WO2020073044A1 - Polythérapie pour le traitement du cancer - Google Patents

Polythérapie pour le traitement du cancer

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Publication number
WO2020073044A1
WO2020073044A1 PCT/US2019/054989 US2019054989W WO2020073044A1 WO 2020073044 A1 WO2020073044 A1 WO 2020073044A1 US 2019054989 W US2019054989 W US 2019054989W WO 2020073044 A1 WO2020073044 A1 WO 2020073044A1
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WO
WIPO (PCT)
Prior art keywords
cancer
cells
her2
lapatinib
drug
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Application number
PCT/US2019/054989
Other languages
English (en)
Inventor
Brian J. Czerniecki
Gary K. Koski
Lori SHOWALTER
Original Assignee
Czerniecki Brian J
Koski Gary K
Showalter Lori
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Filing date
Publication date
Application filed by Czerniecki Brian J, Koski Gary K, Showalter Lori filed Critical Czerniecki Brian J
Publication of WO2020073044A1 publication Critical patent/WO2020073044A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/217—IFN-gamma
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/812—Breast
    • 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

  • sequence listing filed concurrently herewith comprising eight amino acid sequences (SEQ ID NOS: 1-8) and having the filename“sequence_listing.txt” is incorporated by reference in its entirety.
  • breast cancer is no longer considered a homogeneous disease but rather several subtypes distinguishable by gene expression profiling. Perou, C.M., et al., Nature 406:747-52 (2000). Due to the heterogeneity of breast cancers, lO-year progression free survival can vary widely by stage and type, from 98% to 10%. Different forms of breast cancers can have remarkably different biological characteristics and clinical behavior. Thus, classification of a patient’s breast cancer has become a critical component for determining a treatment regimen. For example, along with classification of histological type and grade, breast cancers are routinely evaluated for expression of hormone receptors (estrogen receptor (ER) and progesterone receptor (PR)) and for expression of HER2 (ERbB2). Therefore, a number of treatment options are currently available that respectively target hormone receptors or the HER2 receptor.
  • hormone receptors estrogen receptor (ER) and progesterone receptor (PR)
  • PR progesterone receptor
  • HER2 account for about 20-25% of breast cancers (Meric, F., et al., J. Am Coll. Surg. 194:488- 501 (2002)), is associated with an aggressive clinical course, resistance to chemotherapy, and a poor overall prognosis in breast cancer. See, Henson, E.S., Clin. Can. Res. 12:845-53 (2006) and Wang, G.S., Mol. Med. Rep. 6(4):779-82 (2012). In incipient breast cancer, HER2
  • HER2 -targeted therapies e.g., trastuzumab
  • a vaccine in which a patient’s dendritic cells are pulsed with at least one antigen and activated to generate polarized type-l DCs is an autologous antigen-pulsed activated DC1 vaccine.
  • the antigen is HER2
  • the vaccine is a HER2-pulsed activated DC1 vaccine (also referred to herein as HER2-pulsed DC1 vaccine).
  • Thl cytokines secreted by Thl cells namely IFN-g and TNF-a
  • IFN-g and TNF-a could mediate in vitro many of the effects observed in vaccinated individuals (for example, apparent tumor cell death and loss of HER2 expression) and could be major contributors to DC1 vaccination effects.
  • SMI small molecule inhibitor
  • the anti-cancer agent lapatinib (TYKERB ® ) is an SMI and receptor tyrosine kinase (RTK) inhibitor (SMIRTK) that directly interferes with kinase activity for both EGFR (HER1) and HER2 signaling, and indirectly reduces HER3 signaling, thereby suppressing important downstream events.
  • SMIRTK SMI and receptor tyrosine kinase inhibitor
  • Lapatinib has found use in breast cancer treatment in combination with chemotherapeutic agents (Geyer, C., et al., N. Eng. JMed. 355(26):2733-43 (2006); Guan, Z., et al. J. Clin. Oncol. 31(16): 1947-53 (2013)) as well as with trastuzumab (Baselga, J., et al. Lancet 379(98l6):633-40 (2012); de Azambuja, E., et al., Lancet Oncol. 15(10): 1137-46 (2014)).
  • SMI lapatinib over the monoclonal antibody trastuzumab is its ability to cross the blood brain barrier to gain access to central nervous system (CNS) metastases of HER2 pos breast cancer (Saleem, A., et al., EJNMMI Res. 5(l):30 (2015)), in addition to lower reported incidence of cardiotoxicity.
  • CNS central nervous system
  • a negative attribute of SMIs is the tendency of cancerous cells to develop resistance after initially being responsive to the drug. Chen, F.L., et al., Clin. Cancer Res.
  • a method for treating cancer comprising, administering to a patient in need thereof a combination of: a targeted inhibitor drug (TID) against the cancer, and an anti-cancer agent either capable of inducing the production of at least one Thl cytokine in the patient or that comprises at least one Thl cytokine or its functional facsimile supplied to the patient.
  • TID targeted inhibitor drug
  • the at least one Thl cytokine comprises at least one of interferon-gamma (IFN-g) and tumor necrosis factor-alpha (TNF-a).
  • IFN-g interferon-gamma
  • TNF-a tumor necrosis factor-alpha
  • the anti-cancer agent is a vaccine or other
  • the anti-cancer agent is a dendritic cell (DC) vaccine.
  • the anti-cancer agent is autologous antigen-specific activated DC vaccine.
  • the cancer expresses a HER family receptor tyrosine kinase (RTK).
  • RTK HER family receptor tyrosine kinase
  • the expressed HER family RTK is selected from the group consisting of HER1 and HER2 or any combination thereof.
  • the targeted inhibitor drug is an inhibitor of receptor tyrosine kinases (IRTK).
  • the IRTK is a small molecule inhibitor of receptor tyrosine kinases (SMIRTK).
  • the IRTK is a large molecule inhibitor of receptor tyrosine kinases (LMIRTK).
  • the SMIRTK is selected from the group consisting of lapatinib, genfitinib, erlotinib, neratinib, vandetanib and osimertinib or any combination thereof.
  • the LMIRTK is selected from the group consisting of trastuzumab, pertuzumab, panitumumab cetuximab and
  • the cancer is selected from the group consisting of: a primary or metastatic melanoma, a thymoma, a lymphoma, a sarcoma, a lung cancer, a liver cancer, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, a leukemia, a uterine cancer, a cervical cancer, a bladder cancer, a gastric cancer, a gastroesophageal junction cancer, a kidney cancer, a breast cancer, a prostate cancer, an ovarian cancer and a pancreatic cancer.
  • the cancer is breast cancer.
  • the cancer is HER2 breast cancer
  • the anti-cancer agent is autologous HER2 -pulsed type-l polarized activated DC vaccine
  • the targeted inhibitor drug is lapatinib.
  • Also provided herein is a method of treating cancer in a patient who has natural or acquired resistance to a drug that the patient has been receiving for treatment of the cancer, comprising the steps of: 1) administering an anti-cancer agent either capable of inducing the production of at least one Thl cytokine in the patient or that comprises at least one Thl cytokine or its functional facsimile supplied to the patient; and 2) subsequently administering an additional therapeutically effective amount of the drug to the patient.
  • the at least one Thl cytokine comprises at least one of interferon-gamma (IFN-g) and tumor necrosis factor-alpha (TNF-a).
  • IFN-g interferon-gamma
  • TNF-a tumor necrosis factor-alpha
  • the anti-cancer agent is a vaccine or other
  • the anti-cancer agent is a DC vaccine.
  • the anti-cancer agent is autologous antigen-specific activated DC vaccine.
  • the cancer expresses a HER family RTK.
  • the expressed HER family RTK is selected from the group consisting of HER1 and HER2 or any combination thereof.
  • the drug is a targeted inhibitor drug.
  • the targeted inhibitor drug is an inhibitor of receptor tyrosine kinases (IRTK).
  • IRTK receptor tyrosine kinases
  • the IRTK is a small molecule inhibitor of receptor tyrosine kinases
  • the SMIRTK is selected from the group consisting of lapatinib, genfitinib, erlotinib, neratinib, vandetanib and osimertinib or any combination thereof.
  • the cancer is selected from the group consisting of: a primary or metastatic melanoma, a thymoma, a lymphoma, a sarcoma, a lung cancer, a liver cancer, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, a leukemia, a uterine cancer, a cervical cancer, a bladder cancer, a gastric cancer, a gastroesophageal junction cancer, a kidney cancer, a breast cancer, a prostate cancer, an ovarian cancer and a pancreatic cancer.
  • the cancer is breast cancer.
  • the anti-cancer agent is autologous HER2-pulsed type-l polarized activated DC vaccine
  • the cancer is HER2 breast cancer
  • the drug is lapatinib.
  • Figure 1 shows lapatinib works in conjunction with Thl cytokines to suppress metabolic activity of breast cancer cell lines.
  • Cultured SK-BR-3 cells (left panel), BT-474 cells (center panel), and MDA-MB-468 cells (right panel) were treated either with Thl cytokines (TNF-a plus IFN-g; 20 and l2.5ng/ml respectively), lapatinib (200nM), Thl cytokines plus lapatinib, or left untreated as control (No Rx).
  • Thl cytokines TNF-a plus IFN-g; 20 and l2.5ng/ml respectively
  • lapatinib 200nM
  • Thl cytokines plus lapatinib or left untreated as control (No Rx).
  • resazurin sodium salt was added to each well and further incubated until color change was noted, and optical densities of supernatants read spectrophotometrically at 630nM.
  • Composite data are shown from at least three trials
  • Figure 2 shows lapatinib works in conjunction with Thl cytokines to increase percentage of non-viable cells.
  • Cultured SK-BR-3 cells (left panel), BT-474 cells (center panel), and MDA-MB-468 cells (right panel) were treated either with Thl cytokines (TNF-a plus IFN-g; 20 andl2.5ng/ml respectively), lapatinib (200nM), Thl cytokines plus lapatinib or left untreated as control (No Rx). After 72 hours, cells were harvested, stained with Trypan Blue dye and percentage of viable and non-viable cells enumerated microscopically with the aid of a hemocytometer.
  • FIG 4 shows lapatinib and Thl cytokines suppress HER-family RTK expression and phosphorylation status.
  • SK-BR-3 cells left panels
  • MDA-MB-468 cells right panels
  • Thl cytokines TNF-a plus IFN-g; 20 and l2.5ng/ml respectively
  • Cytokines 200nM lapatinib
  • Thl cytokines plus Lapatinib Bottom
  • No Rx left untreated as control
  • FIG. 5 shows JIMT-l and MDA-MB-453 cell lines display relative resistance to lapatinib.
  • Cultured SK-BR-3, JIMT-l, MDA-MB-453, and HCC1419 cells were treated with increasing concentrations of lapatinib (0-4mM).
  • resazurin sodium salt Almar Blue dye
  • optical densities % of Control
  • Figures 6A and 6B show lapatinib-resistant lines are re-sensitized to lapatinib in the presence of Thl cytokines.
  • FIG 6A cultured SK-BR-3, HCC1419, JIMT-l, and MDA- MB-453 cells were treated either with Thl cytokines (TNF-a plus IFN-g; 20 and l2.5ng/ml respectively), lapatinib (0-4mM), or lapatinib plus Thl cytokines. After 72 hours incubation, resazurin sodium salt was added to each well and further incubated until color change was noted, and optical densities of supernatants read spectrophotometrically at 630nM.
  • Figure 6B shows statistical analysis at 2pm concentration of lapatinib.
  • Cultured HCC1419 cells left panel
  • JIMT-l cells center panel
  • MDA-MB-453 cells right panel
  • Thl cytokines TNF-a plus IFN-g; 20 and l2.5ng/ml respectively
  • Cytokines Thl cytokines
  • lapatinib 200nM [2 pm]
  • Thl cytokines + lapatinib Cytokines + Lapatinib
  • No Rx No Rx
  • FIG. 7 shows lapatinib works in conjunction with Thl cytokines to increase cell death.
  • Cultured HCC1419 cells left panel
  • JIMT-l cells center panel
  • MDA-MB 453 cells right panel
  • lapatinib (2mM)
  • Lapatinib Thl cytokines
  • TNF-a plus IFN-g 20 and l2.5ng/ml respectively
  • Cytokines lapatinib plus Thl cytokines
  • Cytokines + Lapatinib or left untreated for control (No Rx).
  • Figure 8 shows lapatinib works in conjunction with Thl cytokines to maximize indicators of apoptotic cell death in drug-resistant cell lines.
  • Cultured HCC1419 cells left panel
  • JIMT-l cells center panel
  • MDA-MB 453 cells MDA453
  • Thl Cytokines Thl Cytokines
  • lapatinib (2mM) Lapatinib
  • lapatinib plus Thl cytokines Thl cytokines +
  • embodiment or the like means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment.
  • appearances of the phrases“in one embodiment” or“in an embodiment” or the like in various places throughout this specification are not necessarily all referring to the same embodiment.
  • 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
  • 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 article.
  • “an element” means one element or more than one element.
  • “Adjuvant therapy” for breast cancer as used herein refers to any treatment given after primary therapy (i.e., surgery) to increase the chance of long-term survival. “Neoadjuvant therapy” is treatment given before primary therapy.
  • antigen or“ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that 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 readily 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 or 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” or“APC” is a cell that is capable of activating T cells, and includes, but is not limited to, monocytes/macrophages, B cells and dendritic cells (DCs).
  • “Antigen-pulsed” dendritic cell or DC1 or an“antigen-loaded” or antigen specific dendritic cell or DC1 includes a dendritic cell which has been exposed to an antigen and activated by one or a combination of TLR agonists.
  • a dendritic cell may become Ag-pulsed in vitro , e.g., during culture in the presence of an antigen.
  • a dendritic cell may also be loaded in vivo by exposure to an antigen.
  • An“antigen-pulsed” dendritic cell is traditionally prepared in one of two ways: (1) small peptide fragments, known as antigenic peptides, are “pulsed” directly onto the outside of the dendritic cells; or (2) the dendritic cell is incubated with whole proteins or protein particles which are then ingested by the dendritic cell. These proteins are digested into small peptide fragments by the dendritic cell and are eventually transported to and presented on the dendritic cell surface.
  • an antigen-pulsed dendritic cell can also be generated by introducing a polynucleotide encoding an antigen into the cell.
  • Anti-HER2 response or“anti HER2 Thl response” is the immune response specifically against HER2 protein.
  • anti-tumor effect refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition.
  • An “anti -tumor effect” can also be manifested by the ability of binding peptides, polynucleotides, cells and antibodies in prevention of the occurrence of a tumor in the first place.
  • Apoptosis is the process of programmed cell death. Caspase-3 is a protease frequently activated during apoptotic cell death. Likewise, enhanced staining with Annexin V and propidium iodide (PI) and evidence of mitochondrial membrane depolarization is considered evidence of apoptotic cell death.
  • PI propidium iodide
  • autologous refers to any material derived from the same individual to which it is later to be introduced.
  • Binding peptides are small antigenic peptide fragments which are pulsed directly onto the outside of an APC such as a dendritic cell, or otherwise incubated with an APC which ingests the peptide fragment. The peptide fragment then associates with, or“binds” cellular proteins on the APC known as major histocompatibility complex (MHC) proteins which then remain at the surface of the APC, a situation favorable for the initiation of T cell-dependent immune responses.
  • MHC major histocompatibility complex
  • the antigens may include any antigen suitable for pulsing /loading onto an APC to elicit an immune response. See also,“breast cancer binding peptides” and“HER2 binding peptides.”
  • Breast cancer binding peptides are peptides from any form of breast cancer, such as HER2, HER3, C-MET, and EGFR/HER1. See also,“HER2 binding peptides”).
  • the term“cancer” as used herein is defined as a hyperproliferation of cells whose unique trait— loss of normal control— results in unregulated growth, lack of differentiation, local tissue invasion, and/or metastasis.
  • Examples include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, esophageal cancer, pancreatic cancer, colorectal cancer, gastric cancer, renal cancer, liver cancer, brain cancer, bone cancer, head and neck cancer, lymphoma, leukemia, lung cancer, germ-cell tumors, and the like.
  • CD4 + Thl cells “Thl cells,”“Thl cells,”“CD4 + T-helper type 1 cells,”“CD4 + T cells,” and the like are defined as a subtype of T-helper cells that express the surface protein CD4 and produce high levels of the cytokine IFN-g. See also ,“T-helper cells.”
  • 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.
  • “cryomedium” refers to any medium mixed with a cell sample in preparation for freezing, such that at least some cells of the cell sample can be recovered and remain viable after thawing.
  • DC vaccination refers to a strategy using autologous activated dendritic cells to harness the immune system to recognize specific molecules and mount specific responses against them.
  • the term“dendritic cell” or“DC” is an antigen presenting cell existing in vivo , in vitro , ex vivo , or in a host or subject, or which can be derived from a hematopoietic stem cell or a monocyte.
  • Dendritic cells 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.
  • DCs have a characteristic morphology with thin sheets (lamellipodia) extending in multiple directions away from the dendritic cell body.
  • dendritic cells express high levels of MHC and costimulatory (e.g., CD80 (B7-1) and CD86 (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 Toll-like receptor agonist such as lipopolysaccharide“LPS.” An activated DC may or may not be pulsed with an antigen. See also ,“mature DC.”
  • DC-l polarized dendritic cells “DCls” and“type-l polarized activated DCs” refer to mature activated DCs that secrete Thl -driving cytokines, such as IL-12, IL-18, and IL- 23. DCls are fully capable of promoting cell-mediated immunity.
  • HER2 + breast cancer an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result such as, for example, an amount sufficient to effect treatment, prognosis or diagnosis of a HER2 + breast cancer, when administered to a subject.
  • a therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the patient, the severity of the disease condition, the manner of administration and the like, which can be readily determined by one of ordinary skill in the art.
  • Estrogen receptor (“ER”) positive or“ER pos cancer is cancer which tests positive for expression of estrogen. Conversely,“ER negative” cancer tests negative for such expression. Analysis of ER status can be performed by any method known in the art.
  • HER2 is a member of the human epidermal growth factor receptor (“EGFR”) family which includes HER1, HER2, HER3, and HER4. HER2 is overexpressed in EGFR
  • HER2 binding peptides refer to MHC Class II peptides derived from or based on the sequence of the HER2/neu protein, a target found on approximately 20- 25% of all human breast cancers and their equivalents.
  • HER2 extracellular domain“ECD” 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/neu protein within the cytoplasm of a cell.
  • HER2 epitopes or otherwise binding peptides comprise at least one of 6 HER2 binding peptides which include 3 HER2 ECD peptides and 3 HER2 ICD peptides.
  • Preferred HER2 ECD peptides comprise:
  • Peptide 42-56 HLDMLRHLYQGCQVV (SEQ ID NO: 1);
  • Peptide 98-114 RLRIVRGT QLFEDN Y AL (SEQ ID NO: 2); and Peptide 328-345: TQRCEKCSKPCARVCYGL (SEQ ID NO: 3);
  • Preferred HER2 ICD peptides comprise:
  • 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).
  • HLA-A2pos HLA A2.1 positive blood type
  • HER2 MHC class I peptides or epitopes further comprise at least one of
  • Peptide 369-377 KIFGSLAFL (SEQ ID NO: 7); and Peptide 689-697: RLLQETELV (SEQ ID NO: 8).
  • HER2 pos and“HER2 + ” denote 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 by FISH (fluorescent in situ hybridization) assay and 2+ or 3+ on intensity of immunohistochemical staining.
  • FISH fluorescent in situ hybridization
  • HER2 nes is defined by the lack of gene amplification by FISH, and can encompass a range of immunohistochemical staining from 0 to 2+ in most cases.
  • Immunotherapy is any substance or biological agent such as cells, proteins, viruses, genetic materials synthetic materials, or other constructs capable of stimulating or suppressing immune system function for the purpose of correcting or ameliorating a
  • IRTKs inhibitor of receptor tyrosine kinases
  • the IRTK may for example be a small molecule such as a synthetic drug, or a high molecular weight biological agent, such as a monoclonal antibody.
  • IRTKs are not limited by size, since substantial portions of the RTK target protrude from the membrane of the cell. Therefore, inhibitor molecules need not gain access to the interior of the cell, and small size is not a requirement for interaction with the RTK target.
  • LMTRTK Large molecule inhibitors of receptor tyrosine kinases
  • LMTRTK is defined as large molecules, typically proteins which are most often monoclonal antibody -based but in theory could be any pseudo-ligand that interferes with the signaling process of the target by binding with its extracellular domain.
  • LMIRTKs are typically too large to traverse cell membranes and must exert their inhibitory activity through interaction with the extracellular domain of the receptor tyrosine kinases (RTKs). Examples include monoclonal antibodies such as trastuzumab/HERCEPTIN® (anti-HER2) and cetuximab (anti-EGFR/HERl).
  • LMINKs target transmembrane proteins that are not themselves RTKs or other kinases, but nonetheless regulate cellular process through transmembrane signaling.
  • LMINKs could for example modulate-membrane channels or could bind to target transmembrane proteins through their extracellular domains, thereby affecting the activity of cytoplasmic domains of the LMINK target. Such induced changes in the cytoplasmic domain will affect whether the LMINK target can interact with other regulatory proteins (which may themselves be kinases), positively or negatively affecting their activity.
  • LMINKs are large molecules, typically monoclonal antibodies, but could also be any
  • checkpoint inhibitors like pembrolizumab (KEYTRUDA®), niviolumab (OPDIVO®) and antibodies against Semaphorin 4D.
  • LPS lipopolysaccharide, a bacterial cell wall component recognized by dendritic cells through Toll-like receptor 4.
  • MHC major histocompatibility complex
  • Class I MHC, or MHC class I function mainly in antigen presentation to CD8 T lymphocytes (killer T cells).
  • Class II MHC, or MHC class II function mainly in antigen presentation to CD4 T lymphocytes (helper T cells).
  • “Mature DC” as used herein means a dendritic cell that expresses molecules, including high levels of MHC class II, CD80 (B7.1) and CD86 (B7.2) molecules.
  • immature DCs iDCs or“IDCs”
  • iDCs immature DCs
  • IDCs immature DCs
  • “Mature DC” also refers to an antigen presenting cell existing in vivo , in vitro , ex vivo , or in a host or subject that may also be DC1- polarized (i.e., fully capable of promoting cell-mediated immunity.)
  • Receptor tyrosine kinases or“RTKs” are a class of membrane-spanning proteins that consist of an extracellular domain, a short transmembrane domain and a
  • RTKs transfer a phosphate group from adenosine triphosphate (ATP) to a tyrosine residue on a substrate protein. The attachment of the phosphate group either increases or decreases the biological activity of the target protein. RTKs therefore are capable of controlling important aspects of intracellular signaling that regulate cell growth, division and death.
  • ATP adenosine triphosphate
  • sample or“biological sample” as used herein means a biological material from a subject, including but is not limited to blood, organ, tissue, exosome, plasma, saliva, urine and other body fluid.
  • a sample can be any source of material obtained from a subject.
  • A“small molecule inhibitor” or“SMI” is a targeted inhibitor drug (“TID”) that has a relatively low molecular weight that allows it to cross membranes to achieve its inhibitory function, although it is possible that a SMI can act on the extracellular domain of a target molecule such that the SMI need not enter the cell.
  • A“small molecule inhibitor of receptor tyrosine kinases” or“SMIRTK” is a SMI whose activity is directed against receptor tyrosine kinases (RTKs). While RTKs are capable of controlling important aspects of intracellular signaling that regulate cell growth, division and death, SMIRTKs allow these processes to be efficiently interfered with. Examples of SMIRTKs include lapatinib.
  • A“small molecule inhibitor of other kinases” or“SMIOK” is a SMI whose activity is directed against other kinases, such as, for example, those kinases that are neither transmembrane receptors, nor kinases that attach phosphate groups to tyrosine residues on their targets.
  • Such other kinases may be free in the cytoplasm, or at least not bound directly to membranes through proteinaceous membrane spanning domains. They also phosphorylate serine and threonine residues on their targets instead of tyrosine.
  • These kinases like RTKs can control important cellular events like cell growth, division and cell death. Consequently, SMIOKs can interfere with these processes. Examples of SMIOKs include MK2206 (AKT/protein kinase B inhibitor) and AZD1206 (Pirn kinase inhibitor).
  • Small molecule inhibitors of non-kinases are molecules that participate in the regulation of important cellular processes like cell growth, proliferation and death, but which do not possess known kinase activity. Instead, cellular proteins participating in the regulation of these processes may be, for example, membrane channels, proteases, phosphatases or proteins that bind to other target proteins (including kinases), thereby affecting their activity. SMINKs can interfere with these proteins, preventing their activity. Examples of SMINKs include gamma-secretase inhibitor PF-03084014 and Kras antagonist statin drugs (e.g. simvastatin).
  • 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.
  • the patient, subject or individual is a human, and preferably a female in the instance of breast cancer treatment.
  • synergistic or“synergy” as used herein, is known by those skilled in the art as referring to the joint action of agents, for example, drugs in a composition, that, taken together, produce a superior effect than the sum of their individual effects. That is, the combination of two or more active ingredients in a composition which is referred to as synergistic exerts a better effect than the sum of the separate effects of those two or more active ingredients individually.
  • TERT Tumorted inhibitor drug
  • TKI small molecule inhibitors
  • LMI large molecule inhibitors
  • IRTKs inhibitors of receptor tyrosine kinases
  • targeted therapies refers to cancer treatments that use drugs or other substances that interfere with specific target molecules involved in cancer cell growth usually while doing little damage to normal cells to achieve an anti-tumor effect.
  • T-cell or“T cell” as used herein are defined as a thymus-derived cell that participates in a variety of cell-mediated immune reactions.
  • 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 cell or leukocyte) including different cell types identifiable by those skilled in the art.
  • T- helper cells are effector T cells whose primary function is to promote the activation and functions of other B and T lymphocytes and/or macrophages.
  • Helper T cells differentiate into two or more major subtypes of cells including at least“Thl” or“Type 1” and“Th2” or“Type 2” phenotypes.
  • Th cells secrete cytokines, proteins, or peptides that stimulate or interact with other leukocytes.
  • “Thl cell,”“CD4 + Thl cell,”“CD4 + T-helper typel cell,”“CD4 + T cell” and the like as used herein refer to a mature T-cell that has expressed the surface glycoprotein CD4.
  • CD4 + T-helper cells become activated when they are presented with peptide antigens by MHC class II molecules which are expressed on the surface of antigen-presenting peptides (“APCs”) such as dendritic cells.
  • APCs antigen-presenting peptides
  • cytokines such as interferon-g (IFN-g) and TNF-a.
  • IFN-g interferon-g
  • TNF-a TNF-a
  • Thl cytokines or“principal Thl cytokines” refer to at least IFN-g and TNF-a which are soluble factors and cytokine effectors of vaccine-induced immunity, produced by Thl cells which result from a response to autologous antigen-specific activated DC1 vaccine such as HER2-pulsed DC1 vaccine.
  • a compound, formulation, material, or composition as described herein, that when administered to a patient, is effective to achieve a particular biological result.
  • the amount of a compound, formulation, material, or composition described herein, which constitutes a“therapeutically effective amount” will vary depending on the compound, formulation, material, or composition, the disease state and its severity, the age of the patient to be treated, and the like.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.
  • the therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to his/her own knowledge and to this disclosure.
  • TLR Toll like receptor
  • the terms“treat,”“treating,” and“treatment,” refer to therapeutic or preventative measures such as those described herein.
  • the methods of“treatment” employ administration to a subject, in need of such treatment, a composition or method of the present embodiments, for example, a subject afflicted with a disease or disorder such as HER2 + breast cancer, or a subject who ultimately may acquire such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • a disease or disorder such as HER2 + breast cancer
  • the term“vaccine” as used herein is defined as a material used to provoke an immune response after administration of the material to an animal, preferably a mammal, and more preferably a human.
  • the vaccine 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.
  • the vaccine can be autologous antigen- specific activated dendritic cell vaccine that is type 1 polarized (DC1 vaccine/immunotherapy).
  • the dendritic cells are pulsed with antigenic HER2 peptides and is referred to herein as HER2-pulsed DC1 vaccine or HER2-pulsed activated DC1 vaccine and the like.
  • dendritic cells can be pulsed with other antigenic peptides against other cancers desired to be targeted.
  • Ranges throughout this disclosure, various aspects of the embodiments can be presented in a range format. It should be understood that the description in 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 specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 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 provide methods of enhancing the anti-cancer effects of targeted inhibitor drugs (TIDs) used for treatment of cancer in general, and breast cancer in preferred embodiments, by combining such drugs with an anti-cancer agent.
  • TIDs targeted inhibitor drugs
  • the anti-cancer agent is capable of inducing the production of at least one Thl cytokine in a patient, or comprises at least one Thl cytokine or Thl cytokine receptor agonist or their functional facsimiles that are supplied to a patient in such a way that it mimics the biological effects of therapeutic
  • the cytokine effectors of vaccine-induced immunity are at least the soluble factors IFN-g and TNF-a produced by Thl cells.
  • the anti-cancer agent can be an autologous antigen-specific activated dendritic cell vaccine that is type 1 polarized (DC1 vaccine) that changes the immune response to a patient’s tumor.
  • DC1 vaccine type 1 polarized
  • the present embodiments also include other means of supplying at least one Thl cytokine to the patient’s area of disease (tumors) including administration of at least one recombinant Thl cytokine systemically in soluble form or in any other manner that allows therapeutic concentrations of cytokine to traffic to or sequester in the tumor environment.
  • the embodiments also include use of cytokine analogs that may bear no structural similarity to the natural counterparts but could nonetheless act as Thl cytokine receptor agonists, or agonists to downstream components of Thl cytokine signaling pathways, however these agents are delivered.
  • DC1 vaccines generate strong Thl-dominated immunity against targeted antigen. It is believed that Thl cytokines, namely at least IFN-g and TNF-a, secreted by Thl cells are major contributors to the vaccine’s effects, namely at least, the immune cells in the tumor site are more effective in attacking and even killing the tumor cells as well as suppressing HER expression. This therapy may also include improving the migration and activity of immune cells in the tumor site.
  • the present embodiments demonstrate the potency of multiplexed Thl cytokines and offer a consistent explanation of how CD4 + Th cells which cannot recognize tumor cells directly, may nonetheless play a decisive role in their elimination.
  • the targeted inhibitor drug used in the herein embodiments preferably a small molecule inhibitor drug of low toxicity, which is also a small molecule inhibitor of receptor tyrosine kinases and is biochemically targeted to the tumor.
  • a small molecule inhibitor drug of low toxicity which is also a small molecule inhibitor of receptor tyrosine kinases and is biochemically targeted to the tumor.
  • Thl cytokines may exist in addition to IFN-g and TNF-a that contribute to Thl immunity and the present embodiments include any and all such cytokines.
  • the present treatment methods are combination therapies that also use an effective amount of at least one additional anti-cancer agent capable of inducing or otherwise supplying at least one Thl cytokine in a patient which in preferred embodiments is in the form of a vaccine targeted to the cancer and capable of inducing at least one Thl cytokine in the patient.
  • the cancer treated is breast cancer, and more preferably the breast cancer is one that expresses HER family RTKs such as HER1 and HER2.
  • the targeted inhibitor drug is a small molecule inhibitor of receptor tyrosine kinases (SMIRTK) such as lapatinib which inhibits one or more of HER2 and HER1 and indirectly reduces HER3 signaling.
  • the cancer vaccine is autologous HER2-pulsed DC1 vaccine.
  • the present embodiments provide a system of cancer treatment whereby the vaccine-induced or otherwise supplied Thl response can be considered to be a physically-directed delivery system for one set of anti-tumor agents (at least one Thl cytokine, through T cells) to the site of disease to attack the tumor cells while a low toxicity, biochemically targeted drug also attacks the tumor.
  • the resulting synergy between the vaccine or other anti-cancer agent and targeted inhibitor drug enhances tumor kill while limiting toxic side effects to healthy tissue.
  • cytokines to sensitize tumor cells at the site of disease, rendering them more susceptible to conventional, non-targeted chemotherapy agents.
  • drug doses administered to a patient could be lessened to reduce toxic and physical side effects while achieving high killing of cells at the site of disease, since disease sites are affected by the at least one cytokine as well as the non-targeted drug.
  • the danger of drug resistance is concomitantly lessened.
  • the methods and compositions of the present embodiments are based, at least in part, on the discovery that one or more Thl cytokines that are induced by dendritic cell-based vaccine against early breast cancer (such as, for example, autologous HER2-pulsed DC1 vaccine) are able to sensitize vaccinated individuals against HER2.
  • a DC1 vaccine could be formulated against additional targets such as HER1, HER3, CMET or other targets.
  • a suitable targeted inhibitor drug to use in the combination therapies of the present embodiments.
  • the present embodiments can provide methods and compositions of using targeted drug therapy combined with vaccination or other immunotherapy as a treatment regimen to enhance anti-cancer effects.
  • the embodiments herein are not limited to methods for treatment of breast cancers, and include use of any antigen suitable for pulsing onto dendritic cells to elicit an immune response or used in other types of vaccines/immunotherapy.
  • Autologous antigen-pulsed activated DC1 vaccines can use peptides or the like derived from the tumor or cancer cells to be treated.
  • tumor antigens both shared tumor antigens (antigens expressed by many tumors) and those unique to individual tumors (unique tumor antigens that can result from mutations induced through physical or chemical carcinogens and are therefore expressed only by individual tumors) can be used to pulse autologous dendritic cells for a DC1 vaccine.
  • shared tumor antigens antigens expressed by many tumors
  • those unique to individual tumors unique tumor antigens that can result from mutations induced through physical or chemical carcinogens and are therefore expressed only by individual tumors
  • the choice of targeted small molecule inhibitors for various types of cancers would likewise be known to those skilled in the art
  • Vaccines for use in accordance with the embodiments herein can be derived from the tumor or cancer cells to be targeted for treatment.
  • the breast cancer cells can be treated as described at least in US 2013/0183343 to produce a breast cancer-directed DC 1 vaccine.
  • other cancers that can be suitably treated in accordance with the practices of the present embodiments include, without limitation, cancers of the lung, ovary, cervix, colon, head and neck, pancreas, prostate, stomach, bladder, kidney, bone, skin, liver, esophagus, brain, testicle, uterus, germ cell tumors, and the various leukemia's and lymphomas.
  • the administration of a cancer vaccine in accordance with the present combination therapy embodiments is broadly applicable to the prevention or treatment of cancer, determined in part by the selection of antigens for the cancer vaccine and the choice of targeted inhibitor drug.
  • the present embodiments also provide a HER2 breast cancer combination therapy that can use therapeutic agents that are capable of crossing the blood brain barrier of the patient.
  • Brain metastases afflict approximately 37% of patients with HER2 pos breast cancer.
  • Commonly used HER2 therapies entail use of monoclonal antibodies such as trastuzumab (HERCEPTIN®) and pertuzumab (PERJETA®) which are not able to pass an intact blood brain barrier leaving the brain metastases untreated. Therefore, in the present combination therapy embodiments, the use of a small molecule inhibitor such as lapatinib that can cross the blood brain barrier provides an avenue for treatment of brain metastases.
  • Efforts to further improve DC1 vaccine efficacy will entail identification of additional targeted drugs that will work synergistically, or at least additively, with Thl cytokines to enhance apoptosis in breast cancer cell lines. Such drugs could then be rapidly translated into clinical trials to test for their ability to enhance vaccine effects.
  • cellular kinases and their substrates which are often dysregulated in cancer, which accounts for the malignant cell’s uncontrolled growth and proliferation.
  • Small molecule inhibitors of receptor tyrosine kinases (SMIRTKs) have a theoretical capacity to interfere with dysregulated and over- active processes in cancerous cells and may have anti-cancer effects by themselves or in conjunction with vaccines or other immunotherapy.
  • SMIRTKs are usually produced by design to inhibit a rationally-determined and narrow range of kinases, they are considered a form of targeted therapy.
  • the HER family of receptor tyrosine kinases has been linked to deregulation of growth and proliferation for multiple types of cancer. Members of this family have therefore become the focus of many drug and immune- based therapy innovations.
  • the targeted anti-cancer agent, lapatinib is a small molecule inhibitor that directly interferes with EGFR (HER1) and HER2 signaling and indirectly reduces HER3 signaling, thereby suppressing important downstream events.
  • DC1 vaccines are used herein as a means to take advantage of the activity of anti-cancer drugs.
  • cytokine effectors of vaccine-induced immunity namely at least one of IFN-g and TNF-a
  • cytokine effectors of vaccine-induced immunity appears to reverse resistance to anti-cancer targeted inhibitor drugs such as small molecule inhibitors of RTKs in drug resistant lines.
  • HER2 -pulsed DC1 vaccine can be administered to a breast cancer patient who has become resistant to the targeted inhibitor drug lapatinib and can thereafter be treated with additional lapatinib resulting in a degree of restored drug sensitivity, which would contribute to increased tumor destruction.
  • it is believed the presence of at least one Thl cytokine re-sensitizes lapatinib-resistant cells to the drug.
  • the herein methods for treating cancer patients that have resistance, either natural or acquired is a further demonstration that robust immunity in the form of at least one secreted Thl cytokine can be taken advantage of to mitigate or reverse targeted inhibitor drug resistance. While the preferred embodiments herein provide for cytokine-mediated reversal or mitigation of resistance to targeted drugs in treatment of HER2+ breast cancer, the present embodiments are not limited to cancerous cells that develop therapeutic or acquired resistance after initially being responsive to an anti-cancer drug, but also include instances where cancer cells exhibit natural drug resistance.
  • the methods of treating cancer in a patient who has natural or acquired resistance to a drug that the patient has been receiving for treatment of the cancer comprise the steps of administering an anti-cancer agent either capable of inducing the production of at least one Thl cytokine in the patient or that comprises at least one Thl cytokine or its functional facsimile supplied to the patient; and subsequently an additional therapeutically effective amount of the drug is administered to the patient.
  • the at least one Thl cytokine comprises at least one of IFN-g and TNF-a.
  • the embodiments also include use of cytokine analogs that may bear no structural similarity to the natural counterparts but could nonetheless act as Thl cytokine receptor agonists, or agonists to downstream components of Thl cytokine signaling pathways, however these agents are delivered.
  • the anti-cancer agent can be a vaccine or other immunotherapy capable of inducing the production of the at least one Thl cytokine in the patient.
  • the anti-cancer agent is a DC vaccine, more preferably an autologous antigen-specific activated DC vaccine, and even more preferably an autologous antigen-specific type 1 -polarized activated DC vaccine.
  • the cancer expresses a HER family RTK which comprises at least one of HER1 and HER2.
  • the targeted inhibitor drug is an inhibitor of receptor tyrosine kinases and preferably the drug is selected from the group consisting of lapatinib, genfitinib, erlotinib, neratinib, vandetanib and osimertinib or any combination thereof. In particularly preferred embodiments the drug is lapatinib.
  • Exemplary cancers include, but are not limited to, a primary or metastatic melanoma, a thymoma, a lymphoma, a sarcoma, a lung cancer, a liver cancer, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, a leukemia, a uterine cancer, a cervical cancer, a bladder cancer, a gastric cancer, a gastroesophageal junction cancer, a kidney cancer, a breast cancer, a prostate cancer, an ovarian cancer and a pancreatic cancer.
  • the cancer being treated is a breast cancer, and preferably HER2 pos breast cancer.
  • the anti-cancer agent is autologous HER2 -pulsed DC1 vaccine
  • the cancer is HER2 breast cancer
  • the drug is lapatinib.
  • the HER2 -pulsed DC1 vaccine physical, chemical and pharmaceutical properties and formulation are as follows.
  • the product is preferably supplied as cryopreserved cell therapy.
  • the product comprises autologous activated type I-polarized dendritic cells (DCs) pulsed with at least one of an immunogenic MHC class II peptide derived from HER2 neu protein (SEQ ID NOS: 1-6) and optionally additionally pulsed with at least one of an immunogenic MHC class I peptide derived from HER2 neu protein in the case where donors have HLA A2.1 blood type (SEQ ID NOS: 7-8).
  • DCs autologous activated type I-polarized dendritic cells
  • the peptide-pulsed DC is activated with IFN-g and at least one TLR agonist, such as, for example, LPS, a TLR 7/8/agonist, and resimiquod (R848). About 6-8 hours after the LPS or other TLR agonist addition the resulting autologous antigen specific activated DC is cryopreserved in a medium comprising about 5-10% DMSO (v/v). Upon thawing, the DC produces an effective amount of IL-12 which is principally involved in the development and recruitment of IEN-g-secreting Thl cells.
  • TLR agonist such as, for example, LPS, a TLR 7/8/agonist, and resimiquod (R848).
  • TLR agonist such as, for example, LPS, a TLR 7/8/agonist, and resimiquod (R848).
  • TLR agonist such as, for example, LPS, a TLR 7/8/agonist, and resimiquod (R848).
  • Each vial of vaccine contains between about 10-20 million viable activated HER2-pulsed DC1 cells in 1 ml volume that are activated to secreted large quantities of cytokines such as, but not limited to, IL-12, IL-6, and IL-23 and chemokines such as, but not limited to, IP 10 (CXCL10), MIG (CXCL 9), and RANTES (CCL5).
  • cytokines such as, but not limited to, IL-12, IL-6, and IL-23
  • chemokines such as, but not limited to, IP 10 (CXCL10), MIG (CXCL 9), and RANTES (CCL5).
  • the HER2-pulsed DC1 cells express varying amounts of costimulatory molecules, such as, but not limited to CD80, CD86, CCR7, CD83 on the cell surface. Cytokine and chemokine production have been shown to be maintained for up to 24-36- hours post-thaw.
  • HER2-pulsed DC1 vaccine is prepared as described previously. See , Koski, et al., Sharma, A., et al., Fracol, M., et al., Ann. Surg. Oncol. 20(l0):3233 (2013); Lee, M. K. 4th, et al., Expert Rev. 8(1 l):e 74698 (2013); Czerniecki, et al.; Czemiecki, B. T, et al, Cancer Res. 67(l4):653 l (2007); and US 2013/0183343 Al.
  • monocytic DC precursors CD14+ peripheral blood monocytes
  • the monocytes are cultured in an incubator (37°C in 5% C0 2 ) using 6- well tissue culture plates (12c10 6 cells) in macrophage serum-free medium (“SFM”)
  • GM-CSF granulocyte macrophage colony stimulating factor
  • IL-4 IL-4
  • the non-activated DCs are then pulsed separately with each of six antigenic HER2 MHC class II binding peptides derived from or based on the sequence of the HER2/neu protein (peptide 42-56: HLDMLRHL Y QGCQ VV (SEQ ID NO: 1); peptide :98-l 14
  • T QRCEKC SKPC ARV C Y GL (SEQ ID NO: 3) (extracellular domain of HER2), and peptide 776-790: GV GSP YV SRLLGICL (SEQ ID NO: 4); peptide 927-941 : PAREIPDLLEKGERL (SEQ ID NO: 5); and peptide 1166-1180: TLERPKTLSPGKNGV (SEQ ID NO:6) (intracellular domain of HER2)) See, Disis, M. L., et al., Clin. Can. Res.
  • DCls are pulsed with two additional MHC class I binding peptides, peptide 369-377: KIFGSLAFL (SEQ ID NO: 7) and peptide 689-697: KIFGSLAFL (SEQ ID NO: 8).
  • Harvested cells are washed and lot release criteria of >70% viability, negative Gram stain, and endotoxin ⁇ 5 EU/kg are confirmed.
  • a TLR 2 agonist and other TLR agonists selected from among a TLR3 agonist, a TLR4 agonist, and a TLR8 agonist can be used to generate autologous antigen-specific activated DC.
  • the HER2-pulsed DC1 cells are then preferably cryopreserved within 6-8 hours after exposure to LPS (or other TLR agonist) as stated above in about 5-10% DMSO (v/v) in order to maximize IL-12 production upon thawing, prior to use in a patient.
  • LPS or other TLR agonist
  • Human serum and other serum types such as human AB serum and fetal calf serum, and also human albumin may be used in the cryopreservation medium.
  • the cryomedium may include about 20% serum and about 10% DMSO (v/v), and suspended cells can be stored at about - l80°C.
  • Still further embodiments may include medium containing about 55% oxypolygelatine, a plasma expander, about 6% hydroxy ethyl starch, and about 5% DMSO (v/v)
  • the cryomedia comprises about 57% PlasmaLyteA, about 38% human albumin, and about 5% DMSO (v/v).
  • the suspended cells can be aliquoted into smaller samples, such as in 1.8 ml vials, and stored at approximately -70°C or lower.
  • DMSO may be entirely absent in some embodiments, generally however, lower concentrations of DMSO are preferred, such as between about 5% to about 10% and most preferably below 7.5%, the FDA allowable limit for DMSO, and as stated above about 5% DMSO is particularly preferred.
  • any concentration of DMSO that results, after thawing, in cell viability of at least 50% and a cell recovery of at least 50%, and preferably a cell viability 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.
  • Intra-nodal and/or intra-lesion vaccine injection is performed as described by Koski, et al. Injections comprise 10-20 million HER2-pulsed DCls suspended in lml sterile saline, and administered by ultrasound guidance into groin lymph nodes, breast, or both.
  • Immunizations are preferably administered once weekly for 6 weeks or for other administration schedules as would be known to one of skill in the art, in light of the present disclosure.
  • tumor antigens that can be used in the manufacture of DC1 vaccines refers to antigens that are common to specific hyperproliferative disorders.
  • 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, Hodgkin's lymphoma, leukemia's, uterine cancer, cervical cancer, bladder cancer, esophagael cancer, gastric cancer, bone cancer, head and neck 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 also serve as target antigens/peptides for an immune attack. These molecules include, but are not limited to, tissue- specific antigens such as MART-l, 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 HER- 2/Neu/ErbB-2 as described herein above and HER-3/Erb3. Yet another group of target antigens are onco-fetal antigens, such as carcinoembryonic antigen (CEA).
  • CEA carcinoembryonic antigen
  • 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, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.
  • the particular targeted inhibitor drug used in the present embodiments is not limited to small molecule inhibitor of RTKs (SMIRTKs) such as lapatinib and includes drugs of the broader class of inhibitor of receptor tyrosine kinases (IRTKs).
  • An IRTK may be a small molecule such as a synthetic drug (e.g., lapatinib) or a high molecular weight biological agent, such as a monoclonal antibody (e.g., trastuzumab).
  • IRTKs are not limited by size because substantial portions of the RTK target protein protrude from the membrane of the cell.
  • IRTKs which are believed to have effects similar to those of lapatinib in that they also suppress at least one of HER1 and HER2 signaling, include large molecule inhibitors of receptor tyrosine kinases (LMIRTKs) such as monoclonal antibody-based drugs trastuzumab, pertuzumab, panitumumab cetuximab and necitumumab as well as other small molecule inhibitors of receptor tyrosine kinases (SMIRTKs) including but not limited to genfitinib, erlotinib, neratinib, vandetanib and osimertinib.
  • LMIRTKs large molecule inhibitors of receptor tyrosine kinases
  • SMIRTKs small molecule inhibitors of receptor tyrosine kinases
  • HER1 and HER2 inhibition can be used in the methods of the present embodiments, including peptide and nucleic acid aptamers and agents that interfere with the expression of these oncotargets including but not limited to small interfering RNA (siRNA) approaches, and antisense oligonucleotides.
  • siRNA small interfering RNA
  • the targeted inhibitor drug lapatinib effective dose ranges when combined with DC1 vaccination are between 50 mg/day minimum and 2,000 mg/day maximum.
  • a therapeutically effective amount will vary depending upon the patient and the disease condition being treated, the weight and age of the patient, the severity of the disease condition, the manner of administration and the like which can readily be determined by one of ordinary skill in the art.
  • lapatinib The preferred route of administration of lapatinib is oral.
  • transdermal, intravenous, intramuscular, intradermal, subcutaneous and intravenous routes are possible and suitable for effective delivery of the drug to the patient as can be determined by one skilled in the art.
  • Lapatinib supplied first For example, a patient with DCIS can be provided lapatinib drug once daily for 4 weeks. Drug therapy ends with the administration of the first of 6 weekly DC vaccines. After the course of 6 weekly vaccines, any remaining disease is removed surgically. [00132] 3) Vaccine supplied first. A patient with DCIS is supplied with 6 weekly vaccinations prior to scheduled surgical resection of disease. Immediately following the last immunization, a 4-week course of daily lapatinib is supplied ending shortly before surgery.
  • HER2-pulsed DC1 vaccine plus lapatinib could also be supplied after surgical resection of the primary tumor.
  • the timing of vaccine and lapatinib administration could be any combination of the 3 schedules described above, with provision of vaccine and lapatinib partially overlapping.
  • Lapatinib is cell membrane permeable so it enters the cell and binds to the ATP binding cassette of HER2 and EGFR thereby preventing phosphorylation of the receptor tyrosine kinase portion of the proteins themselves which in turn prevents cross phosphorylation with dimerization partners.
  • trastuzumab being a monoclonal antibody, is not cell permeable and there for can only bind to the extracellular domain of HER2 (not EGFR). It has been suggested that this blocks binding with dimerization partners and prevents extracellular cleavage by
  • lapatinib is orally active whereas trastuzumab must be given intraveneously.
  • trastuzumab In a clinical trial lapatinb in combination with trastuzumab showed
  • Lapatinib is capable of crossing the blood-brain barrier making it an effective treatment for patients with brain metastases whereas a monoclonal antibody such as trastuzumab cannot cross the blood-brain barrier.
  • a monoclonal antibody such as trastuzumab cannot cross the blood-brain barrier.
  • Lapatinib is capable of targeting CD44+/CD24- cancer stem cells by inhibiting mammosphere-formation. Gong, C., et al., PLoS One 5(l2):el5630 (2010)
  • Drug resistance is one of the major problems of cancer therapy, and is responsible for the failure of most anti-cancer drug regimens.
  • Malignant tumors are known for their heterogeneity. Because the cells are inherently unstable, they are constantly changing, and any given tumor is composed of malignant cells exhibiting a number of different phenotypes. This provides ample variability for the selection of drug resistance.
  • the tumor microenvironment is composed not only of the malignant cells, but also of untransformed stromal cells, infiltrating immune system cells, extracellular matrix, and the complex intra- and intercellular signaling networks provided by cytokines and other soluble factors (e.g. IL-6, VEGF, NO) as well as cell-cell interactions (adhesion and other membrane- bound molecules linking tumors to each other, stromal cells, etc.).
  • cytokines and other soluble factors e.g. IL-6, VEGF, NO
  • Tumors may come to express large amounts of activated enzymes that may act upon drug targets, converting them into compounds of reduced or absent toxicity to the cell. These are often specific to a drug or class of drugs.
  • Multi-drug resistance A class of so-called ABC transporters are tasked with moving various nutrients across cell membranes. They are also often involved transporting a wide range of anti-cancer drugs from the cancer cell’s cytoplasm (where they exert their anti tumor effect) and out into the extracellular environment where they may become harmless to the malignant cell. Enhanced expression or activity of these ABC transporters can render cells resistant to multiple and broad classes of anti-cancer drugs.
  • - Drug targets can acquire mutations such that the drug can no longer bind.
  • Cancer cells can also greatly over-produce the drug target (e.g. through gene amplification or other regulatory mechanism) so that the concentration of the drug inside the cell is not enough to deal with the amplified target. This mechanism is often selective to a class of drugs that binds to a particular receptor or receptor domain.
  • the HER family of receptor tyrosine kinases has been linked to deregulation of growth and proliferation for multiple types of cancer. Members of this family have therefore become the focus of many drug and immune-based therapy innovations.
  • the targeted inhibitor drug lapatinib is a small molecule inhibitor that directly interferes with EGFR (HER1) and HER2 signaling, and indirectly reduces HER3 signaling.
  • HER1 EGFR
  • HER2 signaling HER2 signaling
  • a recently-developed dendritic cell-based vaccine against early HER2 breast cancer (ductal carcinoma in situ; DCIS) that generates strong Thl-dominated immunity against HER2, i.e., HER2-pulsed DC1 vaccine has induced pathologic complete response in about one-third of immunized individuals. See for example.
  • Thl cytokines secreted by Thl cells could be major contributors to the vaccine’s effects including induction of apoptosis and suppression of HER expression.
  • Thl cytokines IFN-g and TNF-a
  • lapatinib which is a small molecule RTK highly selective for some HER family members to suppress activity of breast cancer lines in vitro.
  • lapatinib-sensitive SKBR3, MDA-MB-468 and BT474 cells were incubated with the Thl cytokines IFN-g and TNF-a, lapatinib, or both. It was found that combined treatment maximized metabolic suppression (Alamar Blue assay), as well as cell death (Trypan Blue) and apoptosis (Annexin V/Propidium Iodide and TMRE staining).
  • the embodiments herein provide methods for combining targeted drug therapy, in preferred embodiments using small molecule inhibitors of RTKs, for example, lapatinib, with vaccination, preferably Thl- polarizing DC vaccine, and more preferably antigen-pulsed DC1 vaccine, for example autologous HER2-pulsed DC1 vaccine, to enhance anti-cancer effects.
  • small molecule inhibitors of RTKs for example, lapatinib
  • vaccination preferably Thl- polarizing DC vaccine
  • antigen-pulsed DC1 vaccine for example autologous HER2-pulsed DC1 vaccine
  • Thl- polarizing DC vaccine such as HER2-pulsed DC1 vaccine provides in the form of at least one secreted Thl cytokine may mitigate resistance to targeted inhibitor drugs.
  • SK-BR-3, BT-474, MDA-MD-468, MDA-MB-453 and HCC1419 cell lines were obtained from American Type Culture Collection (Rockwell, MA). JIMT-l cells were a gift from Pravin Kaumaya, (The Ohio State University, Columbus OH). SK-BR-3 cells were cultured in McCoy’s 5A Media (Gibco) supplemented with 10% v/v fetal calf serum (FBS; BioWhittaker), 100 units/ml of potassium penicillin and 100 pg/ml of streptomycin sulfate (BioWhittaker).
  • McCoy 5A Media (Gibco) supplemented with 10% v/v fetal calf serum (FBS; BioWhittaker), 100 units/ml of potassium penicillin and 100 pg/ml of streptomycin sulfate (BioWhittaker).
  • BT-474 cells were cultured in HybriCare (ATCC), supplemented with 10% v/v FBS, 100 units/ml of potassium penicillin and 100 pg/ml of streptomycin sulfate.
  • MDA-MB- 468, MDA-MB-453 and HCC1419 cells were cultured in RPMI-1640 (BioWhittaker), 10% v/v FBS, 100 units/ml of potassium penicillin and 100 pg/ml of streptomycin sulfate (BioWhittaker), 2mmol/L glutamine (BioWhittaker), lmmol/L sodium pyruvate (BioWhittaker), and 1% non- essential amino acids (BioWhittaker). All cells were maintained in culture at 37°C in 5% C0 2.
  • Alamar Blue Assay Cellular metabolism (consistent with cell viability) was measured by the Alamar Blue assay.
  • Breast cancer cell lines were harvested using trypsin (Lonza), counted and plated at a density of approximately 5xl0 4 cells per well in 48-well cluster plates containing 500pl culture media. Cells were treated the next day with 20 ng/ml TNF-a (Peprotech), 12.5 ng/ml of IFN-g (Peprotech), or 0.2pM lapatinib and incubated for
  • Trypan Blue Exclusion Assay Cells were plated and treated in a manner identical to that described for the Alamar Blue assay. On day 3, cells were harvested by scraping, pelleted at 2l8g for 5 minutes, and resuspended. Live/dead staining using the Trypan Blue dye was assessed by two methods. The first was the traditional microscopy-based technique where cells were resuspended in a 1 : 1 solution of PBS and Trypan Blue. The cells were then counted on a hemocytometer and determined to be dead if they retained stain and alive if they excluded the dye. The second method took advantage of the fluorescent properties of the Trypan Blue dye and was flow cytometry-based.
  • the resuspended cells were resuspended in a 0.002% (w/v) solution of Trypan blue in PBS.
  • Cells were analyzed via an Amnis/Millipore FlowSight flow cytometer using an excitation wavelength of 642nm and emission detection between 642nm and 740nm.
  • Annexin V/Propidium Iodide Apoptosis Assay Cellular apoptosis was assessed via staining with fluorescently-labeled Annexin V and propidium iodide (PI) followed by flow cytometry analysis. Cells were treated in a manner identical to that described above for the Alamar Blue assay with the exception of the cells being plated at a density of 6xl0 5 per well in 6-well cluster plates. On day 3, the cells were harvested using trypsin, washed with PBS, and resuspended in 50m1 Annexin V binding buffer (BD Pharmingen).
  • BD Pharmingen 50m1 Annexin V binding buffer
  • FITC-conjugated Annexin V (BD Pharmingen)
  • 10m1 of PI (BD Pharmingen)
  • Binding was assessed with a FlowSight flow cytometer (Amnis Corporation, EMD Millipore).
  • FITC fluorochrome was excited using a 488nm laser with emission detection between 505 and 560nm.
  • Propidium iodide was excited with a 642nm laser and emission detected between 642 and 740nm.
  • TMRE Mitochondrial Membrane Potential assessment
  • a Bradford protein assay was performed to determine total protein concentration so that 30pg of total protein could be loaded into each well of a 4-15% Mini Protean TGX gel (Bio-Rad). The gels were run at 300V for approximately 15 minutes and then transferred onto a .2pm pore PVDF membrane (Bio-Rad) via 100V for 1 hour. Membranes were blocked with either 1% BSA in PBS or 5% skim milk in TBS-T for 1 hour and then incubated with primary antibodies in blocking buffer (1 : 1000) overnight at 4°C. Membranes were then washed 3 times for 5 minutes each with TBS-T and then incubated with HRP-conjugated secondary antibodies in blocking buffer (1 : 10,000) for 1 hour at room temperature. Membranes were then washed 3 times with TBS-T and bound antibody was detected via SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific) and visualized using an ImageQuant LAS 4000 mini (GE
  • Treatment groups were considered significantly different if the p value was ⁇ 0.05.
  • Thl Cytokines Work in Concert with Lapatinib to Suppress Cellular Metabolism
  • the targeted inhibitor drug lapatinib exerts its pharmacological effects by selectively interacting with the ATP -binding cassette of both HER-2 and EGFR, thereby interfering with transmembrane signaling (Segovia-Mendoza,. M., et al. Am. J. Can. Res.
  • HER-family drug targets SK-BR-3 (HER2 hi /EGFR med /ER neg ), MDA-MB-468 (HER2 neg /EGFR hi /ER neg and BT-474 (HER2 hi /EGFR med /ER pos ) were selected for further study.
  • the study began with the Alamar Blue assay, which estimates levels of cellular metabolism via the reduction of resazurin salt to resofurin; a conversion that can be followed spectrophotometrically.
  • Figure 1 shows mean optical densities of dye solution acted on by breast cancer cells that were either left untreated (control/No Rx)), incubated with Thl cytokines alone (lOug/ml each), lapatinib alone (200nM), or both treatments (Thl cytokines plus lapatinib).
  • Thl cytokines alone lOug/ml each
  • lapatinib alone 200nM
  • Thl cytokines plus lapatinib Treatment alone.
  • TMRE staining is a fluorescent dye that is sequestered in healthy, metabolically-active mitochondria (providing a strong fluorescent signal) but diminished when these organelles lose their membrane potential as a consequence of apoptosis (weak fluorescent signal).
  • the latter assay takes advantage of the ability of Annexin V to bind phosphoserine residues that are flipped to the outer leaf of the plasma membrane in early apoptosis, and the entry of propidium iodide into the nuclei and subsequent intercalation into DNA in late apoptotic cells that lose nuclear membrane integrity. Cells staining positive for both Annexin V and propidium iodide are therefore considered to be in late stage apoptosis.
  • Thl cytokines were able to down-regulate total HER3 but not eliminate it.
  • the addition of lapatinib did not enhance the observed down-regulation of total HER3 by Thl cytokines but it was able to nearly eliminate observed phosphorylation of HER3.
  • Results from these experiments show that Thl cytokines and lapatinib work together to suppress HER signaling; Thl cytokines contribute by lowering the amount of total HER2 and HER3, while lapatinib acts by reducing the
  • HCC1419 cells were also examined which are Herceptin-resistant but lapatinib-sensitive, as well as SK-BR-3 cells which are sensitive to both drugs.
  • HCC1419, MDA-MB-453, and JIMT-l lines were cultured in the presence of increasing doses of lapatinib (0-4mM).
  • lapatinib plus Thl cytokines worked cooperatively to suppress metabolic activity in the lapatinib-sensitive cell lines SK-BR-3 and HCC1419 as seen in Figure 6A, upper panels.
  • cytokine-treated (but not untreated) JIMT-l and MDA-MB-453 cells now displayed a dose-dependent sensitivity to lapatinib for suppression of cellular metabolism as seen in Figure 6A lower panels.
  • Immunotherapy in the form of dendritic cell-based vaccines that target the HER2 protooncogene and induce strong Thl immunity can induce complete pathological responses in up to one third of all HER2 pos ductal carcinoma in situ patients when supplied in the neoadjuvant setting.
  • Sharma, et al. Lowenfeld, et al.
  • this response rate drops precipitously after the cancer becomes invasive.
  • Lowenfeld, et al. This means that in order to improve response rates both for DCIS patients as well as individuals with more advanced cancer, vaccination will likely need to be paired with other treatment modalities such as drug or radiation therapy.
  • Thl cells may be instrumental in controlling some malignancies through the induction of cellular senescence or apoptosis.
  • Lapatinib selectively blocks cross phosphorylation in EGFR and HER-2 by binding to the ATP binding cassette of the kinase domain.
  • HER3 lacking a kinase domain of its own, is dependent on a dimerization partner for phosphorylation and subsequent activation. (Guy, et al.). EGFR/HER1 and HER2 are both known to act as dimerization partners for HER3
  • Thl cytokines could induce down-regulation of both HER2 and HER3 expression on multiple breast cancer cell lines (Namjoshi, et al.) it was hypothesized that Thl cytokines and lapatinib would work well together: cytokines to diminish total HER expression, and lapatinib to block the activation of the remaining expressed HER family RTKs. It can be readily appreciated by those skilled in the art that such a double hit could virtually eliminate the downstream signaling events critical for continued cell proliferation and resistance to apoptosis.
  • HER2 pos DCIS patients vaccinated with IL-l2-secreting dendritic cells pulsed with HER-2 peptides (autologous HER2 -pulsed activated DC1 vaccine) not only generated strong Thl immune responses evident in peripheral blood, but also developed heavy infiltrates of both CD4 pos T cells as well as B lymphocytes into the area of disease in the breast (Czerniecki, et al.).
  • the MHC class II -positive B cells can serve as antigen-presenting cells for the Thl cells, and that T cells, so activated, can secrete Thl cytokines into the tumor beds.
  • These cytokine- secreting T cells unlike traditional drugs, concentrate at the site of disease to disgorge their cargo of senescence- and apoptosis-inducing factors.
  • cytokine effectors of vaccine-induced immunity namely at least one of IFN-g and TNF-a
  • HER2-pulsed DC1 vaccine can be administered to a breast cancer patient who has become resistant to the targeted inhibitor drug lapatinib and can thereafter be treated with additional lapatinib resulting in a degree of restored drug sensitivity, which would contribute to increased tumor destruction.

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Abstract

L'invention concerne une méthode de traitement du cancer, comprenant l'administration à un patient en ayant besoin d'une combinaison : d'un médicament inhibiteur ciblé (TID) contre le cancer, et d'un agent anticancéreux pouvant induire la production d'au moins une cytokine Th1 chez le patient ou qui comprend au moins une cytokine Th1 ou sa copie fonctionnelle fournie au patient.
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WO2008014220A2 (fr) * 2006-07-25 2008-01-31 Irx Therapeutics, Inc. Immunothérapie par vaccin
US20170216421A1 (en) * 2014-07-17 2017-08-03 The Trustees Of The University Of Pennsylvania Manufacturing of multi-dose injection ready dendritic cell vaccines, combination therapies for blocking her2 and her3, and estrogen receptor positive her2 breast receptor positive her2 breast cancer therapy

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Publication number Priority date Publication date Assignee Title
WO2008014220A2 (fr) * 2006-07-25 2008-01-31 Irx Therapeutics, Inc. Immunothérapie par vaccin
US20170216421A1 (en) * 2014-07-17 2017-08-03 The Trustees Of The University Of Pennsylvania Manufacturing of multi-dose injection ready dendritic cell vaccines, combination therapies for blocking her2 and her3, and estrogen receptor positive her2 breast receptor positive her2 breast cancer therapy

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* Cited by examiner, † Cited by third party
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CN113398262A (zh) * 2021-06-18 2021-09-17 北京康爱瑞浩生物科技股份有限公司 用于治疗her2阳性胃癌的组合物及应用

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