assay and 2÷ or 3+ on intensity of pathological staining.
"HER ^' is. defined! by the lack of gene amplificat on by FISH, and can encompass a . range of pathologic staining from 0 to 2+ i most cases.
The term "hyperprolifetative disease" is defined as a disease that results from a hyperproliferation of cells. Exemplary hyperproliferative diseases include, but are not limited to, cancer or autoimmune diseases. Other hyperproliferative diseases may include vascular occlusion, restenosis, atherosclerosis, or inflammatory bowel disease, for example.
The term "inhibit," as used herein, means to suppress or block an activity .or unction, for example, about ten percent relative to a control value. Preferably, the. activity is suppressed or blocked by 50% compared to a control value, more preferably by 75%, and even more preferably by 95%. "Inhibit," as used herein, also means to reduce a molecule, a reaction, an interaction, gene, an mRNA, and or a protein's expression, stability, function or activity b a measurable amount or to prevent entirely. Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., antagonists. As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to
communicate the usefulness of the compositions and methods of the embodiments. The instructional material of the kit of the embodiments may, for example, be affixed to a container which contain the nucleic acid, peptide, arid/or composition of the
embodiments or be shipped together with a container which contains the nucleic acid, peptide, and/or composition. Alternatively, the instnictiona! material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
"Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
"Metrics" of CD4'! Thl responses (or Thl responses) are defined for each subject group analyzed for aoti-HER2 CD * Thl immune response; (a) overall anti~HER2 responsivtty (expressed as percent of subjects responding to >! reactive peptide); (b) response repertoir (expressed as mean number of reactive peptides (n) recognized by each subject group); and (c) cumulative response (expressed as total sum of reactive spots (spot-forming cells "SFC" per 10* cells from iFN-γ ELiSPOT analysis) from 6 MHC Class II binding peptides from each subject group.
By the term "modulating," as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the le vel of a response in the subject in the absence of a treatment or compound, and/or compared wi th the level of a response in an otherwise identical but untreated subject. The term
encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a hunian.
"Neoadjuvant therapy" for breast cancer as used herein refers to treatment given before primary therapy (i.e., surgery). "Adjuvant therapy" is treatment given after primary therapy to increase the chance of long-term survival. As used herein, a "population." includes reference to an isolated culture comprising a homogenous, a substantially homogenous, or a heterogeneous culture of cells. Generally, a "population" may also be regarded as an "isolated" culture of cells.
As used herein, a "recombinant cei l" is a host cell that comprises a recombinant polynucleotide.
" esponsivity" or "antt-HE 2 responsivity" are used interchangeably herein to mean the percentage of subjects responding to at. least I of 6 binding peptides.
"Response repertoire" is defined as the mean n umber ("n") of reactive peptides recognized by each subject group.
"Sample" or "biological sample" as used herein means a biological material from a subject, including but is not limited to organ, tissue, exosome, blood, plasma, saliva, urine and other body fl uid, A sample can be any source of materia! obtained from a subject.
"Senescence" refers to cells no longer capable of dividing but which are stil! alive and metabo!ica!iy active. Hallmarks of senescent cells include an essentially irreversible growth arrest, and expression of SA-p-gal, PI SINK
4B and l6IN 4a.
"Signal 1 " as used herein generally refers to the first biochemical signal passed from an acti vated DC to a T cell. Signal 1 is provided by an. antige expressed at the surface of the DC and is sensed by the. T cell through the T ceil receptor .
"Signal 2" as used herein generally refers to the second signal provided by DCs to T cells. Signal 2 is provided by "costimulatory" molecules on the activated DC, usually CD80 and/or CD86 (although there are other co-stimulatory molecules known), and is sensed by the T cell through the surface receptor CD28.
"Signal 3" as used herein generally refers to the signal generated from soluble proteins (usually cytokines) produced by the activated DC. These are sensed through receptors on the T lymphocyte. The 3rd signal instructs the T cell as to which phenotypical or functional feature they should ac uire to best deal with the current threat. By the term ^specifically binds," as used herein, is meant a molecule, such as an antibody, which recognizes and binds to another molecule or feature, but does not substantially recognize or bind other molecules or features i a sample.
The terms "subject " "patient," "individual." and the like are used
interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.
" T/C" is defined as uastuzumab and chemotherapy. This refers to patients that receive both trastazumab and chemotherapy before/after surgery for breast cancer.
The term "T cell" as used herein is defined as a thymus-derived cell that participates in a variety of cell-mediated immune reactions.
The terms "T~helper cells," "helper T cells," "Th cells," and the like are used herein with reference to cells indicates a sub-group of lymphocytes (a type of white blood ceil or leukocyte) including different cell types identifiable by a skilled person in the art. In particular, T-helper ceils are effector T-cells (such as Th 1, Th2 and Thl7) whose primary function is to promote the activaticm and functions of other B and T lymphocytes and/or macrophages. Helper T cells differentiate into two major subtypes of cells known as "Th!" or "Type I" and "Th2" or "Type 2" phenotypes. These Th cells secrete cytokines, proteins, or peptides that stimulate or interact with other leukocytes. "Th! cell " "CDC Th! cell," "CD4! T-helper type! cell," "CM* T csHw and the like as used herein refer to a mature T-cell that has expressed the surface glycoprotein CD4. CD4' T-helper ceils become activated when they are presented with peptide antigens by MHC class H molecules which are expressed on the surface of antigen-presenting peptides ("APCs" ) suc h as dendr itic cells. Upon acti vation of a CM T helper ceil by the MHC -antigen complex, it secretes high levels of cytokines such as interferon-y flFN-γ"), Such cells are thought to be highly effective against certain disease-causing microbes that Hve inside host cells, and are critical in antitumor response in hum
cancer.
"Thi T celF as used herein refers to a T cell that produces high levels of the cytokine !F -γ and is thought to be highly effective against certain disease-causing microbes that live inside host cells, and cancer as well. "Thl7 T cell" as used herein refers to a T cell thai produces high levels of the cytokines IL~17 and !L-22 and is thought to be highly effective against disease- causing microbes that live on mucousat surfaces.
"Therapeutically effective amount" is an amount of a compound of the embodiments, that when administered to a patient, ameliorates a symptom, of the disease. The amount of a compound of the erabodiements which constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity , the age of the patient to be treated, and the like. The therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.
The terras "treat," "treating," and "treatment," refer t therapeutic or preventative measures described herein. The methods of "treaimenf ' emplo
administration to a subject, in need of such treatment, a composition of the present embodiments, for example, a subject afflicted a disease or disorder, or a subject who ultimately may acquire such a d sease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorat one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
The term "Toll like receptor", or "TLR" as used herein is defined as a class of proteins that play a role in the innat immune system, TLRs are single membrane-spanning, non-catalytic receptors that recognize structurall conserved molecules derived from microbes. TLRs activate immune cell responses upon binding to a ligand.
The term "Toll like receptor agonists", or 4lTLR agonists" as used herein is defined as a ligand that binds to the TL to acti ate immune cell response.
The term "vaccine" as used herein is defined as a material used to provoke an immune response after administration of the materia! to an animal, preferably a mammal, and more preferably a human. Upon introduction into a subject, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies, cytokines and/or other cellular responses. Ranges; throughout this disclosure., various aspects of the embodiment can be presented in a range format, it should be understood that the description i range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the embodiments. Accordingly, the description of a range should be considered to have spec.ifica.lly disclosed all the possible subranges as well as individual numerical values withi that range . For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges soch as from 1 to 3, from 1 to 4, from 3 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example. 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range . fJtescription
The present embodiments include a preparation of DCs. In one embodiment, the DC preparations are greater than 90% pure. In another embodiment, the DC preparations are fully activated. For example, the DCs are activated with a cytokine and''or a Toil like receptor Hgaiich a state which is fully maintained by the
cryopreservation technique of the embodiments. A benefit of the DC preparation of the embodiments is that the cells are efficientl cryopreserved from a single leukapheresis (patient collection) into an initial vaccine plus multiple "booster" doses (e.g., 10 or more) that can be thawed as needed at remote treatment locations without any specialized cell processing facilities or further required quality control testing.
As contemplated herein, the present embodiments provide a method for generating and cryopreservmg DCs with superior functionality in producing stronger signals to T cells, and thus resulting in a more potent DC -based vaccine. By effectively cryopreserving such cells, samples can be stored and thawed for later use, thereby reducing the need for repeated pheresis and elutriation processes during vaccine production. Being able to freeze DCs and then thaw them out later is an advantage because it means that a single round of vaccine production ca be divided into small parts, frozen away, and then administered one at a time to a patient over the course of weeks, months, or years to give "booster" vaccinations that strengthen immunity. In one embodiment, the present embodiments includes an FDA- approved injectable multi-dose antigen pulsed dendritic cell vaccine produced by coileciing DCs in a single patient leukaplieresis. The FDA- approved injectable multi-dose antigen pulsed dendritic cell vaccine comprises an initial immunizing dose and multiple "booster" doses. The FDA- approved injectable multi-dose antigen pulsed dendritic ceil vaccine are cryopreserved and can be shipped to remote medical centers for serial administration to the patient with no special requirements at the administrati site (e.g., FDA mandated QC QA steps).
The present embodiments also relate to the cryopreservatio of these activated DCs in a. manner that retains their potency and functionality in presenting antigen as well as their production of various cytokines and chemokines after thawing, such that the cryopreserved and subsequently thawed activated DCs are as clinically effective as freshly harvested and activated DCs,
The present embodiments also relate to inducing tumor senescence and apoptosis in a cell by blocking one or more of HER-2 and HER-3 in combination with activating anti-HER-2 CD4 Till cells. Accordingly, the embodiments include a combination and method for promoting an anti-oncodriver T i immune response with an oncodri ver blockade for HER-2 in order to promote tumor senescent in HER-2
expressing breast cancers. In one embodiment, promoting an anti-oncodriver Thi immune response comprises TNF~ and IFN-γ, i one embodiment an oncodri ver blockade for HER-2 includes any composition that blocks HER-2 i cluding but is not limited to trastuxumab and pertuzumab.
in one embodiment, there are compositions and methods for the combination of blocking one or more of HER-2 and HER-3 together with, the addition of TNF-a and IFN-γ for inducing senescence of HER-2 expressing breast cancer, in one embodiment, the TNF-a and lFN-y is secreted from CD4 Thi cells,
in one embodiment, HER2 is required in the mechanism of TNF-a and IFN-y inducing senescence and apoptosis in breast cancer cells.
In one embodiment, TNF-a and IFN-γ restores the sensitivity to trastuzuBiah and pertumtmab to breast cancer resistant cells, in one embodiment, the Thi cytokines, IFN~y and TNF-a, revert the resistance to the therapeutic agents that, is affecting cancer patients widely.
DC-Based Immunotherapy
DCs are derived from pluripotent monocytes that serve as antigen- presenting cells (APCs). DCs are ubiquitous in peripheral tissues, where they are prepared to capture antigens. Upon antigen capture, DCs process the antigen into small peptides and move towards secondary lymphoid organs. It is within the lymphoid organs that DCs present antigen peptides to naive T cells, thereby initiating a cascade of signals that polarizes T cell differentiation. Upon exposure, DCs present antigen molecules bound to either MHC class I or class II binding peptides and activate CDS'" or CD4* T cells, respectively (Stehnnan, 1991 , Anna. Rev. Immunol 9:271—296; Banchereaa. et al , 1998, Nature392,245-252; Stemman, ei at, 2007, Nature 449:41 -426; Ginhoux et al, 2007, J. Exp, Med. 204:3133-3146; Banerjee et al, 2006, Blood 108:2655-2661 ;
Sailusto et al, 1 99, J . Exp. Med. 189:61 3 -614; eid et al., 2000, Curr. Opin.
Immunol .12: 1 14—121 ; Bykovskaia et al., 1 99, J. Leukoc. Biol 66:659-666; Clark et at, 2000, Microbes infect. 2:257-272).
DCs are responsible for the induction,, coordination and regulation of the adapti ve immune response and also serve to orchestrate communication, between effectors of the innate arm and the adaptive arm of the immune system. These features have made DCs strong candidates for immunotherapy, DCs have a unique capacity to sample the environment through macropinocytosis and receptor-mediated endocytosis (Gemer et al, 2008, J. Immunol.181:155-164; Sto.itar.ner et al. , 2008, Cancer Immunol Iimmmother 5 ; 1665- 1673; Lanzevecchia A., 1 96, Curr. Opin. Immunol 8:348-354; Delamarre et al, 2005, Science, 307(5715): 1630-1634).
DCs also require maturation signals to enhance their antigen-presenting capacity. DCs upregulate the expression of surface molecules, such as CD80 and CD86 (also known as second signal molecules) by providing additional maturation signals, such as TNF-a. CD40L or calcium signaling agents (Czerniec et al, 1997,. J.
Immunol.159:3823-3837; Bedrosian et al. 2000, L immunother. 23:311-320; Mai!Iiard et at, 2004, Cancer Res.64,5934-5937; Brossart et al, 1998, Blood 92:4238-4247; Jin et al, 2004, Hum. 'Immunol.. 65 93- -103). It has been, established that a mixture of cytokines, including TNF-et, IL-l p, IL~6 and prostaglandin. E2 (PGE2), have the ability to mature
DC (Jonuleit, et a!.5 2000, Arch. Derm. Res. 292:325 --332). DCs can also be matured with calcium ionophore prior to being pulsed with antigen,
In addition to pathogen-recognition receptors, such as PK.R and MDA-5
( alaii et a!., 2008, I Immunol. 181 :2694 -2704; Nallagatla et al, 2008, RNA Biol.
5(3): 140-144), DCs also contain a series of receptors, known as Toll-like receptors
(TLRs), that are also capable of sensing danger from pathogens. When these TLRs are triggered, a series of activational changes are induced in DCs, which lead to maturation and signaling of T cells (BouHart et al. 2008, Cancer Immunol Immunotber.
57(1 1): 1589-1597; Kaisbo et al, 2003, Curr. MoL Med. 3(4):373-385; PuJendran et al.,
2001 , Science 293(5528):253-256; Napolitani et al, 2005, Nat. Immunol. 6(8):769-776).
DCs can activate and extend the various arms of the cell-mediated response, such as natural killer γ~δ T and α~β T cells and, once activated, DCs retain their immunizing capacity (Steinman, 19 1 , Anrai. Rev. Immunol 9:271 -296; anchereau et al., 1 98,
Nature 392:245-252; Reid et al, 2000, Curr. Opin. Immunol 12:1 14-121 ; Bykovskaia et al, 1999, J. Leukoc. Biol66:659 --666; Clark et al, 2000, Microbes infect. 2:257 -272).
The present embodiments include mature, antigen loaded DCs activated by Toll-like receptor agonists that induce clinically effecti ve immune responses, preferably when used earlier in the disease process. The DCs of the present embodiments produce desirable levels of cytokines and cheniokmes, and further have the capacity to induce apoptosis of tumor cells.
In one embodiment, there is provided a method of large scale production of antigen pulsed dendritic celi vaccine. In one embodiment, the method comprises rapidly maturing dendritic cells, cryopreservmg the dendritic cells, and thawing the cryopreserved cells wherein the thawed dendritic cells produce an effective amount of at least one cytokine to generate a T cell response.
In one emhodiment, the maturation, of dendritic cells comprise contacting the cells with IF -gamma and LPS.
in one embodiment, the thawed cells maintain. DO phenotype to drive a
Thi polarized immune response. In one embodiment, the thawed cells maintain the ability to primarily sensitize T cells.
Generation of a loaded (pulsed) immune cell
The present embodiments include a cell that has been exposed or
otherwise "pulsed" with an antigen. For example, an AFC. such as a DC, may become Ag-ioaded in vitro, e.g., by culture ex vivo in the presence of an antigen, or in vivo by exposure to an antigen.
A person skilled in the art would also readily understand that an. APC can be "pulsed" in a manner that exposes the APC to an antigen for a time sufficient to promote presentation of that antigen on the surface of the APC. For example, an APC can be exposed to an antigen in the form of small peptide fragments, known as antigenic peptides, which axe "pulsed" directly onto the outside of the APCs (Mehta-Damaiii t al., 1 94); or APCs can be incubated with whole proteins or protein particles which are then ingested by the APCs, These whole proteins are digested into small peptide fragments by the APC and eventually carried to and presented on the APC surface (Cohen et a!,, 1994). Antigen in peptide form may be exposed to the cell by standard "pulsing" techniques described herein.
Without wishing to be bound by any particular theory, the antigen in the form of a foreign or an autoantigen is processed by the APC of the embodiments in order to retain the immunogenic form of he antigen. The immunogenic form of the antigen implies processing of the antigen through fragmentation to produce a form of the antigen that can be recognized b and stimulate immune cells, for example T cells. Preferably, such a foreign or an autoantigen is a protein which is processed into a peptide b the APC. The relevant peptide which is produced by the APC may be extracted and purified for use as an immunogenic composition. Peptides processed by the APC may also be used to induce tolerance to the proteins processed by the APC.
The antigen-loaded APC, otherwise known as a "pulsed APC" of the embodiments, is produced by exposure of the APC to an. antigen either in vitro or in vi , In the case where the APC is pulsed in vitro, the APC can be plated on a culture dish and exposed to an antigen in a sufficien t amount and for a s uffic ient period of time to allow the antigen to bind to the APC, The amount and time necessary to achieve binding of the antigen to the APC may be determined by using methods known in. the art or otherwise disclosed herein. Other methods known to those of skill in the art, for example immunoassays or binding assays, may be used to detect the presence of antigen on the APC following exposure to the antigen.
in a further embodiment, the APC may be transfected with a vector which allows for the expression of a specific protein by the APC The protein, which is expressed by the APC may then be processed and presented on the cell surface. The transfected APC may then be used as an immunogenic composition to produce an.
immune response to the protein encoded by the vector.
As discussed elsewhere herein, vectors may be prepared to include a specific polynucleotide which encodes and expresses a protein to which an immunogenic response is desired. Preferably, retroviral vectors are used to infect the cells. More preferably, adenoviral vectors are used to infect the cells,
in another embodiment, a vecior may be targeted to an APC by modifying the viral vector to encode a protein or portions thereof that is recogni zed by a receptor on the APC, whereb occupation of the APC receptor b the vector will initiate endocytosis of the vector, al lo wing for processing and presentation of the antige encoded by the nucleic ac id of the viral vector . The nucleic acid which is delivered by the virus may be native to the virus, which when expressed on the APC encodes viral proteins which are then processed and presented on the MHC receptor of the A P
As contemplated herein, various methods can be used for transfecting a polynucleotide into a host cell. The methods include, but are not limited to, calcium phosphate precipitation, lipofection, particle bombardment, microinjection,
electroporation. colloidal dispersion systems (i.e. macromolecute complexes,
nanoeapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes). These methods are understood in the art and are described in published literature so as to enable one skilled in the art to perform these methods.
in another embodiment a polynucleotide encoding an antigen can be cloned into an expression vector and the vector can be introduced into an APC to otherwise generate a loaded APC. Various types of vectors and methods of introducing nucleic acids into a cell are discussed i the available published. literature. For example, the expression vector can be transferred into a host cell by physical, chemical or
biological means. See, for example, Sambrook et al {'2001. Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory. New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York). It is readily understood that the introduction of the expression vector comprising a
polynucleotide encoding an antigen yields a pulsed cell.
The present embodiments include various methods for pulsing APCs including, but not limited to, loading APCs with whole antige in the form of a protein, cDNA or mRNA. However, the embodiments should not be construed to be limited to the speci fic form of the antigen used for pulsing the APC, R ather, the embodiments encompass other methods known in the art for generating an antigen loaded APC.
Preferably, the APC is tranfected with mRNA encoding a defined antigen, mRNA corresponding to a gene product whose sequence is known can be rapidly generated in vitro using appropriate primers and reverse transcriptase-polymerase chain reaction (RT- PCR) coupled with transcripti on reactions. Transfection of an APC with an mRNA provides an advantage over other antigen-loading techniques for generating a pulsed APC. For example, the ability to amplify RNA from a microscopic amount of tissue, i.e. tumor tissue, extends the use of the APC fo vaccination to large number of patients .
For an antigenic composition to he useful as a vaccine, the antigenic composition must induce an immune response to the antigen in a cell, tissue or mammal (e.g., a human). As used herein, an "immunological composition" may comprise an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding an antigen (e.g., an antigen expression vector), or a cell expressing or presenting an antigen or cellular component. In particular embodiments the antigenic composition comprises or encodes all or part of any antigen described herein, or an immunologically functional equivalent thereo In other -embodiments,, th antigenic composition, is in a mixture that comprises m additional immunostimulatory agent or nucleic acids encoding such an agent.
Imm.unostimulatory agents include but are not limited to an additional antigen, an immunomodulatory an antigen presenting cell or an adjuvant, in other embodiments, one or more of 'the additional agentfs) is covalently bonded to the antigen or an
iramimostiraulatory agent, in any combination. In. certain embodiments, the antigenic composition is conjugated to or comprises an HLA anchor motif amino acids.
A vaccine, as contemplated herein, may vary in its composition of nucleic acid and/or cellular components. In a non-!imiting example, a nucleic encoding an antigen might also be formulated with an adjuvant. Of course, it will be understood that various compositions described herein may further comprise additional components. For example, one or more vaccine components may be comprised in a lipid or liposome, in another non-limiting example, a vaccine may comprise one or more adjuvants. A vaccine of the present embodiments, and its various components, may be prepared and/or administered by any method disclosed herein or as would be known to one of ordinary skill in the art, in light of the present disclosure.
It is understood that an antigenic composition of the presen embodiments may be made by a method thai is well known, in the art, including hut not limited to chemical synthesis by solid phase synthesis and purification away from the other products of the chemical reactions by HPLC, or production by the expression of a nucleic acid sequence (e.g., DNA sequence) encoding a peptide or polypeptide comprising an antigen of the present embodiments in an in vitro translation system or in a living cell, in addition, an antigenic composition can comprise a cellular component isolated from a biological sample. The antigenic composition isolated, and extensively dialyzed to remove one o more undesired small molecular weight molecules and/or lyopliilized for more ready formulation into a desired vehicle. It is further understood that additional amino acids, mutations, chemical modification and such like, if any, that are made in a vaccine component will preferably not substantially interfere with the antibody
recognition of the epitopic sequence.
A peptide or polypeptide corresponding to one or more antigenic
determinants of the present embodiments should generally be at least five or six amino acid residues in length, and may contain up to about 10, about 15, about 20, about 25, about 3.0, about 35, about 40, about 45 or about 50 residues or so. A peptide sequence may be synthesized by methods known to those of ordinary skill in the art, such as, for example, peptide 'synthesis using automated peptide synthesis machines, such as those available from Applied Biosysiems, lac, Foster City. CA (Foster City, CA).
Longer peptides or polypeptides also may be prepared, e.g., by .recombinant means. In certain embodiments, a nucleic acid encoding an antigenic composition .and/or a component desc ed herein may be used, for example, to produce an antigenic composition in vitro or in vivo for me various compositions aod methods of the present embodiments. For example, in certai embodiments, a nucleic acid encoding an antigen is comprised in, for example, a vector in a recombinant cell. The nucleic acid may be expressed to produce a peptide or polypeptide comprising an antigenic sequence. The peptide or polypeptide may be secreted from the cell, or comprised as part of or within the cell.
In certain embodiments, an immune response may be promoted by transfectiftg o inoculating a mamma! with a nucleic acid encoding an antigen. One or more cells comprised within a target mamma! then expresses the sequences encoded by the nucleic acid after administration of the nucleic acid to the mamma!. A vaccine may also be in the form, for example, of a nucleic acid (e.g., a cDN A or an RNA) encoding all or part of the peptide or polypeptide sequence of an antigen. Expression in vivo by the nucleic acid may be, for example, by a plasraid type vector, a viral vector, or a viral/plasmid construct vector.
in another embodiment, the nucleic acid comprises a coding region that encodes all or part of the sequences encoding an appropriate antigen, or an
immunologically functional equivalent thereof. Of course, the nucleic acid may comprise and/or encode additional sequences, including but not limited to those comprising one or more immunomodulators or adjuvants.
Antigens
As contemplated herein, the present embodiments may include use o f any antige suitable for loading into an. APC to elicit an. immune response. In one
embodiment, tumor antigens may be used. Tumor antigens can be divided into two broad categories: shared tumor antigens; and unique tumor antigens. Shared antigens are expressed by many tumors, while unique tumor antigens can result from mutations induced through physical or chemical carcinogens, and are therefore expressed only by individual tumors. In certain embodiments, shared tumor antigens are loaded into the DCs of the present embodiments. In other embodiments, unique tumor antigens are loaded into the DCs of the present embodiments.
In the contex of the present embodiments, "inmor antigen" refer to antigens that are common to specific hyperproliferative disorders. I certain aspects, the hyperproliferative disorder antigens of the present embodiments are derived from
cancers, including but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkins lymphoma, leiikeraias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.
Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include, but are not limited to, tissue- specific antigens such as MART- 1 , tyrosinase and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HBR~2/ eu/ErbB~2. Yet another group of target, antigens are onco-fetal antigens, such as carcinoembryonic antigen (CEA). In B cell lymphoma, the tumor- specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor, B cell differentiation, antigens, such as CD 19, CD20 and CD37, are other candidates for target antigens in B cell lymphoma. Some of these antigens (CEA, HER-2, CD 19, CD2G, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.
The tumor antigen and the antigenic cancer epitopes thereof may be purified and isolated from natural sources such as from primary ciinicai isolates, ceil lines and the like. The cancer peptides and their antigenic epitopes may also be obtained by chemical synthesis or by recombinant DMA techniques known hi the arts. Techniques for chemical synthesis are described in Steward et at. (1969); Bodansk et al. (!9?6);
Meienhofer (1983); and Schroder et al. (1965). Furthermore, as described in Renkvist et al. (2001). there are numerous antigens known in the art. Although analogs or artificially modified epitopes are not specifical ly described, a sk lled artisan recognizes how to obtain or generate them by standard means i the art. Other antigens, identified by antibodies and as detected by the Serex technology (see Sahin et al. (1 97) and Chen et al. (2000)). are identified in the database of the Ludwig Institute for Cancer Research.
In yet; another embodiment, the present embodiments may include microbial antigens for presentation by the AFCs. As contemplated herein, microbial antigens may be viral, bacterial, or fungal, in origin. Examples of infectious virus include: Retroviridae (e.g. human immunodeficiency viruses, such as HlV-1 (also referred to as HTLV-IH, LAV or HTLV-III/LAV, or HIV-ΠΙ; and other isolates, such as HIV-LP; Pico navMdae (e.g. polio viruses,, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echov ruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g. corona viruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); FiSoviridae (e.g. ebola viruses); Para yxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthoniyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, plrleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses): Reoviridae (e.g. reoviruses, orbiviruses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);
Papova iridae (papilloma viruses, polyoma viruses); Adenoviridae (most adeiiovimses); i lerpesviridae (herpes simplex virus (BSY) 1 and 2, varicella zoster virus,
cytomegalovirus (CMV), herpes virus); Poxviridae (variola viruses, vaccima viruses, pox viruses); and Iridoviridae (e.g. African, swine fever virus); and unclassified viruses (e.g. the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis
(thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1 ^internally transmitted; class 2=patenteraUy transmitted (i.e. Hepatitis C); Norwaik and related viruses, and astroviruses).
Examples of infectious bacteria include'.. Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophila, Mycobacteria, sps (e.g. M. tuberculosis, M. avium, M. intraceHulare, M. kansasii, M. gordonae). Staphylococcus aureus, Neisseria gonorrhoeae. Neisseria meningitidis. Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus (viridans group). Streptococcus faecalis, Streptococcus bovis,
Streptococcus (anaerobic sps.), Streptococcus pneumoniae., pathogenic Campylobacter sp.s Enterococcus sp., Haemophilus influenzae, Bacillus antbracis, corynebacteriura diphtheriae, corynebacterium sp,, Erysipelothrix rhusiopathiae, Clostridium perfrin.ge.ns, Clostridium tetaai, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bactero des sp., Fusobacterium nucleatum, StxeptobaciUus moniliformis, Treponema Treponema pertenue, Leptospira, and Actinomyces israelii.
Examples of infectious fungi include: Cryptococeus neoformans,
Histoplasma capsulatimi, Coccidioides immitis, Blastomyces dermaiitidis. Chlamydia trachomatis, Candida albicans. Other infectious organisms (i.e., protists) including:
Plasmodium falciparum and Toxoplasma gondii.
Activation of DCs
While traditional DC-based vaccines (that have previously dominated clinical trials) comprise of matured DCs generated from using a cytokine cocktail mixture including combinations of TNF, IL-6, PGE2 and IL-Ι β, which ultimately stimulate aseptic inflammation, the present embodiments instead utilize TLR agonists to mature the DCs and stimulate production of signal.
According to an aspect of the present embodiments, the stimulation of
DCs with a combination of TLR !igands leads to the production of increased amounts of IL-12. Further, activation of DCs with a combination of TLR agonists can yield a more pronounced CD4 and CDS T~eell response (Warger et al., 2006, Blood .108:544-550). Thus, the DCs of the present embodiments can secrete Th l driving cytokines, such as IL- 2, by exposure to these !igands that trigger TLRs. For example, the addition of polyCL'C), a TLR3 agonist to IL-.1 β, TNF-α, and IFN-γ, can generate a potent type-1 polarized DC, characterized by robust levels of IL-12 produc tion (Heifler et al. , 1996, Em. J. Immunol.. 26: 59-668). in certain embodiments, antigen can be loaded into the DC prior to TLR agonist exposure. In other embodiments, antigen can he loaded into the DC subsequent to TLR agonist exposure. Accordin to an aspect of the present embodiments, the injectable multi- dose antigen pulsed dendritic cell vaccine is produced by collecting DCs in a single patient leukapheresis whereby the cells are activated with biomolecules mat simulate bacterial infection (e.g., LPS). This unique activation method endows the DCs with qualities not found in DCs that are matured with a cytokine cocktail of TNF, IL-6, PGE2 and IL-lp (the "traditional maturation"), which also, simulates aseptic inflammation (Lombardi et aL, 2009, J. Immunol. 182:3372-3379).
in one embodiment, the DCs of the present embodiments can be acti vated wit the combination of the TLR4 agonist, bacteria! !ipopo!ysaccharide (LPS), the TLR7/8 agonist, resiniiquod (R848) and/or IFN-y (Amati et aL 2006, Curr. Pbarra, Des 12:4247-4254), By acti vating DCs with a TLR4 agonist and bacterial LPS, DCs are generated that are at least virtually identical (in phenotype) to DCls generated via traditional maturation methods. These DCs have a high expression of surface molecules, including CD83, CD80, CD86 and HLA-DR. In other embodiments, TLR2 agonists, such as lipotechoic acid (LTA), TLR3 agonists, such as polyCLC), and/or other TLR4 agonists, siich as MPL, may be used. As contemplated herein, any TLR agonist, or combination of TLR agonists, can be used to active DCs, provided such ligands stimulate tire production of cytokine and chemokiae signals by the activated DCs. Many other TLR agonists are known in the art and can be found in the published literature for use with the present embodiments.
Even though there are similarities in phenotype between DCs and traditionally matured DCs, the DCs of the present embodiments display many marked advantages. Cryopreservation
After culture initiation and activation, the ceils ar harvested and the vaccine is cryopreserved. For example, peripheral blood monocytes are obtained by leukapheresis. The cells are cultured in se um fee medium with GM-CSF and I L~4 for a period, of time followed by pulsing the cells with a desired antigen. Following pulsing the cells with a desired antigen, the antigen pulsed dendritic ceils are incubated with 1 FN-γ followed by a TLR agonist (e.g., LPS), The activated antigen pulsed dendritic cell is harvested and eryopreserved in a freezing medium and stored in liquid nitrogen. In one embodiment the freezing medi um comprises 55% plasmalyte, 40% human serum
albumin, and 5% DMSO.
The eryopreservation aspect of the embodiments allow for the generation of an FDA- approved injectable multi-dose antigen pulsed dendritic cell vaccine. An advantage of the embodiments is that the multi-dose antigen pulsed dendritic ceils retain their ability to produce signals critical to T cell function after thawing. As contemplated herein, the present embodiments include a variety of eryopreservation techniques and cr omedia, as would be understood by those skilled in the art. For example, in certain embodiments, the freezing medium comprises 55% plasmalyte, 40% human serum albumin, and 5% DMSO. Accordingly, the embodiments provide the ability to produce the multi-dose antigen pulsed dendritic cell vaccine of the embodiments at a centralized area comprising of an initial immunizing dose and multiple "booster" doses. Therefore the multi-dose antigen pulsed dendritic cell vaccine can be shipped to remote medical cemers for serial administration to the patient with no special FDA qualit control/quality assurance requirements at the administration site.
In one embodiment, the dendritic cell vaccine of the embodiments is eryopreserved in a!iquots for multiple doses. For example, the cells are eryopreserved at a concentration of 30x106 cells/mL. For example., a bag of freezing medi um containing a volume equal to the cell volume is prepared. Working rapidly, the freezing medium is added to the cell bag and the cells are transferred to labeled cryovials.. In one
embodiment, the vials are frozen using a rate controlled freezer. For example, cryovials are frozen using an automated rate controlled freezer at C/min and stored in vapor phase nitrogen.
In one embodiment, the vials are frozen using a rate controlled freezer.
The vials are placed in a freezing chamber and liquid nitrogen enters the chamber through an electronic solenoid valve. Since vaporization is almost instantaneous, controlling the rat at which liquid nitrogen enters the chamber directly controls the rate at which heat is absorbed and removed from the freezing chamber and its contents.
As contemplated herein, the present embodiments include a variety of eryopreservation techniques and freezing medium, as would be understood by those skilled in the art. For example, in certain embodiments, the freezing medium for cultured cells can include about 55% pJasraal te, about 40% human serum albumin, and about 5% DMSO. In other embodiments, the cryomedia can be serum-free, In certain, embodiments, controlled rate freezing may be used, while other embodiments can include use of insulated containers in which vials of cells mixed with freezing medium are placed in the freezer, such as at temperatures ranging from about -70*C to -80&C, The present
embodiments provide a method to preserve acti vated DCs in such a manner so as to further facilitate clinical application of such cells., and to reduce the need for extensive and repeated pherisis and . etutriation steps. As contemplated herein, cryopreservatton techniques may be used for both small-scale and large-scale batches.
When considering the extensive utility for activated DC, the ability to provide a steady supply of cryopreserved activated DC represents a significant advantage that can facilitate various therapeutic uses of such cells. For example, a large-scale culture of activated DC may be cryopreserved in aliquots of the appropriate size
comprising an initial immunizing dose and multiple "booster" doses, according to the methods of the present embodiments, such that individual doses of cells can later be used in any particular immunotherapeutic protocol. In certain embodiments, activated DCs can be cryopreserved for 2-24 weeks at temperatures of approximately -70°C or lower. At lower temperatures, such as at about -120°C or lower, acti vated DCs can be
eryopreserved'.for a least a year or longer.
In. one exemplary embodiment, the DCs axe suspended in human serum and approximately 5% DMSO (v v). Alternatively, other serum types, such as fetal calf serum, may be used. The suspended cells can be aliqitoted into smaller samples, such as in 1.8 ml vials, and stored at approximately -70°C or lower. In otlier embodiments, the freezing medium may include about 20% serum and about 10% DMSO, and suspended cells can be stored at about. - I SO^C. Stii! further embodiments may include medium containing about 55% piasrnalyte, and about 5% DMSO. Other exemplary freezing media may include about 12% DMSO and about 25-40% serum.
While the present embodiments as described herein may include specific concentrations of serum, it should be understood by those skilled in the art that the exact amount of serum in the freezing medium may vary, and in some embodiments ma be entirely absent, but will generally be within the range of about 1% to 30%. Of course, any concentration of serum that results in a cell viability of around 50% and/or a cell recovery of around 50% may be used in any D composition of the present embodiments., as well as with any cryopreservation method as described herein. Preferably, cell viability and recovery of at least 60%, more preferably at least about 70%, or even 80% is desired when recovering cryopreserved ceils in the selected freezing medium.
Similarly, while the present embodiments as described herein may include specific concentrations of DMSO, those skilled in the art should recognize that DM SO may be entirely absent in some embodiments, while in other embodiments,
concentrations from about 5% to as high as about 20% may be used in the freezing medium and included within the cryopreservation. methods described herein. Generally, Sower concentrations of DMSO are preferred, such as between about 5% to about 10%. However, any concentration of DMSO that results, after thawing, in ceil viability of at least 50% and a cell recovery of at least 50% . and preferably a cell, viabil ity and recovery of at least 60%, more preferably about 70%, more preferably about 80% and even more preferably about 90% and higher, may be used.
While the present embodiments as described herein may include reference to a rate controlled freezing, it should be understood by those skilled in the art that methods of freezing in a rate controlled or non-rate controlled manner can routinely be employed.
it should also be understood by those skilled in the art that the various cryopreservation media as described herein may either include serum or may be serum free. Examples of serum free media can include XV1VO 10, XVIVO 15, XV1VO 20, StemP.ro, as well as any commercially available serum free media. When utilizing a serum-free freezing medium, the cryopreservation methods of the present embodiments are generall free of infectious agents, antibodies and foreign proteins, which may be antigenic, and any other foreign molecule that may typically be found in serum-based freezing medium.
Cryopreservation '.of antigen loaded, active DCs can occur at an point after activation of the cells with a TLR agonist, in one embodiment, the activated DCs are cryopreserved approximately 6-8 hr after exposure to the TLR agonist. Preferably, the time point chosen to cryopreserve the activated cells should be based on the
maximization of signal production of the cells, particularly IL-12 production.
The present embodiments provide compositions and methods for producing large scale dendritic cell vaccines. In one embodiment, the large scale
production of dendritic cell vaccines allows for the production of an FDA-approved injectable multi-dose antigen pulsed dendritic ceil vaccine for the personalized treatment and prevention of cancer or other disorders. In one embodiment, there are provided compositions arid methods for producing large scale of antigen pulsed type I polarized dendritic eel! vaccine (DO).
in one embodiment, die embodiments provide a method to cryopreserve dendritic ceils that are in an antigen-loaded, pre-activated state in a large scale that is "syringe-ready", i.e. suitable for immediate injection into the patient without the
necessity of any further cell processing that would require (e.g., by FDA mandate) additional facilities and quality control/assurance steps.
In one embodiment, die embodiments provide a method to efficiently produce in a large scale injectable multi-dose antigen pulsed dendritic cell vaccine, preferably injectable multi-dose antigen pulsed type I polarized dendritic cell vaccine that exhibit maximal efficacy.
Packaging of Compositions or Kit Components
Suitable containers for compositions of the embodiments (or kit components) include vials, syringes (e.g. disposable syringes), etc. These containers should be sterile.
Where a composition/component is located in a vial the vial is preferably made of a glass or plastic material. The via! is preferably sterilized before the
composition is added to it. To avoid problems with latex-sensitive patients, vials are preferably sealed with a latex-free stopper, and the absence of latex in all packaging material, is preferred. The vial may include a single dose of vaccine, or it may inc lude more than one dose (a "raultidose" vial) e.g. 10 doses. Preferred vials are made of colorless glass. A vial can have a cap (e.g. a Liter lock) adapted such that a pre- filled syringe can be inserted into the cap, the contents of the syringe can he expelled into the vial, and the contents of the vial can he removed back into the syri nge. After removal of the syringe from the vial, a needle can then be attached and the composition can be administered to a patient. The cap is preferably located inside a seal or cover, such that the seal or cover has to be removed before the cap can be accessed. A vial may have a cap that permits aseptic removal of its contents, particularly for multidose vials.
Where a composition component is packaged into a syringe, the syringe may have a needle attached to it. If a needle is not attached, a separate needle may be supplied with the syringe for assembly and use. Such a needle may be sheathed. Safety needles are preferred. 1-inch 23-gauge, 1-inch 25-gauge and 5/8-inch 25-gauge needles are typical. Syringes may be provided with peel-off labels o which the lot number, influenza season and expiration date of the contents may be printed, to facilitate record keeping. The plunger in the syringe preferably has a stopper to prevent the plunger from being accidentally removed during aspiration. The syringes may have a latex rubber cap and/or plunger. Disposable syringes contain a single dose of vaccine. The syringe will generally have a tip cap to seal the tip prior to attachment of a needle, and the tip cap is preferably made of a butyl rubber. If the syringe and needle are packaged separately then the needle is preferably fitted with a butyl rubber shield,
Containers may be marked to show half-dose volume e.g. to facilitate delivery to children. For instance, a syringe containing a 0,5 ml dose ma have a mark showing a 0.25 ml volume.
Where a glass container (e.g. a syringe or a vial) is used, then it is preferred to use a container made from a borosiiicate glass rather than from a soda lime glass.
A kit or composition may be packaged (e.g. in the. same box) with a leaflet including details of the vaccine e.g. instructions for administration, details of the antigens within the vaccine, etc. The instructions may also contain warnings e.g, to keep a solution of adrenaline readily available in case of anaphylactic reaction following vaccination, etc.
Methods for Treating a Disease The presen t embodiments also encompass methods of treatment and/or prevention of a disease caused by pathogenic microorganisms, autoimmune disorder and/or a hyperproliferative disease.
Diseases that may be treated or prevented by use of the present
embodiments include diseases caused by viruses, bacteria, yeast, parasites, protozoa, cancer cells and the like. The pharmaceutical composition of the present embodi ments may be used as a generalized immune enhancer (DC activating composition, or system) and as such has utility in treating diseases. Exemplary diseases that can. be txeaied and/or prevented utilizing the pharmaceutical compositio of the present embodiments include, but are not limited to infections of viral etiology such as HIV, influenza. Herpes, viral hepatitis, Epstein Bar, polio, viral encephalitis, measles, chicken pox, Papilloma virus etc.; or infections of bacterial etiology such as pneumonia, tuberculosis, syphilis, etc.; or infections of parasitic etiology such as malaria, trypanosomiasis, leishmaniasis,
trichomoniasis, amoebiasis, etc.
Preneoplastic or hyperplastic states that may be treated or prevented using the pharmaceutical composition of the present embodiments (transduced DCs, expressio vector, expression construct; etc. ) of the present embodiments include but are not limited to preneoplastic or hyperplastic states such as colo polyps, Crohn's disease, ulcerative colitis, breast lesions and the like.
Cancers that may be treated using the composition of the present embodiments include, but are not limited to primary or metastatic melanoma,
adenocarcinoma, squamous ceil carcinoma, adenosquamous cell carcinoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leuke.rn.ias, uterine cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, NPC, bladder cancer, cervical cancer and the like.
Other hyperproliferative diseases that may be treated using DC activation system of me present embodiments include, but are not limited to rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas.
hemangiomas, fibronras, vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions (such as adenomatous hyperplasia and prostatic intraepithelial neoplasia), carcinoma in situ, oral hair}' leukoplakia, or psoriasis.
Autoimmune disorders that ma be treated using the composition of the present embodiments include, but are not limited to, AIDS. Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes mel!itus, emphysema, erythema nodosum, atrophic gastritis, glomendonephritis. gout. Graves' disease, hypereosinophiiia, irritable bowel syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, and autoimmune thyroiditis; complications of cancer, hemodialysis, and extracorporeal circulation; viral, bacterial, fungal, parasitic, protozoal, and helminthic infections; and trauma.
In the method of treatment, the administration of the composition of the embodiments may be for either "prophylactic" or '"therapeutic" purpose. When provided prophylactic-ally, the composition of the present embodiments is provided in advance of any symptom, although in particular embodiments the vaccine is provided following the onset of one or more symptoms to prevent farther symptoms from developing or to prevent present symptoms from becoming worse. The prophylactic administration of composition serves to prevent or ameliorate any subsequent infection or disease. When provided therapeutically, the pharmaceutical composition is provided at or after the onset of a symptom of infection or disease. Thus, the present embodiments may be provided either prior to the anticipated exposure to a disease-causing agent or disease state or after the initiation of the infection or disease.
An effective amount of the composition would be the amount that achieves this selected result of enhancing the immune response, and such an amount could be determined as a matter of routine by a person skilled in the art. For example, an effective amount of for treating an immune system defici ncy against cancer or pathogen could be that amount necessary to cause activation of the immune system, resulting in th development of an antigen specific immune response upon exposure to antigen. The term is also synonymous with "sufficient amount." The effective amount for any particular application cm. vary depending on such factors as the disease or condition being treated, the particular composition being administered, the size of the subject, and/or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular composition of the present embodiments without necessitating undue experimentation.
Theraffeutic applications
The present embodiments include the generation of an antigen loaded, activated APC that produces significant levels of cytokines and chernokin.es when thawed from ci opreservation, where the antigen loaded and activated APC is used in
immunotherapy for a mammal, preferably a human. The response to aa antigen presented by an APC may be measured by monitoring the induction of a cytolytic T-cell response, helper T-cell response, and/or antibody response to the antigen using method well known in the art.
The present embodiments include a method of enhancing the immune response in a mammal comprising the steps of; generating immature DCs from monocytes obtained from a mamma! (e.g., a patient); pulsing the immature DCs with a composition comprising an antigenic composition; activating the antigen loaded DCs with at least one TL agonist; cryopreserving the activated, antigen loaded DCs; thawing the activated, antigen loaded DCs and then administering the activated, antigen loaded DCs t a mammal in need thereof. The composition includes' at least an antigen, and may further be a vaccine for ex vivo immunization and/or in vivo therapy in a mammal.
Preferably, the mammal is a human.
Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human). The cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and die cells can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with, respect to the recipient
in one embodiment, peripheral blood monocytes are obtained from, a patient by combined leukapheresis and ehitriation. The monocytes can be cultured in SFM with GM-CSF and IL-4 overnight. The next clay, immature DCs can. he pulsed with antigen, Followed by contacting the DCs with IFN-γ and LPS. The activated DCs can then be suspended in a freezing medium and frozen until ready for use in
immunotherapy.
Cryo preserved D s can be : .cultured ex vivo under conditions effective to generate the percent recovery and percent viabi lity of the cells as compared freshly activated DCs. DCs generated from crvopreserved samples ca show similar stability as compared to freshly prepared DCs. Furthermore., comparisons of cryopreserved mature DCs with those of freshly prepared DCs can show virtually identical phenoiypes as well as signal secretion profiles. As contemplated herein, DCs can be preserved at both small and large scale for approximately 2 to 24 weeks, in the various freezing media described herein, at temperatures of approximately ~70*C to -80 . At temperatures below about - 120°C, the duration of storage can be extended indefinite ly or at least beyond 24 weeks without impacting cell recovery, viability, and functionality of the DCs. For example, in certain embodiments, the activated ceils can be preserved for at least one year and still retain their ability to produce signal after thawing. The present embodiments provide fo effective recovery and viabilit profiles upon thawing the cells, and furthermore the cryopreservation conditions described herei do not affect the ability of DCs to retain their signal profiles as explained herein throughout.
In an exemplary embodiment, cryopreservation.niay ¼ erformed after activation of DCs by re-suspending the ceils in a freezing medium comprising' about 55% plasmalyte, about 40% human serum albumin, and about 5% D SO. The mixture can then be a!iquoted in 1.8 ml via!s and frozen at about -80°C in a cryochamber overnight. Vials can then be transferred to liquid nitrogen tanks the following day. After about 2 to 24 weeks of cryopreservation, the frozen DCs ca be thawed and examined for their recovery and viability. Recovery of such DCs can be greater than or equal to about 70% with a viability of greater than or equal to about 70%. m other embodiments, DCs or even monocytes can be cryopreserved prior to cell activation.
Alternatively, the procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described m U.S. Pat. No. 5,199,942, which is incorporated herein by reference, can be applied to the cells of the present embodiments. In addition to the cellular growth factors described in U.S. Pat No. 5, 199,942, other factors such as flt3-L, IL-l, IL-3 and c-kit tigand, can be used for ci turmg and expansio of the cells.
A variety of cell selection techniques are known for identifying and separating ceils from a population of cells. For example, monoclonal antibodies (or other specific cell binding proteins) can be used to bind to a .marker protein or surface antigen protein found on the cells. Several such markers or cell surface antigens are known in the art..
Vaccine Formulations
The presen embodiments further include vaccine formulations suitable for use in immunotherapy. In certain embodiments, vaccine formulations are used for the prevention and/or treatment of a disease, such as cancer and infectious diseases. In one embodiment, the administration to a patient of a vaccine in accordance with, the present embodiments for the prevention and/or treatment of cancer can take place before or after a surgical procedure to remove the cancer, before or after a che otherapeutic procedure for the treatment of cancer, and before or after radiation therapy for the treatment of cancer and any combination thereof. In other embodiments, the vaccine formulations may be administrated to a patient in conjunction or combination with another composition or pharmaceutical product. It should be appreciated that the present embodiments can also be used to prevent cancer in individual's without cancer, but who might be at risk of developing cancer.
The administration of a cancer vaccine prepared in accordance with the present embodi men ts, is broadly applicable to the prevention or treatment of cancer, determined in part b the selection of antigens forming part of the cancer vaccine.
Cancers that can be suitably treated in accordance with the practices of the present embodiments include, without limitation, cancers of the lung, breast, ovary, cervix, colon, head and neck, pancreas, prostate, stomach, bladder, kidney, bone, liver, esophagus, •gastroesophageal., brain, testicle., uterus and the various leuke ias and lymphomas,
in one embodiment, vaccines can be derived from the tumor -or cancer cells to be treated. For example, in the treatment of lung cancer, the lung cancer cells would be treated as described hereinabove to produce a lung cancer vaccine. Similarly, breast cancer vaccine, colon cancer vaccine, pancreas cancer vaccine, stomach cancer vaccine, bladder cancer vaccine, kidney cancer vaccine and the like, would be produced and employed as im unotherapeutc agents in accordance with the practices tor the prevention and/or treatment of the tumor or cancer cell from which the vaccine was produced.
In another embodiment, vaccines could, as stated, also be prepared to treat various infectious diseases which affect mammals, by collecting the relevant antigens shed into a culture medium by the pathogen. As there is heterogeneci ty in the type of immunogenic and protective antigens expressed by different varieties of organisms causing the same disease, polyvalent vaccines can be prepared by preparing the vaccine from a pool of organisms expressing the different antigens o importance.
In another embodiment, the vaccine can be administered by intranodal injection into groin nodes. Alternatively, and depending on the vaccine target, the vaccine can he i tradermally or snbcu!aneously administered to the extremities, arms and legs, of the patients being treated. Although this approach is generally satisfactory for melanoma and other cancers, including the prevention or treatment of infectious diseases, other routes of administration, such as intramuscularly or into the blood stream may also be used.
Additionally, the vaccme can be given together with adjuvants and/or immune-modulators to boost the activity of the vaccine and t he patient's response. Such adjuvants and/or mununo-modulators are understood by those skilled in the art, and are readily described in available published literature.
As contemplated herein, and depending on. the type of vaccine being generated, the production of vaccine can, if desired, be scaled up by culturing cell s in bioreactors or fermentors or other such vessels or devices suitable for the growing of cells in bulk. In such apparatus, the culture medium would be collected regularly, frequently or continuously to recover therefrom any materials or antigens before such materials or antigens are degraded in. the culture medium.
If desired, devices or compositions containing the vaccine or antigens produced and recovered, in accordance with the present embodiments, and suitable for sustained or intermitteBt release could be, in effect, implanted in the body or topically applied thereto for a relatively slow or timed release of such materials into the body.
Other steps in vaccine preparation can be indi vidualized to satisfy the .requirements of particular vaccines. Such additional steps will he understood by those skilled in the art. For example, certai coilected antigenic materials may be concentrated and in some cases treated with detergent and ultracentriraged to remove transplantation alioantigens.
Combination Therapy
The present embodiments provide an effective therapy to treat cancer wherein the therapy includes changing the immune response in the tumor so that the immune cells in the tumor site are more effective in attacking the tumor cells, m some instances, the effective therapy includes improving the migration and activity of immune cells in the tumor site. In one embodiment, mere are provided compositions and methods of using a dendritic cell vaccine in combination with an inhibitor of one or more of HBR2 and HERS as a treatment regimen to treat cancer. In another embodiment, the treatment regimen comprises the use of a dendritic cell vaccine, an inhibitor of one or more of H0ER2 and HE 3, and a chemokine .modulator. In one embodiment, the chemokine modulator is a chemokine-activating agent. An example of a chemokine-activating agent is a TLR8 agonist.
In. one embodiment, there are provided compositions and methods of using a dendritic cell vaccine in combination with blockage of HER-2 and HER-3 as a treatment regimen to treat cancer, in another embodiment, there are provided
compositions and methods of using a dendritic cell vaccine in combination with blockage of HER-2 and HER-3 with TNF~a and I F -v. In another embodiment, there are provided compositions and methods of blocking both of HER-2 and HER-3 with the addition of TNF~a and IFN-γ as a treatment regimen to treat cancer.
In one embodiment, a treatment regimen can be used to treat cancer and therefore can be considered as a type of anti-cancer therapy, in another' embodiment, a treatment regimen can be used m the context of a combination therapy with another anticancer or anti-tumor therapy including but not limited to surgery, chemotherapy. radiation therapy (e.g. X ray), geae therapy,, immunotherapy, hormone therapy, viral therapy, DMA therapy, R A therapy, protein therapy, cellular therapy, and nanotherapy.
In one embodiment, there is provided a treatment regimen in combination with another cancer medicament for the treatment or prevention of cancer in subjects. The other cancer med cament' is administered in synergistic amounts or in various dosages or at various time schedules with the treatment regimen of the embodiments. The embodiments also relate to kits and compositions concerning the combination of treatment regi mens of the embodiments alone or in combination with a desired cancer medicament.
In one embodiment, a treatment regimen is used prior to receiving another anti-cancer therapy. In another embodiment, a treatment regimen is used concurrently with receiving another anti-cancer therapy. In another embodiment, a treatment regimen is used after receiving another anti-cancer therapy.
I n some embodiments, the present embodiments provide a method of treating breast cancer that is negative for ER in a subject. In some embodiments, there is a method of treating breast cancer that, is negative for ER and positive for HER2 in a subject. In some embodiments, the breast cancer is a metastatic breast cancer, in some embodiments, the breast cancer is at stage Ϊ, stage II, or stage III
In another embodiment, the treatment regimen may be used in combination with existing therapeutic agents used to treat cancer. In order to evaluate potential the rapeutic efficacy of the treatment regimen of the embodiments in
combination with the antitumor therapeutics described elsewhere herein, these combinations may be tested for antitumor activity according to methods known, in the art.
In one aspect, the present embodiments contemplate that a treatment regimen may be used in combination with a therapeutic agent such as an anti-tumor agent including but is not. limited to a chemotherapeutic agent, an anti-cell proliferation agent or any combination thereof.
The embodiments .should not limited to any particular chemotherapeutic agent. Rather, any chemotherapeutic agent can be used with the treatment regimen of the embodiments. For example, any conventional chemotherapeutic agents of the following non- limiting exemplary classes are included in the embodiments: alkylating agents; nitrosoureas; antimetabolites; antitumor antibiotics; plant alkyloids; iaxaoes; hormonal agents; and miscellaneous agents.
Alkylating agents are so named because of their ability to add aikyl groups to many electronegative groups under conditions present in cells, thereby interfering with DNA. replication to prevent cancer cells from reproducing. Most alkylating agents are cell cycle non-specific, In specific aspects, they stop tomor growth by cross-linking guanine bases i DMA double-helix strands. Non-limiting examples include busul&n, earboplatin, chlorambucil, eisplatin, cyclophosphamide, dacarbazroe, ifosfamide, mechlorethamine hydrochloride, melphaian, procarbazine, thiotepa, and uracil mustard.
Anti-metabolites prevent incorporation of bases into DNA during the synthesis (S) phase of the cell cycle, prohibiting normal development and division. on- 1 uniting examples of antimetabolites include drugs such as 5-fliiorou.racil, 6- mercaptopurine, capecitab e, cytosine arabinoside, iloxuridine, tludarabine,
gemcitabine, methotrexate, and thioguanine.
There are a variety of antitumor antibiotics that generally prevent cell division by interfering with enzymes needed for cell division or by altering the membranes that surround cells. Included in this class are the anthracyc lines, such as doxorubicin, which act to prevent cell division by disrupting the structure of the DNA and terminate its function. These agents are cell cycle non-specific, Non-limiting examples of antitumor antibiotics include daetiftomycin, daunorubicin, doxorubicin, idarubicin, mitomyein~C, and mitoxamraiie.
Plant alkaloids inhibit or stop mitosis or inhibit enzymes that prevent cells from making proteins needed for cell growth. Frequently used plant alkaloids include vinblastine, vincristine, vindesine, and vinorelbine. However, the embodiments should not be construed as being limited solely to these plant alkaloids.
The taxanes affect cell structures called microtubules that are important in cellular functions, in normal cell growth, microtubules are formed when a cell starts dividing, but once the cell stops dividing, the .microtubules 'are disassembled or destroyed. Taxanes prohibit the microtubules from breaking clown such that the cancer cells become so clogged with microtubules that they cannot grow and divide. Non- limiting exemplary taxanes include pacHtaxel and docetaxel. Homionai agents and hormone- like drugs are - utilized for certain types of cancer, including, for example, leukemia, lymphoma, and multiple myeloma. They are often employed with other types of chemotherapy drags to enhance their effectiveness. Sex hormones are used to alter the action or production of female or male hormones and are used to slo w the growth of breast, prostate, and endometrial cancers. Inhibiting the production (aromatase inhibitors) or action (tamoxifen) of these hormones c an often be used as an adjunct to therapy. Some other tumors are also hormone dependent.
Tamoxifen is a non-limiting example of a homionai agent that interferes with the activity of estrogen, which promotes the growth of breast cancer ceils.
Miscellaneous agents include chemotherapeuttcs such as bleomycin, hydroxyurea, L-asparaginase, and procarbazine that are also useful in the embodiments.
An anti-cell proliferation agent can further he defined as an apoptosis- induciiig agent or a cytotoxic agent The apoptosis-inducing agent may be a granzyme, a Be 1-2 family member, cytochrome C, a caspase, or a combination thereof. Exemplary granzymes include granzyme A, granzyme B, granzyme C, granzyme D, granzyme E, granzyme F, granzyme G, granzyme H„ granzyme I, granzyme J, granzyme , granzyme L, granzyme , granzyme Ns or a combination thereof. In other specific aspects, the Bcl- 2 family member is, for example, Bax, Bak, Bcl-Xs, Bad, Bid, Bik, Hrk, Bok, or a combination thereo
In additional aspects, the caspase is caspase- 1 ,. caspase-2, easpase-3, easpase-4, easpase~5, caspase-6, caspase-7, easpase~8, easpase-9, caspase- 1.0, caspase- .1 1 , caspase- 12, caspase- 13, caspase- 14, or a combination thereof. In specific aspects, the cytotoxic agent is TNF- , gelonin, Prodigiosan, a rihosome-inhihiting protein (RIP), Pseudomonas exotoxin. Clostridium difficile Toxin B, Helicobacter pylori VacA, Yersinia enterocoiitica YopT, Vlolacein, diemylenetriaminepentaacetic acid, irofulven, Diptheria Toxin, mitogillin, ricin, botulinum toxin, cholera toxin, saponin 6, or a combination thereo
in one embodiment, a treatment regimen is used in combination with an anti-tumor agent wherein the anti-tumor agent is an antitumor .alkylating agent, antitumor antimetabolite, antitumor antibiotics, plant-derived antitumor agent, antitumor platinum complex, antitumor campthotecin derivative, antitumor tyrosine kinase inhibitor, monoclonal antibody, interferon, biological response -modifier, hormonal anti-tumor agent, anti-tumor vital agent, angiogenesis inhibitor, differentiating agent,
FI3K/mTOR/A .T inhibitor, cell cycle inhibitor, apoptosis inhibitor, hsp 90 inhibitor, tubulin inhibitor, DNA repair inhibitor, anti-angiogenk agent, receptor tyrosine kinase inhibitor, topoisomerase inhibitor, taxane, agent targeting Her-2, hormone antagonist, agent targeting a growth factor receptor, or a pharmaceutically acceptable salt thereof. I some embodiments, the anti-tumor agent is citabme, capecitabine, valopicitabrae or gemcitahine. in some embodiments, the anti-tumor agent is selected from the group consisting of Avasttn, Sutent, Nexavar, Recenttn, ABT-869, Axidnib, Irinotecan.
topotecan, pac!itaxel, docetaxel, lapatinib, Berceptin. lapatinib, tamoxifen, a steroidal aromatase inhibitor, a non-steroidal aroraatase inhibitor, Fulvestrant, an inhibitor of epidermal growth factor receptor (EGFR), Cetuximab, Panituraimab, an inhibitor of insulin-like growtli factor I receptor (IGF ), and CP-751871 ,
I n one embodiment, the anti-tumor agent is a chernotherapeutic agent A chemotherapeutie agent as used herein is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN); alkyl sulfonates such as bnsu!fan, iraprosulfan and piposuifan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethyleneme!amine, trietylenephosphoramide, triethiy!enef hipphosphoratnide and trimethylolomelamine; aeetogenins (especially huOatacin and bullatacinone); delta~9~tetrahydrocafmabinol (dronabinol, MARINOL); beta-lapachone; ia achol; colchicines; betiilmic acid; a caniptothecin (including the synthetic analogue topotecan (HYCAMTIN), CPT-l 1 (irinotecan, CAMPIOSAR), acetylcamptothecin, scopolectin, and 9- aminocamptothecin); hryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid;
teniposide; cryptophycms (particularly cryptophycin 1 and etyptophycin 8); dolastatin; duocan.nycin (including the synthetic analogues, KW-2189 and CBl -TMl); eleiithero n; pancrati statin; a sareodictyin; spongistatin; nitrogen mustards such as- chlorambucil, chlornaphazine, cholopfaospharnide, estramustine, ifosfamide, rnecbloretharnine, raecMoretharaine oxide hydrochloride, tnelphalan, noverabichin, phenesterine. precinimustine, trofosfamide, uracil mustard; iikrosureas such as camiusiine,
chlorozotoc n, fotemustine, ionmstine, nimustine, and ranimnusiine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especi ally ealieheamiein gamma Li and ealkheamkin omegall (see, e.g., Agnew, Chem Intl. Ed. Engl, 33: 183-186 (1994)}; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chxomoprotein enediyne antiobioiic chromophores),
aclacinoraysins, actinotnycm, aiiihraraycin, azaserme, bleomycins, cactinoraycin, carabicin, carminomyc n, cmzinophilin, chromomycmis, dactinomycin, daunorabicm, detorubkin, 6-diazo- 5 -oxo-L-norieucine. doxorubicin (including ADRIAMYCI , morpholino-doxorubicin, eyanomo^holino-doxorabicin, 2-pyrroHno-doxorubicin, doxorubicin HC! liposome injection (DOXIL), liposomal doxorubicin TLC D-99
( YOCET), peglylated liposomal doxorubicin (CAELYX), and deoxydoxorubicin)5 epirubkin, esorubkin, idarubicm, marceliomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalanrycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamychi, rodorubicin, streptonigrin, streptozocin, tubercidin, iibenimex, zmostatin, zorubicm; anti-metabolites such as methotrexate, gemciiabine (GEMZAR), tegafur ( IJFTORAL), capecitabiae (XELODA), an epothilone, and S-fluorouracil (5-FU); folic acid analogues such as denopterra, methotrexate, pieropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabme, azacitidine, 6-azauridine, canno&r, cytarabine, dideoxyuodme, doxifl uridine, enocitabine, fioxiuidioe; anti-adrenals such as aminogluteihimide,
mitotane, trilostane; folic acid reple isher such as frolinic acid; aceglatone;
aidophosphamide glycoside; aminolevulinic acid; eniluracii: amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziqtione; elforaithine; elliptinimn acetate; etoglucid; gallium nitrate; hydroxyurea; lentiiian; kmidainine; maytansraoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;
nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone 2-ethyJhydrazide;
procarbazine: PS polysaccharide complex (JHS 'Natural Products, Eugene, Qreg.}; razoxane rhizoxin; sizofiran; spirogermamura; te uazonk acid triaziquone; 2i2',2"~ trichiorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); tirethan; dacarbazme; raai oraustme; mitobroniiol; mitolactol; pipobxoman; gacytosine; arabinoside ("Ara-C"); ioiepa; taxoM, e.g., paclitaxel (TAXOL), albumin engineered nanoparticle formulation of paclitaxel (ABRAX A E), an d doceta el (TAXOTE E); chioranbucil; 6-thioguanine; mercaptopirrme; methotrexate; platinum agents such as cisplatin. oxaliplatin, and carboplatin; vincas, which prevent tubulin polymerization .from forming microtubules, including vinblastine (VELBA ), vincristine (ONCOVIN), vindesine (ELDISIME, FILDESIN), and. vinorelbme ( AVELBINE); etoposide (VP- 16); ifosfamide; mitoxantrone; leucovovin; novantrone; edatrexate;
daunomycin; aninopterin; ibandronate; topoisomerase inhibitor RFS 2000;
difluorometlhyloraifhtee (DMFO); retinoids such as reiinoic acid, including hexarotene (TARGRETIN); bisphosphonates such as clodronate (for example, BONEEOSO or
OSTAC), etidronate (DIDROCAL), NE-58095, zoledronic acid/zoledronate (ZOMETA), alendronate (FOS MAX), pamidronate (A .EDIA), tihidronate (SKELID), or risedronate (ACTONEL); troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated m aberrant cell proliferation, such as, tor example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF'-R); vaccines such as THERATOPE.RTM vaccine and gene therapy vaccines, for example, ALLOVECTIN vaccine, LEUVECTIN vaccine, and VAXID vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN); niiRH (e.g., ABARELIX); BAY439006 (sorafenib; Bayer); SU-1 1248 (Pfizer); perifosine, COX -2 inhibitor (e.g., celecoxib or etoficoxib), proteosome inhibitor (e.g., PS341);
bottezoraib (VELCADE); GCI-779; tipf&mib (Rl 1577); orafeftib, ABT5I0; Be!~2 inhibitor such as oblimersen sodium (GENASENSE); pixantrone; EGFR inhibitors; tyrosine kinase inhibitors; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFQX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN) combined with 5-FU and leucovovin.
in another embodiment combinations.of'immnno.therapie;S are used to treat estrogen receptor -positive/HER -positive (ER¾>0S HERi>os) DCIS breast cancer patients. Anti-estrogen therapy such as, for example, tamoxifen is combined with amti-HER2 dendritic eel! vaccination to improve pathologic complete response. These methods described herein are by BO means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be farther approximated through analogy to compounds known to exert the desired effect.
EXPERIMENTAL EXAMPLES
The embodiments are further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless othenvise specified. Thus, the embodiments should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following ilbstrative examples, make and utilize the present embodiments and practice die claimed methods. The following working examples therefore, specifically point out the preferred embodiments, and are not to be construed as limiting in any way the remainder of the disclosure. Example I : A cryopreserved, pre-aetivated, multi-dose dendritic cell vaccine
To be able to produce these large scale vaccines, a process was developed to produce fully activated DO vaccines pulsed with tumor antigen whereby the fully activated DO vaccines are cryopreserved as multi-dose syringe ready 6 pack DO vaccines. The DO are cryopreserved activated as DO as described elsewhere herein. For example, they are cryopreserved in 55% Plasmalyte medium with 40% Human serum albumin and 5% DM SO. These vaccines have been generated and extensively tested in the laboratory and consistently meet quality standards set by the FDA for administration to■patients.
The materials and methods employed in the experiments and examples disclosed herein are now described. Preparation of fully activated DC for cryopresefy fioe Freshly elutriated myeloid monocytes were cultured in 6 well microplates (12 l.06 cells/well). Culture medium consisted of Serum Free Med um ( SFM Invitrogen Carlsbad CA). The final concentration of added GMCSF was SOng ml and of IL4 is 1000 U/ml. Cells were cultured overnight at 37" C in 5% CO2. In some batches, the cells were pulsed with the adequate peptides after 16-20 br and cultured, for additional 6-8 far, after which lOOOU/ml IFN-γ was added. Dendritic cells were matured with TLR agonist LPS (TLR .4, lOng/nii) or R848 (TLRS, 1 μ«½1). The maturation time was at least about 6hr. After that, the TLR agonist-activated DCs were ready for cryopreservation or immediate use.
To induce the production of the Thi-polarizing cytokine IL- i .2, the DCs are activated with combinations of the cytokine IFN.-y, or the TLR agonists bacterial LPS and/or R848. This should induced T ceils that produce IF -γ. Alternatively, the DCs can be activated with combinations of ATP, bacterial LTA, LPS and prostaglandin E2
(PGE2), This . can cause lL-23, IL-6 and IL-ίβ to be amplified, leading an immune response dominated by IL~I7 and iL-22-seereting Thl ? cells.
DCs were harvested by gentle scraping. All medium and the cells were kepi at wet ice at all times. Cells were gently washed, by centrifogation at about 80GRPM for 10 min. Cells (e.g., 10 x l O6 cells were cryopreserved in freezing medium of piasmalyte 55%, human serum a lbumin 40% wit h 5% DM SO and stored in liquid nitrogen. The results of the experiments presented herein are now described.
Multi-dose DC! vaccines
Experiments were perf rmed to assess the recovery, viability and- sterility of the cells. Briefly, peripheral 'blood monocytes were obtained by combined
leukapheresis the counter current elutriation and were cultured in serum free medium with GM-CSF and 1L-4 overnight. The next day they were pulsed with HER-2 peptides then IFN-γ followed by LPS. The DCl were harvested at 40 hours and cryopreserved in freezing medium ofplasraalyte 55%, human serum albumin 40% with 5% DMSO and stored in liquid nitrogen. Following 1 week they were thawed and release criteria were obtained including viability, yield, endotoxin testing, and sterility cultures. All 12 cultures had no bacterial growth and <0.1 EU endotoxin. Figure 1 demonstrates the viability and yield of cryopreserved DCl . As can be seen in Figure 1 , recovery of cells was on average 89% and viability was 95% when cells were directly thawed and counted These data demonstrate the cryopreservaiiosi and viability of the DC l vaccine is
maintained m medium containing below the FDA allowable 7.5% DMSO.
It was observed that cell function was maintained following crypreservation. IL-12 and Thl chemokines were produced for 36 hours post thaw from multi-dose DCl vaccines. Thawed cells produced high levels of IL-12 from about 6 hoors post thaw through 36 hours. These levels of IL-12 are comparable to prepared DCl vaccines made from cryopreserved monocytes.
The results presented herein demonstrate that cryopreserved, preactivated, multi-dose dendritic ceil vaccine sensitized against me HER-2 tumor target antigen on breast cancer is therapeutic. However, the embodiments are applicable to a variety of additional pathophysiological conditions.
The multi-dose syringe ready pack DCl vaccines can be used in HER-2 non-expressing breast cancer. HER-2 is expressed on approximately 25% of ail breast cancers. Breast cancers that, do not produce detectable levels of HER-2 may not be susceptible to vaccination. To address this limitation, additional target proteins can be added to the vaccine. For example, many breast cancers that do not produce high levels of HER-2 instead produce other, related proteins including HER-i and HER-3. Without wishing to be bound by any particular theory, it is believed, that adding these other proteins to the vaccine would allow the targeting of these other breast cancer phenotypes.
The multi-dose syringe read pack DCl vaccines can be used in other cancer types, besides- breast cancer. Anticipated target proteins such as HER-i , HER-2 and HER-3 can also be present on otiier types of cancer including ovarian, prostate, pancreatic, colorectal, gastric, head and neck and non-small cell lung carcinoma, as well as other common cancers. The multi-dose syringe ready pack DO vaccines can be vised to treat chronic infectious diseases, including but not limited to, chronic infections like HIV or hepatitis virus C. Here, proteins specific for these viruses would replace the HE -2 or other cancer proteins to mobilize the patient's immune response against these persistent infections. It is possible that the enhanced immunity would greatly reduce viral load and attendant disease symptoms and progression, or could possibly help clear the infection entirely.
The multi-dose syr inge ready pack DC I vaccines can. be used to treat autoimmune diseases. Diseases like rheumatoid arthritis- and Lupus occu when the immune system mistakenly attacks the body's own normal tissues. Whereas the current vaccine inimunotherap formulation is designed to initiate and strengthen immune responses, without wishing to be bound by any particular theory, it is believed that in vitxo signals can be provided to the DCs during vaccine production that induce these cells to switch off pathological immune responses.
Dendritic cell-based vaccine therapy is a promising directed therapy against a variety of cancers. While a variety of strategies have been employed to mature DCs to a phenotype that optimizes sensitization of CD4+ and CD8+ T cells to recognize tumor epitopes and elicit antitumor immunity, experiments were designed to utilize a method that employs the rapid maturation of monocytes in serum free media (SFM) using interferon gamma (lFN-γ) and lipopol saccharide (LPS), a toll-like receptor (TLR) 4 agonist, resulting in mature DCs capable of polarizing the immune response to a Thl- type response and eliciting sensitization: via an IL-12 dependent mechanism. Results demonstrate the potential for this vaccine strategy to be used as an adjunct therapy in early breast cancer. Cryopreservation of dendritic cells (DCs) in a matured state permits easier production of and accessibility to personalised, therapy.
Rapid maturation of dendritic cells Both, freshl matured DCs (DC Is) and DCs eryopreserved in a mature state (cryoDCs) and subsequently thawed were compared for viability and recover as well as for phenotypic maturity as determined by expression of cell surface markers by flow cytometry. Function of dendritic cells was determined by measuring production of various cytokines, in particular, interieukin 12p70 (1L~12p70)« The ability of these cells to stimulate naive CD4+ and CD8+ tells was also assessed.
There was no significant difference in the viability (p~ 4807), and recovery (p~ 1220) (Figure 2).
Both populations had similar initial (7 hours post LPS addition) IL~ 12 p70 secretion (p=.0768), The populations continued to exhibit comparable secretion ieveis of IL-12 p70 over a 30 hour observation period with no significant differences between the populations. (Figure 3),
There was no significant difference between populations and production of iL-ibeta (p-0.7690), 0,-1 alpha (p-0.0841), Rantes (p-0.902), MDC (p;::0.1514), 1L- 10 (p-.1937), ! alpha (p- 2673), IP- 10 (p-0.7366), lL-6 (p-0.24), IL-5 (p-0.0735), TNF-beta (p-0,9422), 1L-I5(p-0.S878); MlP-lbeta (ρ=0.92ί 7), TNF-alpha (p-0.8972), IL-8(p-0.7844). (Figure 4).
Cell surface markers denoting DC maturity between DC i s ami cryoDCs exhibit no significant difference in expression. Both populations elicited nonspecific alioantigen CD4+ T cell responses as well as antigen-specific CDS- T cells recognition with a Thl polarized response. CD80, S3, and 86 demonstrate DC. maturation, expression revealed no significant difference. Functional abilities were induced by CD4+ T cells from different donors co-cultured with each population resulting in a nonspecific alioantigen response and CD4+ T cells secreting INF-gamma (DC Is 107.40 ng/niL and cryoDC ls 129.23 ng/niL). Antigen specific sensitizations initiated by co-cutturing the two populations with tumor antigen specific CDS- T cells elicit comparable IFN-y secretions for both groups and result in a THI polarized response. (Figure 5).
The resul ts presented herein demonstrate that rapid maturation .method of DO ca be cryopreserved functionally matured and. maintains, pfaenotype and function, thus can be used to manufacture syringe ready DC 1 for use world-wide in cancer therapy. In conjunction with maintaining the DO phenotype to drive a ThJ polarized immune response, the results presented herein demonstrate that the cryoDCs maintained the ability to primarily sensitize T cells. This also likely relates to the maturation strategy, as the DCis matured with IFN-gamma and LPS exhibit an enhanced ability to primarily sensitize CD 4+ T cells compared to cytokine matured DCs.
The present protocol for eryopreserved mature DC preparations can be easily adapted to current good manufacturing practice guidelines thereby increasing the availability of emerging therapy. Example 3: Cytokines from CD4 T cells and Hereeptm make high HE -2-expressi.ng breast cells susceptibl to killing by CDS T cells
It has been demonstrated that that high MER-2~expressing breast cancer cells down-regulate molecules on the cell surface that make these cancer cells visible to
CDS T cells and allow them to be killed by these immune cells. It has been shown that the cytokines interferon-gamma (IFN-y) and tumor necrosis factor-alpha (TMF-a) produced by CD4 ceils, when combined with Herceptin, cause the intermediate and high
HER-2 -expressing breast cancer cells to increase their Class I molecule expression. As a result of this, the CDS T ceils are able to better see the breast cancer cells and kill them or to produce cytokines to kill them.
Trials were designed to assess the therapeutic effects of using Herceptin in combination with dendritic cell vaccines (e.g., DO vaccines),
A phase I DCIS vaccine trial combining Herceptin with DC 1 vaccines was designed. For example, a Phase I trial was designed for patients with high HER-2- expressing DCIS to receive a DO vaccine combined with 2 doses of Herceptin at week 1 and week 4. Without wishing to be bound by any particular theory, it is believed that this combination will increase the complete response rate from 30% to greater than 50% in patients with HE -2-expressmg DCIS,
in addition, a Phase III DCIS vaccine trial was designed. For example, a vacci ne trial was de vel oped to prevent recurrence of breast cancer i patients with estrogen independent <ER*a*¾<l,ive)> l iER-2"t,osa! ¾ DCIS. There are three treatment arms in the study: 1) a standard therapy (surgery and radiation), 2) receiving a DC I vaccine before surgery, and 3) receiving a DC I vaccine plus Herceptin before surgery. Without wishing to be bound by any particular theory, those patients who have complete responses to the treatment can avoid radiation after surgery. It is also believed that this trial serves demonstrate the prevention of recurrence in patients with DOS using a vaccine.
in addition, a phase 1 neoadjuvant DC I vaccine in combination with
Herceptin in patients with early invasive HER-2~posmvi: breast cancer was designed. For example, a Phase I trial was designed to test whether the combination of Herceptin and vaccines along with a chemokine modulator (e.g., a cheniokme-activating agent) can eliminate small HER-2-expressing invasive breast cancers prior to surgery and avoid the need for chemotherapy. Without wishing to be bound by any particular theory, it is believed that this neoadjuvant (before surgery) stxategy, with the possibility of adding an immune antibody that takes the brakes off the immune response, may eliminate the need for toxi c chemotherapies for the treatment of breast cancer and therefore make imm une therapy the standard of care for this disease. That is, the treatment regimen disclosed herein provides a step forward in the quest to eradicate breast cancer using the natural immune response, which can be restored with vaccines regimens of the embodiments. The regiments discussed herein can drive immune cells into the tumor by changing the immune response in the tumor and enable the immune cells to work longer by taking the brakes off the cells. It is believed that combining DC 1 vaccine with Herceptin and also adding the chemokine modulator improves the migration and activity of the immune cells within the tumor in the breast.
Without wishing to be bound by any particular theory, it is believed that in many cancers, if there are large numbers of good inmiune-fighting cells present, those patieiits do better wi th any type of therapy. This means that if one gets immune cel ls into the tumor to fight it before it is removed, the patient likely does better and responds better to other therapies, including surgery, chemotherapy, and radiation.
Without wishing to be bound by any particular, an. effective therapy to treat cancer would include agents that rapidly change the immune response in the tumor prior to surgery to improve outcomes for patients with breast cancer and other types of cancer. This strateg can be applied to a variety of cancers including but is not limited to colon cancer, melanoma. Sung, brain, pancreas, prostate, esophagus, and the like.
Experiments were designed to develop immune fluorescence assays to measure and quantify the types of immune cells that migrate into the breast after vaccination, the types of chexnokines that are made to bring cells in, and the molecules they express to help eliminate cancer cells. These assays allow multiple cell types to he visualized at the same time and show where they are located in the tumor
n«croenvironment. it has been observed that at least in some of the vaccmated patients, their tumors produced chemokines to recruit cells into the environment to kill the tumor cells.
Example.4 ;, Combinati on of Blockade of HEK-2 and H EE- 3
The results presented herein demonstrate that the combination of an effective an.ti-HER2 GD4+ Thl response combined with. HER2 and HER3 blockade is extremely effective in causing tumor senescence and apoptosis in HER-2 expressing breast cancers.
It was observed that the combination of blockade of HER-2 and HER-3 together with the addition of combinations of Thl cytokines TNF~ and lFM-γ from CD4 Thl cells causes significant senescence and apoptosis of HER-2 expressing breast cancer, This has been verified in several different cells lines of both high and intermediate
HER -expressing breast cancer cells. The results demonstrate that this combination can be potent for prevention and prevention of recurrence.
METHODS
Cell culture and treatments
Human breast cancer cell lines SK-BR-3, BT-474, MCF-7, Τ-47Ό. HCC- 1.419 and MDA-MB-231 were obtained from the American Type Culture Collection (Manassas, VA) and grown in RPMI-1640 (Life technologies. Grand island, NY) supplemented with .10% FBS (Cellgro, -Herndon, VA), IiMT- 1 cells were a kind gift from Dr. Pravio Kaumaya (Ohio State University, Columbus, OH) and were grown in Dulbecco's modified Eagle's medium (DMEM) (fovitrogen, Waiham, MA) supplemented with !ø% FBS, Normal immortalized MCF-10 cells were obtained from the armanos Cancer Institute (Detroit, MI) and. grown in DMEM/F 2 (Invitrogen) supplemented with 10 mM HEPES, 10 pg/ml insulin, 20 ng/m! EGF, .100 ng/ml cholera toxin, 30 mM sodium bicarbonate, 0.5 pg/rnl hydrocortisone, and 5% fetal horse serum. All cells were grown at 37°C in a humidified 5% C02 incubator.
Three hundred thousand of breast cancer cells were treated for 5 days with the indicated concentrations of human recombinant TNF-a (R&D Systems, Minneapolis, MM) and human recombinant IFN~y (R&D Systems) and then cultured for 2 more passages in absence of cytokines. Cells were subjected to senescence associated β- ga!actosidase enzyme (SA-P~gai) detection or lysed and subjected to western blot analysis for pl5JN 4b, pl.61NK.4a. AND CLEAVED CASPACE-3.
In the some eases, the ceils were treated with 10 ng/ml trastuzumah
(HERCEPTiN™) and pertuzumab (PERJETA™) (both, Genentech, Sa Francisco, CA) for the indicated times. This treatment was combined with cytokines or with human recombinant heregultn (R&D Systems).
Fifty thousand cancer cells were also cultured in the lower chamber of a traiiswell system (BD Biosciences, San Jose, CA) with co-culture of 10x 10' human CD4; T-cells and 10s mature (i.e. type 1. polarized) or immature human dendritic cells (DCs) in the upper chamber, DCs and CD4 T-cells were obtained from select trial subjects
(Sharma et a!., 20.12 Cancer 1 18; 4354-4362). Mature and immature DCs were pulsed with Class ll-deri ved HER2 or control irrelevant (B AF and survivine) peptides (20 pg rnl) for 5 days at 37*C Control wells contained CD4 ' T~eells only. In addition,:
0.5x105 cells were incubated in the presence of DC/CD41 T-eell co-culture su ematants tor 5 day at 37°C In both approaches, cells were then .cultured for 2 more passages in absence of cytokines and subjected to senescence studies (SA-p-gal activity at pH 6 and pl51NK4b and pl6!N 4a western blot) or apoptosis studies (cleaved caspase-3 western blot). The following antibodies were added to cells 60 min before incubation with the co- culture of DC and CD4'5' T-cells to neutralize Thl -elaborated cytokines: polyclonal goat IgG anti-human TNF-a (0.06 pg/ml per 0.75 ng/ml TNF-a) and IFN-y (0.3 ng/ l per 5 ng/ml IF -y), and goat IgG isotype as the corresponding negative control (all. from. R&D Systems). Plasmid transections
MDA-MB-231 cells were transiently transieeted for 48 fa with 2 μ¾ of the wt HER2 expression vector (pcDNAHER2). As a control, cells were transfeeted with 2 μ of the empty vector (pcDNA3). Both vectors were kindly provided by Dr. Mark Greene (University of Pennsylvania, Philadelphia, PA). The cells were transfeeted in complete .med um' ithout antibiotics with Tmbofect (1¾emio Scientific, Waltham., MA). Ttansfection efficiency was evaluated by western blot 48 h after transfectton. Fort eight hours later, transfeeted ceils were transferred to complete culture medium containing 0.4 mg ml G418 (Life Technologies). After 15 days of culture, colonies resistant to G418 were selected by limiting dilution. Transfection efficiency was evaluated by westera blot.
RHA interference ( NAi) transfeetions.
Small interfering RNA (siRNA) SMART Pool: ON TARGET Plus HER2 siRNA, HERS siRNA and SMART Pool: ON-TARGETplus Non-targeting Pool were purchased from GE Dharmacon-iLafayette, CO). The following target sequences were used: BER2: UGGAAGAGAUCACAGGUUA (SEQ ID NO: 9%
GAGACCCGCIJGAACAAUAC (SEQ ID NO: 10), GGAGGAAUGCCGAGUACUG (SEQ ID HO: 1 1), GCUCAUCGCUCACAA.CCAA (SEQ ID NO: 12); 1IER3:
GCGAUGCUGAGA ACCAAUA (SEQ ID NO: 13), AGAUUGUGCUCACGGGACA (SEQ ID NO: 14), GCAGUGGAUUCGAGAAGUG (SEQ ID NO: 15),
UCGUCA.UGUUGAACUAUA (SEQ ID NO: 1 ); Non-targeting:
UGGUUUACAUGUCGACUAA (SEQ ID NO: 17), UGGUUUACAUGUUGUGUGA (SEQ ID NO : 18), UGGUUU ACAUGUUUUCUGA (SEQ ID NO: 1.9),
UGGUUUACAUGUUUUCCUA (SEQ I D NO: 20), Three hundred thousand cells were transfecied with siRN A sequences (25 nM) using R Ai Max Lipofeetamine (life Technologies) in serum free medium, and after 1 h the medium was supplemented with 10% FBS. Sixteen hours later, cells were subjected to 48 h of serum starvation followed by various designated treatments and western blot to check expression levels. SA- -gat activity at pH 6
Cells were washed twice in PBS, fixed in 3% formaldehyde, and washed again in PBS. The ceils were incubated overnight at 3 °C (without COa) with freshly prepared senescence associated acidic β-galactosidase (SA- -gal) staining solution from Millipore (Billerica, M A) per manufacturer's instaictio s. The percentage of SA-$-gal- positive (blue) cells in each sample was determined after scoring 300 cells using a bright- field microscope (Eyes Corexr,, Bothel, WA 40X/ 2048 x 1536, 3,2 mu pixel; 3,1 MP COLOR/ Captured images: Color TIFF, PNG, JPG or B P-2048 x 1536 pixels) Western blot analysis
Lysates were prepared from MCF-lOA, SK-BR-3, and CF-7, T-47 D or MDA-MB-231 cells. Cells were lysed in a buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 m.M EDTA, 1 mM EGTA, .10% glycerol, 70% Tergitol, 0.1% SDS, 1 mM gsCl and protease inhibitor cocktail Sigma-Aldrieli (St. Louis, MO). Lysates were centrifuged at .12,OO'0xg for 15 mln i4 C. Proteins were soluhiiized in sample buffer (Life Technologies) and subjected to SDS-PAGE, Proteins were eleetrohiotted onto PVDF. Membranes were immunob lotted with the following antibodies: pl.51NK4b (K- 18), pl6INK4a (50.1 ), IFN-yRa (C-20), HER3 (C- 17) all from Santa Cruz Biotechnology (Santa Cruz, CA); VincuHn (V913 I) from Sigma-Aidrich; HER2 (2908), cleaved caspase-3 (Aspl 75) and TNP-R1 (C25C1) and phospho-Akt (Ser473) from Ceil
Signaling Technologies (Danvers, MA), After washing, membranes were incubated with HRP-conjugated secondar antibody (Bio-Rad, Hercules, CA). Bands were visualized and quantified by using the enhanced eherailuminescence (ECL) western blot detection system and the image Reader LAS- 1000 Lite version 1.0 softwar (Fuji). Quantification of western blots was performed using ImageJ software.
SK-BR-3 cells were untreated, treated with IFN-y (.1 0 U/ml.) and T F-a (l Ong/ml), treated with trastuzumah (l Opg/ml) and pettoznmab (1 Opg/mi), or treated with a '■combination of IFN-y, T F- , trastuzumah, and pertuzumab, for 24 hours. After incubation, apoptosis induction was determined using FITC-Annexin 'V apoptosis detection kit (BD biosciences) according to manufacturer's instructions. Briefly,
untreated and treated SKBR-3 cells were collected, washed with PBS and resuspended in Annexin V binding buffer at a concentration of 1 xLO6 cells/ml ΙΟΟμΙ of ceil suspension was incubated with 5 1 PI and 5μ1 FETC-annexm V for 15 mm at room temperature in the dark. After incubation, 150μΙ annexin V binding buffer was added and apoptosis
induction was analyzed using a BD Accuri€6 flow cytometiy (BD Biosciences) and data was analyzed with CFlow Plus software. UV-irradiated cells were used as positive controls. Tumori genesis studies
For xenograft experiments, SK-BR-3 (2 > 10i! cells/mouse in 200 μΐ PBS) were injected into the flanks of six-week old female oathymic (nude) mice (F'axmw, Ffarlam Laboratories, 5 mice/group). When the tumors were palpable, the animals were treated s.c. with trastuzumab and pertuzumab (30 ug/kg) and then injected .v. c. twice a week with hrTNF-a and hrEFN-y ( 10 ng/kg). Tumor formation was monitored by palpation and tumor volume in was determined with a caliper twice a. week: width2 x !ength/2. Ail animal experiments were carried out in compliance with the institutions guidelines.
Unpaired Student's t-test (two-tailed) analysis was performed using GraphPad Prism■(GraphPad Software, La jolla, CA, USA). A P value of 0.05 or less was considered significant *P < 0.05, **P < 0.01 , ***P < 0.001. RESULTS.
Th j . c jo nes TNF-a
S -BR-3 cells were incubated with human recombinant tumor necrosis factor alpha (T F- ) and interferon gamma (If -y) alone or combined for 5 days at 37°C to study if the elaborated cytokines produced by the immune system cells could induce a specific senescence response in tumor cells. The cells were then cultured for 2 more passages and subjected to senescence studies. The combination of both cytokines resulted in senescence induction of SK-BR-3 cells, evidenced by increased senescence associated acidic β-galaetosidase (SA-p-gaS) staining (Figure 6A) and higher expression of the senescence-associated markers pi ,5IN 4b and pl 6IN 4a (Figure 6B) compared to the control untreated cells or each cytokine alone detected by western blot. Similar results were obtained in BT-474, MCF-7 and T-47D breast cancer cell lines (data not shown). Thus, T-47D cells were treated with several concentrations of TNF-a, from 10 to 100 ng/rni, and lFN-γ, from 100 to 1000 'Όίτχύ, or in combination, it was observed that the induction of the senescence phenotype was dose dependent (Figure 6C). Similar results were found in SK-BR-3. BT-474 and MCF-7 cells. inverse correlation between the HER2 expression level in breast cancer cells and the Thl cytokines TNF- and IFN-y doses requited to induce senescence
Experiments were designed to determine whether the HER2 expression level plays a role in the senescence induced by the treatment with TNF-a and IFN-y for .5 days at 37°C followed by 2 more passages without the cytokines in SK-BR-3, BT-474, MCF-7, T-47D and MDA-MB-231 breast cancer cells, in fact, the amount of SA-β- gal positive cells was augmented in the high HER2 expressing cell lines (SK-BR-3, Figure 6D and BT-474, data not shown), with lower doses of TNF-a and IFN-y compared to the intermediate HER2 expressing cell lines (T-47D, Figure 6D and MCF-7, data not shown) with higher cytokines concentrations. However even highest concentration of TNF-a and IFN-y could not induce senescence in the low HER2~expressing cell line MDA-MB-23 ί (Figure 6D). These results clearly evidence a correlation between the TNF~a and IFN-y Inducing senescence and the HER2 expression level.
HE 2 is eq uired lor Thl cytokines T F-a a d lFN-γ tnediated senescence and apoptosis in MDA-MB-231 breast cancer cells
Onl when MDA-MB-23 ! cells were stably transfected with a wild type I-IER2 plasmid (pcDNA HER2) and treated with high concentrations of TNF-a (200 ng l) and IFN-y (2000 U/ral) for 5 days at 37' C followed by 2 more passages in absence of cytokines, there was a strong increase in SA~[Vgai positive cells (Figure 7 A) and in pl5INK4b expression (Figure 7B). Notably, since it was observed that not only there was a relatively higher number of blue senescent cells when HER2-MDA-MB-231 cells were cultured with highest concentrations of TNF~a and IFN-γ, but also there was a significantly lower amount of cells, the experiment was repeated and the cells were subjected to western blot to detect active caspase-3 to study apoptosis (Figure 7B). The treatment of cells transfected wi th die control empty vector (pcDNA3) with increasing concentrations of TNF-a, from 75 to 200 tig/ml, and IFN-y, from 750 to 200 TJ/n L in combination in the same conditions described above, had no effect on senescence induction assessed by $Α~β-§¾1 staining (Figure 7A) or pl5i!SiK4b and cleaved caspase 3 expressions (Figure 7B). This finding reinforces that HER.2 is required in the mechanism of TNF-a and IFN-y inducing senescence and apoptosis in breast cancer cells.
Cytokine receptors are expressed in sirni jar le els i breast eel 1 1 ines
The high HER2 -expressing ceil lines SK-BR-3. and BT-474, the
intermediate MCF-7 and T-47D and the low HER2-expressing MDA- B-231 breast cancer cell Sines, like the low HER2 normal immortalized MCF-10 breast cell line showed similar IFN-y and TNF-a receptor expression by western blot analysis (Figure 12). This result demonstrates that the expression level of these two cytokine receptors is independent of the HER2 expression level. It is in accordance with reports that describe the action of these cytokines in the different phases of the normal br east . TNF-a has bee in vol ved hi proliferation, development: and branching morphogenesis of the normal mammary gland (Lee et a!., 2000 Endocrinology 141 : 3764-3773). The receptor TNFR1 expression mediates TNF~a~induced proliferation of mammary epithelial cells, and TNFR2 activation induces casein accumulation (Varela et aL 1.996 Endocrinology .137; 4915-4924). Similarly, the active form of IFN-y interacts with its receptor expressed on the surface of almost all normal cells (Ealick et aL, 1991 Science 252: 698-702; Farrar et al., 1993 Annu. Rev. Immunol. 1 1 :571-611). Combined HER2 and HE 3 blockage expressio enhances Th I cytokines TNF-a and IFN-γ senescence induction in breast cancer ceils. The previous results showing the enhancement of senescent and apoptosis phenotypes in high and intermediate HER2-expressing cell lines treated with the combination of cytokines led to studying the effect of knocking down HER2 and HER3 with siRNA. The therapeutic benefit of blocking HER2/HER3 signaling in breast cancer has been demonstrated by several studies (Lee-Hoefiich et al.} 2008 Cancer Res.
14:5878-87: Berghoff et aL 2014 Breast 14: 80960-9776). Although the combined treatment of T F- and IFN-γ in ER3 -depleted cells did iot significantly enhance the number of senescent or apoptosis cells, the double knock down with HER2 and HERS siRNA strongly increased the SA-p-gai staining (Figure 8A) and p 15iN 4b and active caspace-3 expression level (Figure SB) in SK-BR-3 ceils. At the same time, it was observed that a higher apoptosis induction of the cells treated with the double knock down and the cytokines by western blot of active caspase-3 (Figure 8 A). Similar results were found in MCF-7 cells (Figure 13), BT-474 and T47D cells. Combined HER2 inhibition and HER2-HER3 dimerization inhibition enhances. Thl cytokines
cgncer .cells
Trastuzumab and pertuzuraab are antibodies that have bee widely used in the clinic to treat HER2- ositive breast cancer. To study the Thl cytokines-induced senescence and apoptosis in a translational approach, experiments were designed to pretreat SK-BR-3 cells with trastuzumab and pertuztimab and then, the ceils were additionally treated with TNF-a. and IFN-y for 5 days at37°€ followed by 2 more passages without the cytokines and antibodies. It 'was observed that the amount of blue cells was highly increased i the ceils tha received the complete treatment compared to the cells treated with cytokines only, as measured by SA-p-gal staining ( Figure 9A) and l51NK4b increased expression by western blot (Figure 9B). interestingly , the double treatment also had a significant effect on the induction of apoptosis, both by increased active caspase 3 expression by western blot (Figure 9B) and by increased amount of anexin v and propidiura iodide positive cells by flow cytometry analysis (Figure 9C). CD4' Th l -media ted senescence and apoptosis of HE 2-ovexpr¾ssing human breast cancer cel ts
Experiments were designed to co-culture. HER2 Class II peptide-pri ed CD4 ! T-cells with SK-BR-3 breast cancer cells using a transwell culture system to confirm that the Cytokines produced by die immune system cells in vivo could also induce a specific senescence and apoptotic response in tumor cells. The cells were co- cultured for 5 days at 37: "C and then cultured for 2 more passages in complete medium without immune system ceils. The co-culture resulted in senescence and apoptosis of SK- BR-3 cells, evidenced by increased amount of SA-j3-gal staining (Figure lOA) and increased expression of I5IN 4b and cleaved caspase 3 (Figure I OB) detected by westera blot. CD4+ T-cells primed either with immature dendritic cells (DCs) or mature DCs plus irrelevant Class II (BRAF or survivme) peptides were not able to induce either senescence, or apoptosis of SK-BR-3 eel! (Figure 1.0).
The senescence and apoptosis observed was highly augmented in SE-BR- 3 when the cells were co-cultured with HER2 Class II pepti de-primed CD4 ' T-cells in presence of trastuzumab and pertuzunrab in the transwell. This result was clearly evidenced by increase SA-pVgal staining (Figure 10A) and pl5i 4b and cleaved caspase 3 expression levels (Figure 10B).
As another approach, SK-BR-3 cel ls were co-cultured with the supernatant from CD4+ T cell-mature DC co-culture and a similar specific senescence response was observed. The Thl -elaborated cytokines IFN-y and TNF-a obtained from CD4'S" T cell- mature DC co-culture supematants were previously confirmed using ELISA. By both experimental approaches, SK-BR- senescence and apoptosis could be partially rescued by neutralizing ΙΡΝ-γ and TNF-a blocking antibodies (Figure 14).
It was also observed that the effect was dose-dependent as increasing number of immune system cells induced higher SA-p-gal staining, pI 5INK4b and cleaved caspase 3 expression levels in SK-BR-3 cells by both approaches.
Thl cytokines TNF-a and IFN-y sensitize trastuzumab and pertuzumab resistant breast cancer cells to senescence and apoptosis induction To continue unraveling the mechanism by which the lit ! cytokines could induce senescence and apoptosis, it is believed thai TNF~a and IFN-γ could restore the sensitivity to trastuzumab and pertoziimab to breast cancer resistant ceils, it was observed, that the treatment with trastuzumab and pertuzumab could not prevent the acti vation of A T in two resistant cell lines HCC-1419 (O'Brien et t, 2010 Mo! Cancer Ther,
6:1489-502) and JIMT-l (O'Brien et al„ 2010 Mol Cancer Ther. 6: 1489-502; Tanner el al., 2004 l Cancer Ther. 12:1585-92) contrary to T-47D sensitive cells (Figure 16).
The treatment with TNF~a and IFN-y induced senescence and apoptosis in a dose dependent manner in HCC-1419 and JIMT-l cells. When the cells were treated with trastuzunrab and pertuzumab, senescence and apoptosis could not be evidenced even at higher doses (data not shown). However, the double treatment with cytokines and antibodies induced senescence by SA-[3-ga! assay (Figure 11 A) and increase expression of p!5INK4b (Figure 1 1 B) in HCC-1419 and JIMT-l cells. Moreover, the combination of cytokines and antibodies effectively induced ceil death (Figure 1 I B) in HCC-1419 and JIMT-l cells. This result demonstrates that the Thl cytokines IFN-γ and TNF~a could revert the resistance to the therapeutic agents that is affecting cancer patients widely.
DISCUSSION;
Herein it has been demonstrated that TNF-o: and IFN-y induce senescence and apoptosis in breast cancer cells in a dose dependent manner. Also revealed is an inverse correlation between the HER2 expression level in breast cancer cells and the doses of TNF-o: and IFN-γ required to induce senescence i those cells. It has also been shown thai cytokine receptors are expressed in similar levels in all the breast cell lines tested, implicating that this is not the cause of the differential response. Furthermore, only w hen MDA-MB-231 ceils (low HER2 level) were stably transfected with a wild type HER2 plasmid, high doses of Thl cytokines were able to induce senescence and apoptosis.
Thus, it seems that HER2 signaling is required to induce senescence and apoptosis by till cytokines, because in cells that lack HER2 or express very low levels is not possible to induce senescence or apoptosis even with high doses of cytokines. However, in cells that express high or intermediate levels of HBRZ knocking down the gene induces
senescence and apoptosis through a phenomenon called oncogene addiction, it has additionally' been shown that combining HER2 and HER3 siRNA enhances even more of the senescence and apoptosis induced by TNF- and IF -y in breast cancer cells, taking a step ahead of HER3 over counting for the lack of HER2 signaling.
Example 5; Ami -Estrogen Therapy and Anti~HER2 Dendritic Cell 'Vaccination Improves Pathologic Complete Response in ERp0S HER2i>08 DCIS
The antigen-presenting capacity of dendritic cells ("DCs") has led to enthusiasm for their use in anti-tumor vaccination. The present group designed a HER2 peptide-pulsed autologous DC vaccine uniquely engineered to promote anti-HER2 T l sensitization and attraction. See, United States Ser. No. 14/658,095, filed March 13, 2015; United States Ser. No. 14/985,303, filed. December 30, 2015, the disclosures of which are incorporated by reference herein in their entireties; Datta, 1, et al.,
Oncolmmuno gy 4:8 el 022301 (2015) 001:10. 1080/2162402X.2015. 1022301 , and Datta, J., et al., Breast Cancer Res. 17( 1):71 (2015). The feasibility, the safety, and the preliminary clinical results following the neoadjuvant use of the anti-HE 2 vaccine in patients with HE 2P"S DCIS (HER2pfiS ductal carcinoma in siiti) has been reported.
Complete tumor regression (pathological complete response ("pCR")) was induced in 1 % of patients; however, these results were concentrated in atients with hormone independent (ERas¾) DCIS, suggesting that patients with ERi>0S CiS were less responsive to the anti-HER2 DC vaccine man are those patients with hormone independent DCIS (S arma, A., et al.? Cancer 1 18(17):4354-62 (2012)). The extensive bidirectional crosstalk between the BBR2 and ER signaling pathways leads to enhanced eaneereeil proliferation and survival (Arpino, G., et al. Endocrine Reviews 29(2):217-233 (2008) and Prat, A., et al,, Nat. d . Prac. Oncol 5(9): 531-542 (2008)). Combined targeting of both of these two receptors may prevent them from continuing to activate each other , i t is hypothesized that adding aati-estrogen ("AE") therap to anti-HER2 DC vaccination treatment would improve the response in patients with HER2 iVE p0S DCIS. In the studies reported herein comparisons were .made between the clinical and the immune responses in anti~HER2 D vaccinated patients with JER"* DCIS ("B ^), B ^ DCIS patients who received the anti~HE 2 DC vaccine alone (!tERlws without AE"), and ER550* DCIS patients who received both the anti-HER2 DC vaccine and concurrent anti-estrogen therapy fER^with AE"). The results presented herein demonstrate that concurrent neoadjuvant anti- estrogen therapy and anii-HER2 DC! vaccination increases the immune response in the local sentinel lymp nodes and the rate of pathologic complete response in
HER2pf>7ERp0S DCIS. These results further support individualized, targeted, combination therapy.
METHODS
Methods Summary: Eighty-one patients with HERS5*** DCIS received a neoadjuvant a»ti~HER2 DC vaccine. Clinical response was measured in the resected surgical specimen. Immune response -- anti-HER2 CD4 Thl response - was measured in the peripheral blood pre- and post- vaccination and in the sentinel lymph nodes post- vaccination. Clinical and immune responses were compared between ER8*8 patients who underwent anti-HER2 vaccination alone, ER?0S patients who underwent anti-HER2 vaccination alone, and ERp0S patients that received anti-estrogen therapy concurrently with anti-HER2 vaccination.
The methods carried out are set forth below in detail
Breast cancer cell lines, SKBR3 (HER2sXtS 3+ / ER ) and MCF? (HERS**8 2r / ERpos), were treated with Thl cytokines (IFNy and TNFa), a tamoxifen metabolite (4~ hydroxytamoxifen, "4HT"), or both for 72 hours. Metabolic activity was measured vi Alarnar Blue assay.
Trial Design
After approval by the Institutional Review Board of the Uni versity of
Pennsylvania, two neoadjuvant clinical trials of a HER2 peptide pulsed DO vaccine (NCT00107021 1 and NCT02061332) were conducted. The primary objectives were to evaluate the feasibility, safety, and efficacy of DC! vaccination. The secondary objective was to assess clinical and immune responses. Preliminary results of these trials showed that the vaccine is safe, well tolerated, and induces decline or eradication of HER- 2 expression (Shamia, A., et al. Cancer 118(17);4354~62 (2012) ("Sharrna, et a! ")).
Further re view of the preliminary resul ts showed drat vaccination was more effective in hormone independent (ER***) patients [20, 21], Based oa the preclinical data and the preliminary resul ts of the clinical trial, an addendum was approved to treat subsequent E pos patients with hormone dependent (E IK*) DCIS with concurrent AE therapy. Patient .Selection
Female patie nts older than 18 years o f age with biopsy-proven HER2|,0S DCIS and an ECOG Performance Status Score of 0 or 1 were eligible for the trial. All tissue specimens were reviewed by a single pathologist for eligibility; HER-2 positivity was defined as >5% of cells expression 2+ or 3+ intensity of the HER-2 protein . Women of childbearing age were required to have a negative seann pregnancy test and were required to use a medically acceptable form of birth control. Women with cardiac dysfunction, H V, HepC, coagulopathies, or a pre-existing medical illness or medications which might interfere with the study were excluded, from the trial. Women, who had received definitive treatment or whose DCIS was eliminated by exctsional biopsy at diagnosis were not eligible for the trial. Eighty-one women were enrolled in the trial and completed the vaccination treatment. All 81 patients underwent surgical resection with pathologic examination of the resected specimen. The immune responses were measured in patients in the second trial (NCT0206 332) ~ S3 of 54 patients had pre and post- vaccination CD4+ immune responses measured in the peripheral blood; 4 patients had post, vaccination CD4+ immune responses measured in the sentinel lymph nodes; and 22 HLA-A23** patients had pre- and post-vaccination CD8+ immune responses measured in the peripheral blood.
Vaccination Procedure
Vaccine preparation and delivery have been described in detail previously. See, for example, Sharma, et al, Czeraieeki, BJ,, et al. Cancer Res. 67(4): 1842-52 (2007) (Czemiecki, et al.), and Koski, G.K., et al, J. Jmmunother. 35(1) 54-56 (2012). The vaccination procedure is shown in Figure 16. Briefly, monocytic dendritic cell precursors were obtained f om patients via tandem ieukapheresis/coiraierctarent centrifugal eiutriation. Monocytes were cultured at 37°C in serum free medium. (SFM) (Inviixogen, Carlsbad, CA) with granulocyte-macrophage colony-stimulating factor (GM-CSF) (Amgen, 'Newbury Park, CA) and lL-4. The following day, the cells were pulsed with six HER2-derived major histocompatibility complex (MHC) class II binding peptides
(American Peptide Corporation. Sunnyvale, CA) ~ three extracellular domain (ECO) peptides (peptide 42-56: HLD LRHLYQGCQVV (SEQ ID NO: 1 ); peptide 98-1 14; RLRiVRGTQLFEDNYAL (SEQ ID NO: 2); and peptide 328-345:
TQR.CE CSKPCAR.VCYGL (SEQ ID NO: 3» and three intracellular domain (ICO) peptides (peptide 776-790: GVGSPYVSRLLGICL (SEQ ID NO: 4);. peptide 927-941 : PAREIPDLLEt GERL (SEQ ID NO: 5); and peptide 1 166-1 180
TLERPK FLSPGKNGV (SEQ ID NO: 6)). After 8-12 hours, lOOOU/mL of IFN-gamma (Intermune, Brisbane, CA) was added, and 6 hoars before harvest, lOmg mL of clinical grade LPS (gift from Dr Anthony Suffredini at the National Institute of Health (M1H)) was added to complete the rapid maturation to a type-I dendritic cell (DC 1). For HLA- A2pws patients, the monocyte pool was pulsed with MHC class I binding peptides 369-377 and 689-697.
Four to six weekly injections of 10-20 million MBR-2~peplide pulsed DC i s were administered into the breast, the groin lymph nodes, or both the breast and the groin lymph nodes.
Patients were monitored for adverse effects for a minimum of 1 -2 hoars following each weekly vaccination. All adverse events were classified by National Cancer Institute Common Toxicity Criteria (NCI~CTC version 3.0), were assessed at least weekly during vaccination, and were monitored until their resolution.
Clinical Monitoring
Pathologic response was examined at the time of surgical resection ~ lumpectomy (n - 48} or mastectomy (n - 33). A pathologic complete response to immunization was defined as no residual DOS or invasive breast cancer at the time of surgical resection. Patients were monitored after surgical resection for the development of subsequent breas events. A subsequent breast event was defined as a lesion - DCIS or invasive breast cancer, identified in either the ipsilateral or contralateral breast.
Anti-Estrogen Therapy BR positive patients were treated with ao.ti~estrogen therapy concurrent with four to six weekly a«ii-BBR2 DCl vaccinations. A physician investigator decided which of the following ami-estrogen therapies was best suited for each patient: Tamoxifen (4- hydroxytamoxifea ("4HT") (NOLVADEX™)); Letrozole (FEMARA'™); Anastrozole (ARMIDEX™); Exemestane (AROMASiN™); Raloxifene (EVISTA™); or any other suitable anti-estrogen agent that blocks or modifies the ac tions of estrogen.
Immune Monitoring CD4 i- T-cel!s
Systemic anti-HER2 CD4+ T-cell responses were generated from autologous peripheral blood mononuclear cells (PB C) pulsed with six HER2-derive major histocompatibility complex (MHC) class 11 binding peptides peptides. Localized ami-
HER2 CD4+ T -cell responses were measured in the locoregional sentinel lymph nodes (SEN) in 40 patients who underwent SEN biopsy. IFN-γ production was quantified via enzyme- 1 inked immunosorbent spot (EL!SPOT) assays as previously described in detail. (FracoL M„ et al.} Am. Surg. Oncol. 20(i 0):3233-9 (2013)) Briefly, PVDF membrane plates (Mabfcech, Cincinnati, OH) were coated overnight with aati-IFN-γ capture antibody (IDI K), The following day, alter the plates were washed with PBS (Mediatech, Manassas, VA) and blocked with 10% human serum DMEM, 2x 10s PBMCs on SEN cells were plated in each well either unstimulated, ulsed with HER2-derived Class I peptides (4p.g) (42-56, 98-1 1 , 328-345, 776-790, 927-94.1 , 1166-1 1 SO), or pulsed with anti-human CD3 and CD28 antibodies (0.5pg/mL) (positive control, BD Pharmingen, San Diego, CA), and incubated at 37°C+ 5%C02 for 24-36 horn s. After the plates were washed with PBS, lOOpJL of detection antibody ( Img/mL; 7 B6~l~biotra) was added to each well and the plates were incubated for 2 hours. After the plates were washed again with PBS, ϊΟΟμΕ of 1 : 1000 diluted streptavidin-HR was added to each well and the plates were incubated for another hour, TMB substrate solution was added to reveal spot formation. Spot forming cells (SFC) were counted using an automated reader
(toinuaoSpot CTL),
A positive response to an individual HER2 Class II peptide was defined as minimum of 20 SFC/2. J O5 cells after subtracting the unstimulated background and at least a two-fold increase over the unstimulated background. Three metrics were used to quantify the CD4+ Thl response (I) overall response rate (the proportion of patients responding to 1 peptide), (2) response repertoire (the number of peptides to which a patient responds), and (3 ) cumulative response (the sum of the SFCs across all 6 peptides).
CDS+ T-cells
Systemic anti-HER2 CD8+ T-cell responses were measured m twenty two HLA- A2pos(HLA.2.1) patients . Anti-HER.2 CD8+ T~ceil responses were generated by in vitto sensitization assays as previously described in detail by Czemieeki, et ai.. Briefly, CD8+ T-cells were selected from the cryopreserved 120-140 lymphocyte cell fractions via negative selection (StemCell Technologies., Vancouver, BC). Autologous dendritic ceils were suspended in serum free medium (SFM) (Invitrogen, Carlsbad, CA) with GM-CSF (I Qng mi), pulsed with a class 1 HBR2 peptide (lOug/ml) (369-377), and co-cultured with the C 8+ T-cells at a ratio of 10: 1 . IL-2 (30 JU/ml) was added on day 2, On day 10, T- ceSIs were harvested and were tested against T2 target ceils pulsed with either the Class I HER2 peptide or irrelevant controls (p53 and colon cancer peptide). After 24 hours, the supernatant was harvested and analyzed by enzyme-Huked immunosorbent assay
(ELISA). A positive response to the BER2 Glass I peptide wa defined as a two-fold increase in CD8+ T-cell IFNy production compared to the irrelevant peptide controls.
Statistical Methods
Descriptive statistics and univariate logistic regression was used to evaluate demographic and clinical data. P-values <0.05 were considered statistically significant. Development of subsequent breast events was compared using Kaplan Meier analysis. All analyses were performed with STATA 12.0 IC statistical software (STATA Corp, College Station, TX).
RESULTS
Results Summary: Patients with E "¾ DOS and patients with. E *0* DCIS who were treated with anti-estrogen therapy had a similar rate of pathologic complete response (31.4% vs 28.6%, p - 1.00); and both rates were significantly higher than the rate of pathologic complete response found in patients with E ^* DOS that did not receive anti-estrogen therapy (4.0%, p ~ 0.035). The anti-HER2 Thl immune response measured in the peripheral blood increased significantly following vaccination., but was similar across all three groups. In the sentinel lymph nodes, however, the aati~HER2 Thl immune response was significantly higher in the patients with ERpf,s DOS who were treated with combination anti~HER2 vaccination and anti-estrogen therapy compared with the patients with ERpos DOS who were treated with anti-HER2 vaccination alone.
The results are set forth below in detail.
Pre-Climcal Experiments
The S BR3 breast cancer cell line (ERriS¾) increased anti-tumor activity in response to Th l cytokine treatment, but not in response to anti-estrogen treatment.
Furthermore, adding anti-estrogen treatment to the Thl cytokine treatment had no effect on the metabolic activity as shown in Fiaure 1.7 A.
Conversely, the MCF7 breast cancer cell One (ERpos) did not increase anti-tumor activity in response to either Th l cytokine treatment or anti -estrogen treatment; however, the combination of Th l cytokine treatment and anti-estrogen treatment together resulted in an increase in metabolic activity as show in Figure 1 B.
Xrial;...P. ig.nLS
Of the 81 patients who participated in the clinical 'trial, the median age was 55 (interquartile range (IQR) 47-60), median BMl was 25.9 (IQR 22.4-31.0). and a majority of patients were postmenopausal (a - 68; 84.0%) and white (n ~: 65, 80.2%). All eligible patients were diagnosed with DCS S at the time of biopsy; however, a minority of patients were found to have early invasive breast cancer (Stage I) at the time of final surgical resection (n ~ 16, 19.8%). Ail eligible patients were diagnosed with 2+ (n - 28, 34,6%) or 3÷ (n ~ 53, 65.4%) HER2po$ DOS. Vaccination was administered into groin lymph nodes in 47 patients (58%), into the breast in 18 patients (22.2%), and into both the groin lymph nodes and the breast in 16 patients (20%). Surgical resection was completed via lumpectomy in 48 patients (59.3%) and via mastectomy in 33 patients (40.7%). Of those patients who underwent lumpectomy, 37.5% received post-operative radiation therapy.
The vaccine was well tolerated with only grade 1-2 adverse events. The most commonly reported adverse events associated w th the vaccine were fatigue (n ~ 41, 50.6%), injection site reaction (n - 34, 42.0%), and chilis/rigors (n = 26, 32.1%). No patients were unable to complete the trial doe to the side effects.
Figure 18 shows that in the overall cohort, 35 patients (43.2%) had E ueg disease and 46 patients (56.8%) had ERpoif disease. Of those patients with ER*** disease, 25 patients (54.3%) received the DC! vaccine alone and 21 patients (45.7%) received the DO vaccine and concurrent AE therapy. Demographic and. clinical characteristics of these treatment groups are summarized in Table 1 below, and did not show any significant difference between the groups. Table 1 ; Demographic and clinical characteristics comparing patients by Ell status and
Comorbidity
Score 27 (77.1 ) 21 (84.0) 21 (100.0) 0.07
<2 8 (22.9) 4 (16.0) 0 (0.0)
>3
Stage
0 29 (82.9) 17 (68.0) 19 (90.6) 0.14
1 6 (17,1) 6 (32.0.) 2 (9,5) HER2/rseu
2+ 14 (40.0} 3 (12.0} 1 1 (62.4) 0.01
Surgical Resection
Lumpectomy 19 (54.3} 13 (52.0) 16 (76.2) 0.18
^^aste^to^^
ER (estrogen receptor), AE (anti-estrogen), 1QR {interquartile range), HER2 (human epidermal growth factor receptor 2).
1 hold indicates statistical significance
a following 1 umpectomy
Clinical Response Rates to HER2 Vaccination
The clinical response was available for ail 81 patients. Overall, 18 of the Si immunized patients (22.2%) were found to have no residual disease identified in the resected surgical specimen; these patients were considered to have a pathologic complete response (pCR). As shown in Figure , pathologic complete response in the ER group was higher than in the ERiX s group. ore specifically, pCR in the ERut¾ group (n = 11 , 31,4%) and the ER*** group that received AE therapy (n ~ 6, 28.6%) were similar (p = LOO); however, the rate of pCR in the ERpti" group that did not receive AE therapy (n - 1, 4.0%) was significantly lower than the rate of CR i the ER group (p = 0,01) and the ERpi,s group that received AE therapy (p -- 0.04) (Figure .19).
Pathologic coi.npl.ete response .correlates with a decreased risk of recurrence See, for example, Tanioka, M.5 ei al', Br, J, Cancer 103(30:297-302 (2010) Subsequent breast lesions, defined as either DC S or invasive breast cancer identified in either breast, occur red in 6 vaccinated patients (7.4%) . All of the patients who experienced subsequent breast even ts had residua! disease identified at the time of surgical resection, < pCR (Figure 20 A). Two of the patients had ERne DCIS and four of the patients had ERT!,% DCIS but did not receive AE therapy. None of the patients who had E Fm DCIS and received AE therapy have experienced a subsequent breast event (Figure 20B). EE***' patients enrol!ed later in the trial were treated with concurrent vaccinatio and AE therapy, and, therefore, media follow up was shorter for these patients. Of course, these patients will continue to be monitored.
As shown in Table 1 , the rate of lumpectomy was similar across all three groups; however, the rate of radiation following lumpectomy was lower in. the group of patients with ERpos DOS who received AE therapy (18.75%) than in the group of patients with ER >' DOS who did not receive AE therapy (53.8%, p ------ 0.06). Patients with ER¾WS DOS who received AE therapy have a decreased rate of subsequent breast events despite the lower rate of radiation. immune Response Rates to HER2 V accinati o
CD4 ±, . Thl . Resgmme:. Sw mic. - Peripheral. Bkmd
The pre-vaceination and post-vaccination immune responses were measured in.53 patients. Overall, responsiviiy increased significantly from 36.25% pre~vacci.n.ation to
56.25% post-vaccination (p = O.001). Median response repertoire also increased significantly from Ϊ (IQ 0-2) pre- vacci ation to 2.9 (IQR 2-4) post-vaccination (p :::
<Q.0Ol). Finally, median cumulative response increased significantly from 56.1 (IQR
23.3- i 3 .1.7) pre- vaccination to 133.1 (iQR 75.6-240.3) post- vaccination (p - 0,002).
Responsiviiy: Following vaccination, responsivii increased significantly in each group (ER!J<¾ 58.3% to 87.5%, p < 0,01; ERiJ<)A without AE treatment 50.0% to 75.0%, p < 0,01 ; ER with AE treatment 57.1 to 90.5%, p < 0.01). Pre-vaceination responsivity rates were similar across all three groups (ERii¾i 58.3%, EE5™* without AE treatment 50.0%, EW* with AE treatment 57.3%; p. ~ 0.9). Post-vaccinatio responsivity rates were also similar across all three groups (ER1X* 87.5%, ERpos wiihout AE treatment 75.0%, ER*** with AE treatment 90.5%; p = 0.5. Figure 21 A).
Response Repertoire : Following vaccination, response repertoire increased in eac group (ER**8 1 to 3, p » 0,05; ER*08 without AE treatmeni 0 to! .5, - 0.1; ERpi!S with AE treatment 1 to 3. p - 0.03 ). Pre-vaceination median response repertoire was similar across ail three groups (ERBeg 3 (iQR 0-2), ERt, S without AE treatment 0 (IQR 0- 1.5), ERr",s with AE treatment 1 (iQ 0-2); p - 0,5). Post- vaccinatio median response repertoire was also similar across all three groups (ERaeg 3 (IQR 2-4.5), ER^ without AE treatment 1.5 (IQR 0.5-3.5), with AE treatment 3 (IQR 2-5); p - 0.4. Figure 2 I B).
Cturtulati ye Response: Following vaccination cumulative response increased in each group (ERi,£¾ 56.3 to 149.7, p < 0.01; ERpl s without AE treatment 4Ϊ .1 to 178.7, p < 0.01 ; ERpftS with AE treatment 58.6 to 100.9, p < 0.0 Ϊ). Pre- vaccination median
cumulative response was similar across ali three groups (ER.**8 56.3 (IQR 26.1-1.16.2), E ** without AE treatment 41.1 (IQR 9.6-168.6), ER*** with AE treatment 58.6 (IQR 24.2-87.3); ~ 0.886). Post-vaccination median cumulative response was also similar across ail three groups (E "¾ 149.7 (IQ 97.7-246.7), E ^ without AE treatment 178.7 (IQR 64.7-278.3), E ^ with AE tr atment 100.9 (IQR 67.5- 174.4); p - 0.5. Figure 21.C).
( 7)4 j Th ! Response; Local -■ Sentinel Lymph Node The post-vaccination immune responses were measured in 40 patients. Overall, responsivity was 80%, median response repertoire was 2 (IQR 1-5), and median
cumulative response was 76.5 (IQR.23-197).
EgSROi yil - Responsivity rates were significantly higher in patients with
HER2t]")S/ERp0:S DCIS who received with AE treatment compared to patients with
HER ^/E P"* DC S who did not receive AE treatment (92.3% vs 43%; p - 0.03. Figure
22A). Median response repertoire was also significantly higher in patients with HER2pos ERpos DCIS who received with AE treatment compared to patients with HER2pos ERpos DC∑S who did not receive AE treatment (4 (IQR. 2-6} vs 0 (IQR 0-5): p - 0.05. Figure 22B).
Cumulative.Resffon^ Median cumulative response was also significantly higher in patients with HER2lw7ERiW* DCIS who received with AE treatment compared to patients with HER2p0S/ERpiJ* DCIS who did not receive AE treatment (102 (IQR 69-354) vs 23 (IQR 1-100); p - 0.05. Figure 22C). CDS r Response: Systemic - Peripheral Biooci
The pre- vaccination and post-vaccination CDS- immune responses were
measured in 22 HLA-A2+ patients. Overall, responsivity increased significantly from 13.3% pre- vaccination to 72.7% post- vaccination fp ~ <0.0002). Following vaccination, responsivity increased in each group (ERe*8
12.5% to 75%, p - 0.04; ERp0S without AE treatment 0% to 100%, p < 0.03; ER^ with AE treatment 20% to 60%, p - 0.17). Pre-vacc nation responstvity rates were similar across all three groups (ER^ 12,5%, ER90* without AE treatment 0%, ERpi)¾ with AE treatment 20%; p - ). Post-vaccination responsivity rates were also similar across ail three groups (ERB¾- 75%, ER** without AE treatment 100%, ERpilf' with AE treatment 60%; p ~. Figure 23).
CONCLUSIONS : The study described herein clearly shows a»ti-HER2 DO vaccines are clinically effective in ER^/HER5* DCIS patients. Anii-HER2 DC. I vaccine are also shown to he safe when combined with anti-estrogen therapy. It was shown that concurrent neoadjuvant anti-estrogen therapy and anti-HER2 DO
vaccination increases the immune response in the local sentinel lymph nodes and the rate of pathologic complete response in HER2p0S/ERsw* DCI S patients The combination therapy with anti-estrogen may also reduce subsequent Breast Events. These results may offer a personalized approach in DCIS therapy. These results .further support
individualized targeted, combination a.oti-i-!ER2 DC! vaccine and anti-estrogen, treatment. One skilled in the art can appreciate that other anti-pro!iferative combinations of anti-HER.2 treatments such as Trastuzumab and Pertuzumab, and others, are possible. These approaches may limit the need to extensive surgery, eliminate radiatuion therapy, and reduce long term hormonal treatment.
Example 6:A Novel Dendritic Cell Vaccine Targeting Mutated BRAF Overcomes Vemurafemb Resistance and Synergi stically Improves Survival in BR AF-Mutant M urine Melanoma
BRAF inhibitor vemurafemb (PLX) improves survival in B AF-mutant
(BRAF 6im) melanoma, but resistance is common. A BRAF 600f -pulsed type!. -polarized dendritic cell vaccine (BRAFV<¾H¾;~DC 1 ) induces antigen-specific CDS* T-cells that impact murine BRAP <>!)t>E melanoma. We investigated' if combinations of BRAF '600t~ DO and PLX elicit a synergistic clinical response. METHODS
A transplantable BRAF ¾ft"EPTEN ' ^ melanoma model was developed in the C57BS/6 background. DC I were generated from bone marrow precursors using FH3, IL~ 6, GM-CSF, lL-4, CpG and LPS, and pulsed with class I BRAFVS aE peptide. In addition to untreated and ovalbumin- DC 1 controls, BRAFvtitffi:-DCi (2x weekly injections) and PLX were administered alone or in designated combinations to tumor-bearing mice
(η~ίΟ each). Tumor growth and survival were determined. Induction, of B AFVf,i,0i'- specific CDS''" T-celi responses from splenocytes were assessed by lFN-γ ELISA.
Cytokine niRNA quantification in tumor micrqenvitonments (TME) was performed by RT~qPCR.
RESULTS
Figure 24 shows mice receiving B AF 6u0h-DCRPLX combinations, either initiated concurrently or after B AF¾&00fe-DCI induction, demonstrated dramatically delayed tumor growth (P<0.0Q1) and improved median survival (86d and 733d,
respectively) vs BRAFV600E-DC 1 (42d), PLX (43.5d). ovalbomin-DC 1 (28d), or untreated (24d) cohorts (P<0.00i); 35% were rendered disease-free following BRAFveMB- DCRPLX therapy, and remained immune to BRAFV60!>E tumor rechallenge. BRAF 6,,0i:- DC1 +PLX, compared with individual, treatments induced synergisticaily improved systemic CD8* T-ceil recognition of BRAF t,, - -pulsed antigen-presenting cells and BRAFV600E tumor cells (p<0.001 ) measured by IFN-y release in vitro. I TME,
BRAFv^-DCi+PLX generated highsr niRNA levels of Thl (IFN^f ΓΝΡ-α) and T-eeil homing (CXCL CCL5) cytokines, while attenuating PD-Ll expression: CDS f TIL trafficking was augmented by BRAFV¾00£-DC1 +PLX. In conclusion BRAFWM¾~DC I vaccines overcome vemurafenib resistance in
BRAFV<¾ * melanoma, and synergisticaily improve immune and clinical responses. Such combinations will find use by those of skill in the art. The disclosures of each, and every patent* patent application, aid
publication cited herein are hereby incorporated herein by. reference in their entirety. While these embodiments ha been .disclosed with reference to specific, embo iment , i is apparent that other -embodiments and variations of these embodiments ma be devised by others skilled in the art without departing from the true spirit and scope of the embodiments. The appended claims are intended to h constnied to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:
1. A method of generating an injectable multi-dose antigen pulsed dendritic cell vaccine, comprising:
contacting at least one antigen to a dendritic cell (DC); activating said DC with at least one TLR agonist;
cryopreserving said DC in multiple doses wherein the multiple doses comprise an initial immunizing dose and multiple booster doses, wherein when said DC is thawed, and said DC produces an effective amount of at least one cytokine to generate a T cell response.
2. The method of claim 1, further comprising thawing said DC, wherein said DC produces an effective amount of at least one cytokine to generate a T cell response.
3. The method of claim 1, wherein said antigen is a tumor antigen.
4. The method of claim 1, wherein said antigen is a viral antigen.
5. The method of claim 1, wherein said TLR agonist is LPS.
6. The method of claim 1, comprising activating said DC with IFN-γ.
7. The method of claim 1, wherein said cryopreserving comprises freezing said DC in a freezing medium comprising about 55% plasmalyte, about 40% human serum albumin, and about 5% DMSO.
8. The method of claim 7, wherein said cryopreserving comprises freezing said DC at a temperature of about -70°C or lower.
9. The method of claim 1, wherein the recovery and viability of the DC after thawing is greater than or equal to about 70%.
10. The method of claim 1, wherein the recovery and viability of the DC after thawing is greater than or equal to about 80%.
11. The method of claim 1, wherein said DC are cryopreserved for at least about one week.
12. The method of claim 1, wherein said cytokine is IL-12.
13. The method of claim 1, wherein said DC exhibits a killer function whereby said DC are capable of lysing targeted cells.
14. A cryopreserved injectable multi-dose antigen pulsed dendritic cell vaccine for eliciting an immune response in a mammal, wherein the injectable multi-dose antigen pulsed dendritic cell vaccine comprises:
a DC loaded with at least one antigen;
wherein said DC has been activated by exposure to at least one TLR agonist; and wherein said DC produces an effective amount of at least one cytokine to generate a T cell response.
15. The injectable multi-dose antigen pulsed dendritic cell vaccine of claim 14, wherein said antigen is a tumor antigen.
16. The injectable multi-dose antigen pulsed dendritic cell vaccine of claim 14, wherein said antigen is a viral antigen.
17. The injectable multi-dose antigen pulsed dendritic cell vaccine of claim 14, wherein said TLR agonist is LPS.
18. The injectable multi-dose antigen pulsed dendritic cell vaccine of claim 14, wherein said DC has been activated by exposure to IFN-γ.
19. The injectable multi-dose antigen pulsed dendritic cell vaccine claim 14, wherein said vaccine has been cryopreserved at a temperature of about -70°C or lower in a freezing medium comprising about 55% plasmalyte, about 40% human serum albumin, and 5% DMSO.
20. The injectable multi-dose antigen pulsed dendritic cell vaccine claim 14, wherein the recovery and viability of the DC after thawing is greater than or equal to about 70%.
21. The injectable multi-dose antigen pulsed dendritic cell vaccine of claim 14, wherein the recovery and viability of the DC after thawing is greater than or equal to about 80%.
22. The injectable multi-dose antigen pulsed dendritic cell vaccine of claim 14, wherein the composition is cryopreserved for at least about one week.
23. The injectable multi-dose antigen pulsed dendritic cell vaccine of claim 14, wherein said cytokine is IL-12.
24. A method of eliciting an immune response in a mammal, comprising
administering a dose of said cryopreserved injectable multi-dose antigen pulsed dendritic cell vaccine of claim 14 to a mammal in need thereof.
25. A method for treating a subject having, or at risk of developing, a cancer, comprising administering to a subject in need of such treatment a dendritic cell vaccine and an inhibitor of HER-2 in an effective amount to treat the cancer or to reduce the risk of developing the cancer.
26. The method of claim 25, further comprising administering a chemokine modulator to said subject.
27. The method of claim 26, wherein said chemokine modulator is a TLR agonist.
28. The method of claim 26, wherein said chemokine modulator is a TLR8 agonist.
29. The method of claim 25, further comprising administering a cancer medicament in an effective amount to treat said cancer or to reduce the risk of developing said cancer.
30. The method of claim 29, wherein said cancer medicament is selected from the group consisting of surgery, an anti-cancer agent, a chemotherapeutic agent, an immunotherapeutic agent, and a hormone therapy.
31. The method of claim 25, wherein said 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.
32. The method of claim 25, wherein said dendritic cell vaccine comprises an activated dendritic cell that has been contacted with at least one antigen and at least one TLR agonist.
33. The method of claim 25, wherein said dendritic cell vaccine comprises an activated dendritic cell that has been contacted with an agent that elevates the
intracellular calcium concentration in said dendritic cell and an activating agent.
34. The method of claim 33, wherein said agent elevates the intracellular calcium level by blocking the export of calcium out of the cytoplasm.
35. The method of claim 34, wherein said agent comprises a calcium ionophore.
36. The method of claim 35, wherein said calcium ionophore is selected from the group consisting of A23187 and ionomycin.
37. The method of claim 25, wherein said dendritic cell vaccine is in the form of an injectable multi-dose antigen pulsed dendritic cell vaccine.
38. A method of improving the migration and activity of immune cells in a tumor site of a subject, comprising administering to said subject a dendritic cell vaccine and an inhibitor of HER-2 in an effective amount to change the immune response in said tumor so that the immune cells in the tumor site are more effective in attacking tumor cells.
39. The method of claim 38, further comprising administering a chemokine modulator to said subject.
40. The method of claim 39, wherein said chemokine modulator is a TLR agonist.
41. The method of claim 40, wherein said chemokine modulator is a TLR8 agonist.
42. The method of claim 38, further comprising administering to said subject a cancer medicament in an effective amount to treat said cancer or to reduce the risk of developing said cancer.
43. The method of claim 42, wherein said cancer medicament is selected from the group consisting of surgery, an anti-cancer agent, a chemotherapeutic agent, an immunotherapeutic agent, and a hormone therapy.
44. The method of claim 42, wherein said 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.
45. The method of claim 38, wherein said dendritic cell vaccine comprises an activated dendritic cell that has been contacted with at least one antigen and at least one TLR agonist.
46. The method of claim 38, wherein said dendritic cell vaccine comprises an activated dendritic cell that has been contacted with an agent that elevates the
intracellular calcium concentration in said dendritic cell and an activating agent.
47. The method of claim 46, wherein the agent elevates the intracellular calcium level by blocking the export of calcium out of the cytoplasm.
48. The method of claim 47, wherein said agent comprises a calcium ionophore.
49. The method of claim 48, wherein said calcium ionophore is selected from the group consisting of A23187 and ionomycin.
50. The method of claim 38, wherein said dendritic cell vaccine is in the form of an injectable multi-dose antigen pulsed dendritic cell vaccine.
51. The method of claim 38, wherein said dendritic cell vaccine and the inhibitor of HER-2 is administered to said tumor site.
52. The method of claim 39, wherein said dendritic cell vaccine, said inhibitor of HER-2, and said chemokine modulator is administered to said tumor site.
53. A method for treating a subject having, or at risk of developing, a cancer, comprising inhibiting one of more of HER-2 and HER-3 in said subject thereby causing tumor senescence in HER-2 expressing breast cancers.
54. The method of claim 53, wherein inhibiting one or more of HER-2 and HER-3 comprises administering an inhibitor to said subject, wherein said inhibitor is an inhibitor of both HER-2 and HER-3 or a combination of a HER-2 inhibitor and a HER-3 inhibitor.
55. The method of claim 54, wherein said inhibitor is selected from the group consisting of a small interfering RNA (siRNA), a microRNA, an antisense nucleic acid, a ribozyme, an expression vector encoding a transdominant negative mutant, an antibody, a peptide, a chemical compound and a small molecule.
56. The method of claim 53, further comprising administering a dendritic cell vaccine to said subject.
57. The method of claim 53, further comprising administering TNF-a and INF-γ to said subject.
58. The method of claim 56, further comprising administering TNF-a and INF-γ to said subject.
59. A neoadjuvant treatment for a subject having estrogen receptor-positive/HER2- positive ductal carcinoma in situ breast cancer ("ERpoVHER2pos DCIS") comprising, administering at least one dose of an antigen-pulsed DC1 vaccine derived from said subject's monocytic dendritic cell (DC) precursors which are pulsed with six HER2- derived MHC class II binding peptides and said HER2-pulsed DC precursors are matured to type-1 dendritic cells (DC Is) in combination with anti-estrogen therapy.
60. The treatment of claim 59, wherein said six HER2-derived MHC class II binding peptides comprise peptide 42-56: HLDMLRHLYQGCQVV (SEQ ID NO: 1); peptide 98-114: RLRIVRGTQLFEDNYAL (SEQ ID NO: 2); peptide 328-345: TQRCEKCSKPCARVCYGL (SEQ ID NO: 3); peptide 776-790: GVGSPYVSRLLGICL (SEQ ID NO: 4); peptide 927-941 : PAREIPDLLEKGERL (SEQ ID NO: 5); and peptide
1166-1180: TLERPKTLSPGKNGV (SEQ ID NO: 6).
61. The treatment of claim 59 wherein said anti-estrogen therapy comprises administration of an anti-estrogen agent selected from the group consisting of tamoxifen, letrozole, anastrozole, exemestane, raloxifene, and any combination thereof.
62. The treatment of claim 59, wherein if said patient is HLA-A2p0Sltlve the monocytic DC precursors of said patient are pulsed with MHC class I binding peptides comprising peptide 369-377:KIFGSLAFL (SEQ ID NO: 7); and peptide 689- 697:RLLQETELV (SEQ ID NO: 8).
63. The treatment of claim 59, wherein systemic anti-HER2 CD4+ T-cell responses are generated pre- and post- vaccination from said subject's peripheral blood mononuclear cells (PBMC) pulsed with said six HER2-derived MHC class II binding peptides which are then plated and treated to reveal spot formation of spot forming cells (SFCs) and to cause IFN-γ production which is measured.
64. The treatment of claim 62, wherein systemic anti-HER2 CD8+ T-cell responses are generated pre- and post-vaccination from said subject's peripheral blood mononuclear cells (PBMC) pulsed with HER2-derived MHC class II binding peptide 367-377 which are then plated and treated to reveal spot formation of spot forming cells (SFCs) and IFN-γ production which is subsequently measured.
65. The treatment of claim 59, wherein anti-HER2 CD4+ T-cell local regional immune response is measured in the sentinel lymph nodes of said subject post- vaccination.
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