Classifications

• • 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
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • 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
  • A61K39/0005—Vertebrate antigens
  • A61K39/0011—Cancer antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • 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
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • 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
• • 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
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • 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
  • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
  • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
• • 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
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6056—Antibodies
• • 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
  • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to compositions comprising a peptide that activate the immune response and methods of using the compositions for immunotherapy, including for the treatment of cancer.
• the immune system comprises the innate immune system and the adaptive immune system.
• the innate immune response recognizes pathogens in a non-specific way, for example through pattern-associated molecular patterns that distinguish pathogens from host molecules
• the adaptive immune system is directed to specific antigens. Specificity of the adaptive immune response is taught by interaction with antigens, which are presented as a complex with major histocompatibility molecules (MHC) to adaptive immune cells.
• MHC major histocompatibility molecules
• T cell receptor TCR
• Class I MHC can be expressed on nearly every nucleated cell of the body, and it interacts with CD8, which is predominantly expressed on the cytotoxic class of T cells. These cells can induce the death of the cells presenting the antigen that resulted in the activation of the cell, so they are heavily regulated to prevent tissue damage.
• Activation of cytotoxic T cells requires strong MHC complex signal or additional activation provided by helper T cells. Helper T cells are characterized by CD4 expression, so they interact with the class II MHC.
• naive helper T cells results in the release of cytokines that can activate antigen-presenting cells or activate cytotoxic T cells.
• Thl or Th2 helper T cells enhance immune responses to different types of antigens.
• a Thl response which is characterized by the release of interferon- ⁇ (IFNy)
• IFNy interferon- ⁇
• a Th2 response which is characterized by the release of interleukin-4 (IL-4) leads to the responses mediated by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins and certain antimicrobial peptides.
• the immune system also provides for immune suppression to limit the damaging potential of an immune response.
• the immunosuppressive regulatory T cell sub-population of T cells attenuates a cytotoxic T cell response, and normally protects against over-stimulation and development of autoimmunity.
• a group of T cell precursors differentiate into natural regulatory T cells in the thymus by moderate interaction with the self-peptide MHC complex.
• Regulatory T cells also include inducible regulatory T cells developed from CD4 + T cells outside of the thymus. Whereas natural regulatory T cells suppress T cell activation by interaction with antigen-presenting cells to produce negative signals for T cell activation, inducible regulatory T cells produce cytokines that inhibit T cell proliferation.
• Imbalance between the active and suppressive immune response can result in diseases and conditions such as cancer, immunodeficiency (e.g., acquired immunodeficiency syndrome), autoimmune diseases, or hypersensitivity reactions or worsen diseases and infections, for example, tuberculosis, Leishmania, or malaria.
• a strong, anti-cancer immune response requires antibodies (the adaptive, humoral arm) and an active cell-mediated arm. The interplay between these two arms is driven by activation of dendritic cells (the primary antigen-presenting cell type, APCs) and subsequent production of antibodies by B cells. Destruction of cancer cells then occurs by two processes: a cytotoxic cellular response by neutrophils, natural killer cells, cytotoxic T cells, and macrophages, and an antibody-dependent cellular cytotoxicity (ADCC) performed by activated macrophages and neutrophils. Often tumor cells can be made more susceptible to digestion for antigen presentation by radiation treatment or chemotherapy.
• ADCC antibody-dependent cellular cytotoxicity
• a goal of immunotherapy is to restore the ability of the immune system to overcome these diseases.
• Traditional immunotherapy targets have been antigens specific to the targeted cells, such as tumor-associated antigens (e.g., T n antigen or T f antigen) or glycosylation groups expressed on the surface of viruses or bacteria or on cells infected by the viruses or bacteria.
• T n antigen or T f antigen tumor-associated antigens
• glycosylation groups expressed on the surface of viruses or bacteria or on cells infected by the viruses or bacteria.
• vaccinations using these antigens induce endogenous production of antibodies against these antigens to mount an immune response.
• the traditional approach also uses adjuvants to aid the stimulation of the immune system, but greater understanding of immune responses have expanded the potential candidates for factors that activate immune cells to stimulate the immune system either directly or indirectly to mount an immune response.
• immunotherapy to target proteins involved in regulating the balance of the immune response, for example, to suppress or enhance the population of regulatory T cells.
• immunotherapy is now able to 1) induce endogenous production of antibodies, 2) provide exogenous antibodies that manipulate the type of immune response to be mounted, and 3) activate or suppress specific immune cells by factors from the immune system checkpoint.
• the present invention is directed to combinations of these approaches for improved immunotherapy, for example, to boost the immunogenicity of tumors.
• the present invention is directed to the use of peptides in combination with antibodies, such as exogenous antibodies against immune checkpoint proteins or against cancer markers.
• Peptides that mimic sugars and bind to regulatory lectin-type receptors expressed by key cells of the immune system can enhance the immune responses, which can support the therapeutic benefits of the exogenous antibodies.
• compositions and kits that activate the adaptive immune response comprising a therapeutically effective amount of a peptide that has an active peptide sequence represented by SEQ ID N0: 1 ( VQ ATQ SNQHTPR; also referred to herein as "svL4") and a therapeutically effective amount of a first antibody.
• the peptide may be tetravalent.
• the peptide has the structure [(VQATQSNQHTPRGGGS) 2 K] 2 K- H 2 (SEQ ID NO:3).
• the first antibody is against an immune checkpoint protein.
• the compositions and kits may comprise a second antibody, wherein the second antibody is against an immune checkpoint protein of a different immune checkpoint pathway as the first antibody.
• Embodiments of the invention may further comprise a pharmaceutically acceptable carrier.
• the immune checkpoint proteins are selected from at least one of cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1), or ligands to PD-1, such as programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2) in some embodiments.
• CTLA-4 cytotoxic T lymphocyte antigen 4
• PD-1 programmed death 1
• ligands to PD-1 such as programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2) in some embodiments.
• the therapeutically effective amount is an amount sufficient to activate the immune response in a subject.
• the therapeutically effective amount is an amount sufficient to treat cancer in a subject.
• the cancer may be bladder cancer, brain cancer, breast cancer, colon cancer, head and neck cancer, liver cancer, lung cancer, pancreatic cancer, prostrate cancer, ovarian cancer, kidney cancer, or skin cancer, for example, colorectal adenocarcinoma, glioblastoma, hepatocellular carcinoma, hormone-refractory prostate cancer, epithelial ovarian carcinoma, ovarian adenocarcinoma, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, or renal cell carcinoma.
• the therapeutically effective amount is an amount sufficient to suppress the population of regulatory T cells. In another embodiment, the therapeutically effective amount is an amount sufficient to increase the population of effector T cells. In still another embodiment, the therapeutically effective amount is an amount sufficient to increase the levels of anti-cancer cytokines such as IL-2, IL-12p70, IL-21, IL-27, T Fa, and ⁇ . In some aspects, the level of anti-cancer cytokines increases by several folds.
• the therapeutic amount of the first peptide and the second peptide is between about 0.1 nmol/kg body weight to about 1500 nmol/kg body weight, between about 1 nmol/kg body weight to about 1 1000 nmol/kg body weight, or about 1 nmol/kg body weight.
• the present invention further provides methods of activating the adaptive immune response in a subject by administering a therapeutically effective amount of a peptide to a subject, wherein the peptide has an active peptide sequence represented by SEQ ID NO: l; and administering a therapeutically effective amount of an antibody, wherein the antibody is against an immune checkpoint protein.
• the antibody comprises antibodies against immune checkpoint proteins of two distinct immune checkpoint pathways.
• the peptide administered is tetravalent.
• active peptide sequence is connected to the core by a linker sequence.
• the core is a tri-lysine core, and the linker sequence is -GGGS- (SEQ ID NO:2).
• a tetravalent peptide has the structure [(VQATQSNQHTPRGGGS) 2 K] 2 K- H 2 (SEQ ID NO:3).
• the method comprises administering the peptide before administering the antibody, for example, at least three days before the administration of the antibody. After which, the peptide is administered concurrently with the administration of the antibody. In other implementations, the peptide and the antibody are administered together at the start of the treatment. In some implementations, the peptide is administered on alternating days or on a weekly basis. In some embodiments, administration of the peptide is continued after the period of antibody treatment.
• Figure 1 depicts the structure of tetravalent svL4, with the active sequence presented as SEQ ID NO: l (VQATQSNQHTPR).
• A Schematic design of peptide. Arms with active sequences (1, 2) are connected via a spacer sequence (3) to a tri-lysine core (4).
• B Chemical structure of the peptide with a C-terminal amide.
• the peptides are composed entirely of naturally occurring L-amino acids, where A is alanine, G is glycine, H is histidine, K is lysine, L is leucine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, and V is valine.
• the sequence -GGGS- (SEQ ID NO:2) is a linker between the active 12-mer sequence and the tri-lysine core. Accordingly, the depicted tetravalent svL4 has the structure [(VQATQSNQHTPRGGGS) 2 K] 2 K- H 2 (SEQ ID NO:3).
• Figure 2 depicts UPLC trace of svL4. Chromatography was performed by CBL with a C8 column with a gradient of acetonitrile in 0.1% TF A/water. Purity of the peptide is > 95%.
• FIG. 3 depicts electrospray ionization (ESI) mass spectrum of svL4. Ionization state of the peptide for each signal is indicated.
• ESI electrospray ionization
• Figure 4 depicts elution of svL4 from a column of CM-Sephadex C50.
• svL4 was passed through a column of DEAE-Sephadex A25 in 25 mM NaCl. The concentration of NaCl was then raised to 100 mM and the sample was applied to the CM-Sephadex column. The column was washed with 25 mL 100 mM NaCl, then with 50 mL 200 mM NaCl, and then with 500 mM NaCl, beginning at fraction 24.
• Fractions (1 : 100 dilution) were monitored by OD at 210 nm for peptide content and at 405 nm for endotoxin by the Limulus amebocyte lysate (Lonza, Walkersville, MD, USA) colorimetric assay method.
• Figure 5 depicts the binding activity of svL4 to (left-to-right) CLEClOa, Langerin, ASGPR1, Dectin-1, CLEC9a, DC-SIGN, CD44, IL-4R, and Siglec-1.
• Figure 6 depicts fold increase in the high-staining populations for the markers on peritoneal cells from C57BL/6 mice dosed Q2D with 1 nmol/g svL4. Untreated values were normalized to 1.0 (grey bars) to show fold increase with svL4 administration (black bars) and to compare responses between cell types at day 3 of treatment.
• Figure 7 depicts the increases in peritoneal cell types found in C57BL/6 mice treated with 1 nmol/g svL4 in a time course experiment.
• the three bars in each group indicate data from day 1, day 3, and day 5, i.e., one day after each subcutaneous injection on day 0, day 2 and day 4.
• Cell numbers of macrophages are indicated in panel A, activated macrophages (F4/80 CDl lb CD86) in panel B, DCs (CDl lc) in panel C, NKT cells (NK1.1 CD3 + ) in panel D, activated NKT cells (NK1.1 CD3 + CD69) in panel E, activated NK cells (NK1.1 CD3 " CD69) in panel F, activated Th cells (CD4 CD69) in panel G, cytotoxic T cells (CD 8 CD69) in panel H, B cells (CD19) in panel I, and of B memory cells (CD19 CD73 CD80 CD273) in panel J. The data are expressed as actual cell counts in the analyses.
• Figure 8 depicts the percent Treg cells in dissociated B16 melanoma tumors (A) and the ratio of effector T cells to regulatory T cells in tumors (B).
• svL4 was administered subcutaneously on alternate days at a dose of 1.0 nmol/g body weight.
• Antibody against cytotoxic T-lymphocyte associated molecule-4 (a-CTLA-4) was injected intraperitoneally every third day at a dose of 100 ⁇ g per animal. The bars show averages of two flow analyses.
• Figure 9 depicts flow analyses of tumor cells from animals implanted with B16 melanoma cells and the treatments each animal received as described in the legend to FIG. 8.
• Figure 10 depicts the tumor size at day 19 after implantation of glioma cells in the brain of mice (A) and survival of mice implanted with glioma cells in the brain (B).
• a low dose of radiation was given on day 7 and 9, followed by subcutaneous injection of svL4 (1 nmol/g body weight) on alternate days thereafter.
• Figure 11 depicts a study of the effect of dose of svL4, given subcutaneously on alternate days, on tumor size in mice implanted with glioma cells in the brain. Treatment was with peptide alone in this experiment. Tumor size is expressed as a percent of the tumor in untreated control mice.
• Figure 12 depicts the effect of treatment on mice implanted with an ovarian cancer cell line.
• A Average body weight of groups of 8 C57BL/6 mice treated with 0.1 nmol/g svL4 subcutaneously from day 45 to day 98 (lower line) or untreated (upper line). The rapid drops in the average body weight of control mice near day 90 indicated death of animals. Interestingly, when treatment was terminated, ascites seemed to increase.
• B Survival curves of untreated and treated mice after end of treatment at day 95. Whereas all untreated animals died by day 129, four of the eight treated animals were alive at day 150 in the 0.1 nmol/g group, which is 8 weeks after the termination of treatment.
• Figure 13 depicts a comparison of the survival of dogs with a variety of cancer when treated with chemotherapeutic drugs (listed) plus svL4 or only svL4 after termination of other drugs. Injections of 1 mg were given intravenously on a weekly schedule.
• innate immune response or "innate immunity” as used herein refers to the response of immune cells to an acute threat from a pathogen. The responses are often inherent reactions to pathogen recognition factors.
• adaptive immune response refers to the response of antigen-specific lymphocytes (e.g., T cells and B cells) to antigen, including the development of immunological memory. Adaptive immune responses are generated by clonal selection of lymphocytes.
• effector T cell refers to T cells that can mediate the removal of pathogens or cells without the need for further differentiation. Thus effector T cells are distinct from naive T cells and memory T cells, which must differentiate and often proliferate before they become effector cells.
• regulatory T cell refers to T cells that inhibit T- cell responses, particularly by the suppression or down-regulation of effector T cell induction or proliferation. Thus these cells can induce immunological tolerance. Expression of at least one of CD25, CD39, CD73, and Foxp3 is indicative of regulatory T cells. While the majority of regulatory T cells are CD4 + , they may also be CD8 + . Another indication of regulatory T cells is high expression of cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) or glucocorticoid- induced TNF receptor (GITR).
• CTL-4 cytotoxic T-lymphocyte associated molecule-4
• GITR glucocorticoid- induced TNF receptor
• immune checkpoint refers to an inhibitory pathway in the immune system that is crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage.
• immunotherapy refers the treatment of a disease or condition by inducing, enhancing, or suppressing an immune response.
• pathogen refers to anything that can produce disease.
• infectious agents such as viruses, bacteria, prions, fungi, or protozoans, along with host cells transformed to neoplastic cells.
• the present invention relates to a combination immunotherapy approach to activate or enhance the immune response.
• An optimal adaptive immune response includes the presence of antibodies to mark pathogens and activated cellular components, including macrophages, cytotoxic T cells, natural killer cells and dendritic cells.
• the invention may inhibit the immunosuppressive responses, enhance the effector T cell responses, or both.
• the invention enhances the efficacy of effector T cell function, for example by increasing the ratio of effector T cells to regulatory T cells within the tumor and the tumor microenvironment.
• the invention suppresses the population of regulatory T cells within the tumor and the tumor microenvironment.
• the invention alters the cytokine profile of the subject to enhance to efficacy of effector T cell function, for example, by inducing a several-fold increase of IL-2, IL-12p70, IL-21, IL-27, TNFa, and IFNy in the serum.
• the increase in serum levels of these cytokines occurs within around 4 hours of administration of the combination therapy.
• the invention treats diseases and conditions in a subject where the immune response mounted by the subject's immune system without outside aid is not able to defend the subject against the disease or condition.
• diseases and conditions may be cancers and persistent infections.
• cancers that may be treated by the invention are bladder cancer, brain cancer, breast cancer, colon cancer, head and neck cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, ovarian cancer, kidney cancer, or skin cancer.
• the cancers are colorectal adenocarcinoma, glioblastoma, hepatocellular carcinoma, hormone-refractory prostate cancer, ovarian adenocarcinoma, epithelial ovarian carcinoma, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, or renal cell carcinoma.
• persistent infections include viral infections, mycobacterial infections, and parasitic infections.
• the viral infection is caused by retroviruses, such as HIV.
• the mycobacterial infection may be tuberculosis.
• parasitic infection may be Leishmania or malaria.
• the backbone regime is a foundational peptide regime comprising a peptide that is a mimetic of sugar ligands, for example, the terminal sugars of complex glycans.
• Peptides are uniquely suited to immunotherapy. Peptides are flexible in design, easily synthesized on a large scale, water soluble and relatively stable, and bind selectively to receptors with high avidity.
• the peptides of the invention bind to regulatory lectin-type receptors of particular benefit for the amplification of the immune response.
• the peptide mimics N-acetylgalactosamine (GalNAc), which is found on CD45, for example, the peptide is svL4 (VQ ATQ SNQHTPR; SEQ ID NO: l) (see Examples 1-7).
• GalNAc N-acetylgalactosamine
• svL4 VQ ATQ SNQHTPR; SEQ ID NO: l
• the phosphatase activity of CD45 a widely expressed and abundant cell surface protein, is required for lymphocyte activation and development [Trowbridge and Thomas, 1994].
• CD45 removes an inhibitory phosphate from Src family signal transduction kinases [Roskoski, 2005; McNeil et al., 2007], which in T cell activation is expressed as increased TNFa secretion [van Vliet et al., 2006].
• Human antigen-presenting cells such as macrophages and immature DCs express the receptor CLEClOa (CD301), which is specific for GalNAc [van Vliet et al., 2008].
• CLEClOa a strategic target for activation of immune cells, promotes internalization of antigens by DCs, presentation of antigens to CD4 T cells and differentiation of IFNy-producing CD4 + T cells [Streng-Ouwehand et al. 2011].
• Ligand binding to CLEClOa results in enhanced antigen- specific, IFNy-producing CD8 + T cell responses and tilts naive CD4 + T cells towards Thl cells, with increased proliferation of T cells.
• DCs mediate activation and proliferation of NK cells [Degli-Esposti and Smyth, 2005].
• Trans binding of CLEClOa on DCs to a GalNAc residue on CD45 on T cells results in T cell inhibition [van Vliet et al., 2006].
• Introduction of a GalNAc-containing factor frees CD45 and allows dephosphorylation (inactivation) of inhibitory receptors, removal of inhibitory phosphate groups from signal transduction kinases, and T cell activation.
• Inhibitory receptors that may be inactivated by CD45 signaling include CTLA-4 and PD-1.
• the combination therapy of the invention comprises an immunotherapy agent, for example, an antibody.
• an antibody is a monoclonal antibody.
• the antibody is a monoclonal antibody against an immune checkpoint protein.
• Immune checkpoint blockade is a new and promising strategy to induce tumor regression, stabilize disease, and prolong survival by manipulation of the immune system [Weber, 2010].
• Immune checkpoint proteins may be expressed by the T cell or by the antigen- presenting cell.
• T cell immune checkpoint proteins may be, for example, CTLA-4 and PD-1.
• Antigen-presenting cell immune checkpoint protein may be, for example, PD-Ll and PD-L2.
• the antibody is an antibody against CTLA-4 (a-CTLA-4).
• a-CTLA-4 an antibody against CTLA-4
• a-CTLA-4 The use of the fully human, monoclonal antibody against CTLA-4, ipilimumab or Yervoy (Bristol Myers Squibb), has become a primary immunotherapeutic approach to cancer treatment. Studies in the mouse have demonstrated that T cells within a tumor have a high percentage of Treg cells, which have high expression of CTLA-4, and thus abrogate the immune response to the tumor cells. Introduction of a-CTLA-4 tags Treg cells and marks them for antibody-mediated destruction by macrophages. So a-CTLA-4 reduces the population of Treg cells.
• the antibody is an antibody against PD-1 (a-PD-1), which inhibits the interaction of PD-1 with its ligands to prevent inhibition of T cells.
• PD-1 is a member of the extended CD28/CTLA-4 family of T cell regulators [Ishida et al., 1992].
• Monoclonal antibodies targeting PD-1 that boost the immune system have been developed for the treatment of cancer [Weber, 2010].
• the FDA has granted accelerated approval to Keytruda (pembrolizumab; MK-3475), Merck's anti-PD-1 drug for advanced or unresected melanoma that no longer responds to other drugs.
• Melanoma is a skin cancer originating in pigment-producing cells called melanocytes.
• the disease is particularly swift and deadly in cases where it metastasizes to other sites in the body, particularly the brain.
• Keytruda was intended for use after ipilimumab (Yervoy; Bristol-Myers Squibb), but currently is used clinically in combination with Yervoy, which blocks another T-cell receptor called CTLA-4 (see above).
• Bristol-Myers Squibb received approval in Japan for their a-PD-1 drug, Opdivo (nivolumab), leading to speculation that it might be the first to be approved in the U.S.
• Extensive studies have been performed to show durable tumor remission with nivolumab [Topalian et al., 2014].
• the antibody may be an antibody against PD-1 ligands in another embodiment.
• PD- Ll and PD-L2 two ligands of PD-1, are members of the B7 family [Freeman et al., 2000; Latchman et al., 2001].
• Many tumor cells express PD-Ll, an immunosuppressive PD-1 ligand; inhibition of the interaction between PD-1 and PD-Ll can enhance T-cell responses in vitro and mediate preclinical antitumor activity.
• PD-Ll is expressed on almost all murine tumor cell lines, including PA1 myeloma, P815 mastocytoma, and B16 melanoma upon treatment with ⁇ .
• PD-L2 expression is more restricted and is expressed mainly by DCs and a few tumor lines.
• PD-L1 protein is up-regulated in macrophages and dendritic cells (DC) in response to lipopolysaccharide (LPS) and GM-CSF treatment and in T cells and B cells upon TCR and B cell receptor signaling [Iwai et al., 2002].
• LPS lipopolysaccharide
• GM-CSF GM-CSF
• the antibody of some implementations of the invention attacks one immune checkpoint pathway
• the antibody of other implementations of the invention may attack more than immune checkpoint pathway.
• the combination therapy may comprise a peptide with antibodies against the CTLA-4 pathway and the PD-1 pathway, which are two distinct immune checkpoint pathways.
• the combination therapy of the present invention targets a combination of immune modulation avenues.
• the antibodies act by binding to checkpoint proteins on the cell surface
• stimulation of phosphatase activity by CD45 and other phosphatases cause dephosphorylation of CTLA-4 and PD-1 and the accessory protein SFIP-1 through which these proteins act
• the combination therapy comprising the peptides of the invention and the antibodies also inactivate the inhibitory receptors from within the cell.
• the combination therapy of the present invention may target greater combinations of immune modulation avenues with the use of multiple peptides and/or multiple antibodies.
• the combination therapy comprises a therapeutically effective amount of at least one peptide and a therapeutically effective amount of at least one antibody.
• the peptide of the combination therapy has an active peptide sequence represented by SEQ ID NO: 1 (VQATQSNQHTPR).
• the peptide may be tetravalent.
• the active peptide sequences of the tetravalent peptide is connected to the core by a linker sequence.
• the core is a tri-lysine core and the linker sequence is -GGGS- (SEQ ID NO:2).
• An exemplary tetravalent peptide has the structure [(VQATQSNQHTPRGGGS)2K]2K- NH2 (SEQ ID NO:3).
• the peptides are different mimetics that target the same regulatory lectin-type receptor.
• both peptides target CLEClOa but each is a mimetic of either GalNAc or Gal.
• the peptides are different mimetics that target different regulatory lectin-type receptors.
• the antibodies target different immune checkpoint pathways, for example, the antibodies target the CTLA-4 checkpoint pathway or the PD-1 checkpoint pathway.
• the antibodies of the combination therapy may comprise a- CTLA-4 and one of a-PD-1 or a-PD-Ll .
• compositions and dosage forms of the invention comprise the active ingredients disclosed herein.
• the notation of "the pharmaceutical agent” signifies the compounds of the invention described herein or salts thereof.
• Pharmaceutical compositions and dosage forms of the invention can further comprise a pharmaceutically acceptable carrier.
• the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which an active ingredient is administered.
• Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
• the pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
• other excipients can be used.
• composition, shape, and type of dosage forms of the invention will typically vary depending on their route of administration and subject being treated.
• a parenteral dosage form may contain smaller amounts of one or more of the active ingredients it comprises than an oral dosage form used to treat the same disease.
• Typical pharmaceutical compositions and dosage forms comprise one or more excipients.
• Suitable excipients are well known to those skilled in the art of pharmacy, and non-limiting examples of suitable excipients are provided herein. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient.
• oral dosage forms such as tablets may contain excipients not suited for use in parenteral dosage forms.
• the suitability of a particular excipient may also depend on the specific active ingredients in the dosage form. For example, the decomposition of some active ingredients may be accelerated by some excipients such as lactose, or when exposed to water.
• compositions and dosage forms that comprise one or more compounds that reduce the rate by which an active ingredient will decompose.
• compounds which are referred to herein as “stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.
• the dosage is determined empirically, using known methods, and will depend upon facts such as the biological activity of the particular compound employed, the means of administrations, the age, health and body weight of the host; the nature and extent of the symptoms; the frequency of treatment; the administration of other therapies and the effect desired.
• the dosage is determined empirically, using known methods, and will depend upon facts such as the biological activity of the particular compound employed, the means of administrations, the age, health and body weight of the host; the nature and extent of the symptoms; the frequency of treatment; the administration of other therapies and the effect desired.
• the actual dosages and method of administration or delivery may be determined by one of skill in the art. Frequency of dosage may also vary depending on the compound used and whether an extended release formulation is used.
• compositions of the invention that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups).
• dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).
• Typical oral dosage forms of the invention are prepared by combining the active ingredients in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration.
• excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents.
• excipients suitable for use in solid oral dosage forms include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.
• tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.
• a tablet can be prepared by compression or molding.
• Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient.
• Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
• excipients that can be used in oral dosage forms of the invention include, but are not limited to, binders, fillers, disintegrants, and lubricants.
• Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, Natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.
• Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof.
• a specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581.
• Suitable anhydrous or low moisture excipients or additives include AVICEL-PH-103 and Starch 1500 LM.
• fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
• the binder or filler in pharmaceutical compositions of the invention is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.
• Disintegrants are used in the compositions of the invention to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to form solid oral dosage forms of the invention.
• the amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art.
• Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, preferably from about 1 to about 5 weight percent of disintegrant.
• Disintegrants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.
• Lubricants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof.
• calcium stearate e.g., magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc
• hydrogenated vegetable oil e.g., peanut oil, cottonseed oil
• Additional lubricants include, for example, a syloid silica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore, Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Piano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, Mass.), and mixtures thereof. If used at all, lubricants are typically used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated.
• AEROSIL 200 a syloid silica gel
• a coagulated aerosol of synthetic silica marketed by Degussa Co. of Piano, Tex.
• CAB-O-SIL a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, Mass.
• a preferred solid oral dosage form of the invention comprises an active ingredient, anhydrous lactose, microcrystalline cellulose, polyvinylpyrrolidone, stearic acid, colloidal anhydrous silica, and gelatin.
• This invention also provides a method for administering the combination therapy of the invention where the peptide and the antibody are administered separately or in a formulation in which the combined drugs can be administered as a single application.
• the invention provides for the administration of svL4 and the monoclonal antibody a- CTLA-4 in a single application.
• administration of the combination therapy comprises starting the administration of the peptide at the same time as the antibody.
• the combination therapy to activate the adaptive immune response is administered by first administering the peptide to prime the immune system before administering the antibody.
• the peptide is administered at least two weeks, at least ten days, at least one week, at least five days, at least three days, or at least one day before the administration of the antibody.
• administration of the peptide is continued even after the administration of the antibody.
• the administration of peptide is also concurrent to the course of administering the antibody.
• ipilimumab is usually injected intravenously every 3 weeks, so for the duration of the iplimumab treatment, the peptide is also administered.
• administration of the peptide is continued after the period of antibody treatment.
• a benefit of this method is maintaining the therapeutic benefit of the antibody treatment even without subsequent treatment with the antibody.
• ipilimumab is usually injected intravenously over a period of 90 minutes, and each unit dose is given every three week with a single course being up to four doses.
• the concurrent administration of the peptide and the antibody may be at a frequency of every week.
• the unit dosage of the peptide may be administered on alternate days or on a weekly basis.
• Single unit dosage forms of the peptide of the invention are suitable for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular, or intra-arterial), or transdermal administration to a patient.
• dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.
• the peptide is preferably administered via a parent
• the peptide may be administered in unit dosage amounts of about 0.1 nmol/kg body weight to about 1500 nmol/kg body weight, which corresponds to about 0.7 ⁇ g/kg body weight to about 10 mg/kg body weight.
• the unit dosage of the peptide may also be about 100 nmol/kg body weight to about 1500 nmol/kg body weight, about 100 nmol/kg body weight to about 1000 nmol/body weight, about 3 nmol/kg body weight to about 1500 nmol/kg body weight, about 3 nmol/kg body weight to about 1000 nmol/kg body weight, about 3 nmol/kg body weight to about 10 nmol/kg body weight, about 1 nmol/kg body weight to about 1000 nmol/kg body weight, or about 0.1 nmol kg body weight to about 1 nmol kg body weight.
• the peptide may be administered in a unit dosage amount of less than about 1500 nmol/kg body weight, for example, about 1000 nmol/kg body weight, about 500 nmol/kg, about 100 nmol/kg, about 10 nmol/kg, about 1 nmol/kg, or about 0.1 nmol/kg.
• the peptide may be administered in unit dosage amounts of about 5 nmol, about 10 nmol, about 15 nmol, about 25 nmol, about 30 nmol, about 50 nmol, about 75 nmol, about 100 nmol, about 225 nmol, about 250 nmol, about 500 nmol, about 750 nmol, about 1 ⁇ , about 10 ⁇ , or about 50 ⁇ .
• Parenteral dosage forms can be administered to patients by various routes including, but not limited to, subcutaneous, intravenous, bolus injection, intramuscular, and intraarterial.
• the parenteral dosage form is suitable for subcutaneous delivery.
• the parenteral dosage forms of the invention are preferably sterile or capable of being sterilized prior to administration to a patient.
• Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
• Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include, but are not limited to: water for Injection USP; aqueous vehicles such as, but not limited to, phosphate-buffered saline, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-mi scible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Examples
• a peptide mimetic of GalNAc was found in a screen of a phage display library with the GalNAc-specific lectin from Helix pomatia [Eggink and Hoober, 2009, 2010].
• the tetravalent form of the peptide has the structure [(VQATQSNQHTPRGGGS) 2 K] 2 K- H 2 (SEQ ID NO:3, FIG. 1).
• the peptide was synthesized by standard solid-phase chemistry and prepared endotoxin-free at >95% purity by chromatographic procedures (FIGS. 2-4). 2.
• FIG. 5 presents the binding activities of svL4 to various regulatory lectins. Of the lectins tested, svL4 binds strongly to CLEClOa, Langerin, ASGPR-1, and Dectin- 1.
• mice (strain C57BL/6, male, 6-8 weeks old) were given subcutaneous injections of 1 nmol/g tetravalent svL4 on days 0, 2, 4 and 6. Expression of biomarkers with cells stained with antibodies against the markers is shown in FIG. 6. Dramatic changes occurred in cells obtained from the peritoneal cavity of these animals, a major site of immune function. Maturation of immune cells was found in several populations of cells after two treatments as illustrated in FIG. 6, which showed an increase in highly stained cells in treated C57BL/6 mice at day 3. Thus subcutaneous administration of svL4 enhances maturation of immune cells in the peritoneal cavity.
• IL- ⁇ With a few proteins, such as IL- ⁇ , IL-2, IL-6 and TNFa, the values were very low in untreated animals and upon treatment with svL4 were increased but still nearly undetectable on the array blots. Treatment generally resulted in increases with many of the proteins, typically in the range of 3- to 5-fold.
• cytokines that increased but were still low in amount included GM-CSF, CCL4, IGFBP-1, IL-21, lymphotoxin- a, and IL-17.
• Major proteins included CCL1, CCL8, CCL28, Endostatin, Fas, HVEM, IL-1 1, IL-12p70, IL-16, IL-27, IL-28, MIP-2, MMP-9, NOV, Soggy- 1, SPARC, TFMP-2, TLR2 and VEGF-B.
• the higher dose caused a higher amount of the protein, whereas with several proteins the amount was lower with the higher dose.
• the extent of changes suggested dose-dependent responses in this range.
• a number of soluble proteins were identified as cell-surface receptors, such as Fas, HVEM and TLR2, which indicated significant shedding by activated cells. Soluble HVEM was a major protein in tumor-bearing mice, as in healthy mice, but the level in control mice was greater in mice with breast cancer, which led to only a 2-fold increase after treatment with svL4.
• Pentraxin3 ⁇ 4 Mononuclear phagocytes, dentritic cells, and 0.21
• TIMP-2 2.9 Monocytes and placenta (metastasis suppressor) 0.45
• TLR2 4.2 Activated monocytes, dendritic cells, 0.37
• cytokines/chemokines are expressed by a variety of cell types, including activated CD4 and CD8 + T cells, dendritic cells, and macrophages, and are nearly all involved in regulation of the immune system.
• the pattern of expression of cytokines/chemokines provides a strong anticancer immune response.
• cytokines and chemokines increase in the serum in response to treatment, a pattern typical of a toxic "cytokine storm" did not occur.
• a-CTLA-4 is ineffective against melanoma in the mouse unless a source of GM-CSF was added, which routinely was provided by injection of irradiated, engineered GVAX cells [Quezada et al., 2006].
• the inhibitory receptor, CTLA-4 was expressed at high levels by Treg cells within tumors, destruction of cells with bound a-CTLA-4 required macrophages that expressed FcyRIV [Simpson et al., 2013]. Because administration of svL4 causes proliferation and maturation of monocytes, we tested whether svL4 could replace GM-CSF.
• mice Male C57BL/6 mice were implanted on their right flank with 1.5 x 10 5 B16 tumor cells. Subcutaneous injections of 1 nmol/g tetravalent svL4 were initiated on day 0 and given Q2D throughout the experiment. Intraperitoneal injections of 100 ⁇ g a-CTLA-4 monoclonal antibody (clone 9H10 from BioXCell) were given Q3D starting on day 3. Combination treatment retained these injection frequencies.
• Tumor growth was measured on alternate days. Most rapid increases in tumor volume occurred within the group treated with a-CTLA-4 alone, whereas the slowest increases occurred in the group treated with a-CTLA-4 with 1 nmol/g tetravalent svL4 (Table 2).
• mice When the maj ority of the tumors in the untreated group reached a maximum volume of 1,500 mm 3 , the mice were sacrificed and primary tumors from two representative animals from each group were excised and analyzed by flow cytometry. Effector T (Teff) cells were defined as CD3 + CD4 + CD25 " CD39 " while Treg cells were defined as CD3 + CD4 + CD25 + CD39 + [Dwyer et al., 2010]. Uncharacterized CD25 + /CD39 " and CD257CD39 + cells were excluded from the Teff: Treg calculation. A summary of the T cell analyses is shown in FIG. 8. (Examples of the flow cytometry data plots are shown in FIG. 9).
• svL4 complemented the activity of a-CTLA-4 by reducing the population of Treg cells.
• svL4 decreased the number of Treg cells by an a-CTLA-4-independent mechanism.
• a possible explanation for the strong antibody-independent decrease in Treg cells is provided by cytokine data with sera from Balb/c mice bearing breast cancer tumors (Table 1).
• svL4 induced a several-fold increase in IL-12p70, IL-21 and IL-27 within 4 h. These cytokines are implicated in suppression of Treg cell proliferation and a decrease in IL-2 receptor expression [Zhao et al., 2012].
• svL4 did not induce an increase in IL- 10, an inhibitory cytokine secreted by Treg cells.
• the decrease in Treg cells appears more significant for a positive therapeutic outcome than the TeffTreg ratio [Sim et al., 2014].
• the decrease in Treg cells in the groups treated with svL4, along with maturation of macrophages, dendritic cells, CD8 + cytotoxic T cells and natural killer cells that we observed in response to svL4 administration would support a powerful enhancement of the immune system's attack on cancer.
• svL4 is effective in the mouse and is also expected to be effective in humans.
• Antibodies against CTLA-4 are effective in only 20 to 30% of patients, and efficacy is approximately doubled when combined with nivolumab, an antibody against another checkpoint marker PD-1 [Wolchok et al., 2013]. These combinations have increased efficacy because they target different inhibitory receptors.
• addition of svL4 to a treatment with a-CTLA-4, a-PD-1, or a-PD-Ll should enhance benefit.
• the invention satisfies the need expressed by oncologists for a non-toxic backbone or foundational therapy that activates a range of cells and 'primes' the immune system.
• These checkpoint blockade antibodies then can provide focused therapies at lower doses and with less toxicity.
• svL4 Treatment of cancer is usually begun after diagnosis, which is often at the point of advanced disease.
• svL4 is given on alternate days by subcutaneous injection. Activation of immune cells is detected in the mouse after the second or third injection, i.e., day 3 or 5 after the start of treatment.
• Ipilimumab is usually injected intravenously every 3 weeks.
• the continued alternate day administration of svL4 would maintain the immune system at an elevated state of activation and thus establish conditions for the immune system to achieve maximal benefit from the injections of a-CTLA-4.
• a similar method should be feasible with a-PD-1.
• mice (strain C57BL/6, female, 6-8 weeks old) were implanted with glioma cells (murine GL261 cell line) into one hemisphere of the brain.
• tetravalent svL4 was injected subcutaneously at a dose of 1 nmol/g on alternate days for two weeks [Kushchayev et al., 2012a, 2012b].
• mice (strain C57BL/6, female, 6-8 weeks old) with implanted glioma cells
• tetravalent svL4 was administered routinely at a dose of 1 nmol/g on alternate days, which we consider to be a maximal effective but not necessarily the optimal dose. In other experiments we found greater effectiveness at doses of 0.1 to 0.2 nmol/g (0.68 to 1.4 mg/kg). In an experiment with mice into which glioma cells were implanted in the brain, a dose of 0.1 nmol/g alone on alternate days was more effective in causing reduction in the size of glioblastoma tumors than higher doses (FIG. 1 1).
• Ionizing radiation has a synergistic effect with peptide on the immune system, as shown in FIG. 10. Indeed, success in cancer treatment has been found to be largely contingent upon synergy of radiotherapy with the host's immune response.
• high-dose radiation defined as greater than 1 Gy, is immunosuppressive. Nevertheless, radiation up-regulates stress proteins in cancer cells, which improves the ability of antigen-presenting cells to initiate an antitumor response to clear damaged cells by phagocytosis or cytolytic activity [reviewed in Manda et al., 2012].
• PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat. Immunol. 2: 261-268.
• CD45 an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development. Annu Rev Immunol 12:85-116.
• Antibody-based immunotherapy for ovarian cancer where are we at? Ann Oncol 25:322- 331.

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