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  • 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
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• • A—HUMAN NECESSITIES
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  • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/75—Agonist effect on antigen

Definitions

• a pharmaceutical combination for use in the treatment of cancer is provided.
• the present invention relates to the field of pharmaceutical combinations, specifically to a pharmaceutical combination based on a combination of tumor immunotherapy with direct attack of tumor cells by cationic antimicrobial peptides and use thereof for the treatment of cancer, in particular for the treatment of solid tumors and their metastases.
• checkpoint inhibitors are used in the field of cancer immunotherapy.
• Attention is also focused on the use of CAR-T lymphocytes.
• the focus is shifting from anti-CTLA- 4 antibodies to PD-1/PD-L1 intervention and the combination of checkpoint inhibitors with other immuno-, chemo- and radio therapeutic approaches.
• the use of checkpoint inhibitors is effective only in about 20 per cent of patients in a limited number of diagnoses.
• the main reason for the low effectiveness of checkpoint inhibitors is that the defense of tumors against immune attack is very complex and consists of a number of often independent mechanisms. Checkpoint inhibitors and CAR-T resolve only partial issues in this mosaic.
• an immunotherapy composition has been developed that includes Toll-like receptor ligands so called TLR ligands and compounds that label tumor cells as a target for innate immune cell attack.
• This therapeutic mixture has shown good results in the treatment of melanoma (Caisova V, Vieru A, Kumzakova Z et al. (2016) Innate immunitybased cancer immunotherapy: B16-F10 murine melanoma model.
• a suitable therapeutic approach has been very difficult to find because many therapeutic options, such as chemotherapy or radiotherapy, suppress the immune system, and thus their combination with immunotherapy to support the functions of innate immunity is counterproductive. It is therefore necessary to choose another mechanism independent of immunity, which, however, will not disrupt the primary and necessary attack of the immune system.
• the tumor is attacked by components of the immune system; this attack is sufficient for a primary solid tumor, but in the case of metastatic disease, the immune response must be strengthened by another mechanism.
• the object of the invention is therefore to find a pharmaceutical combination that would further improve the tumor immunotherapy with direct attack of tumor cells providing higher effectivity in more patients comparing to the known pharmaceutical combinations.
• the object of the invention is achieved by a pharmaceutical combination of active substances for immunotherapy comprising at least one TLR ligand, at least one compound that labels tumor cells as a target for immune cell attack, at least one anti-CD40 antibody, and at least one cationic antimicrobial peptide.
• Cationic antimicrobial peptides kill directly tumor cells. These substances can be administered simultaneously or sequentially. In this case, the natural anti-apoptotic properties of the tumor are inhibited. On one hand, the tumor is then attacked by the immune system, and on the other hand, its anti-apoptotic defense mechanisms, which make it almost immortal, are weakened. Due to this effect on apoptosis, tumors can respond better to immune therapy while receiving signals leading to their death.
• TLR ligands are substances responsible for the infiltration of tumors by innate immune cells and for their activation. Furthermore, TLR ligands contribute to the formation of the Th1 antitumor environment and promote the formation of co-stimulatory molecules. In this way, TLR ligands promote efficient antigen presentation and the involvement of adaptive immunity.
• the TLR ligand is a substance selected from the group comprising resiquimod (R-848), polyinosinic: polycytidylic acid in the form of free acid or in salt form (poly (I :C)), lipoteichoic acid in the form of free acid or in salt farm (LT A).
• the TLR ligand is a combination of at least two substances selected from the group comprising resiquimod (R-848), polyinosinic:polycytidylic acid in the form of free acid or in salt form (poly (I :C)), lipoteichoic acid in the form of free acid or in salt farm (LTA).
• acid salts designate salts of an acid and a pharmaceutically acceptable cation, in particular alkali metal cations, alkaline earth metal cations, and ammonium cations.
• the compounds labeling tumor cells as the target of innate immune cell attack are mainly ligands of cell phagocytic receptors, such as ligands of dectin- 1 , MR, MBL, CR3, CR4 receptors and scavenger receptors SR-A1 , SR-A2 and MARCO.
• the compound that labels tumor cells as the target of innate immune cell attack is preferably a mannan-biocompatible anchor for membrane (mannan-BAM) conjugate. These compounds bind to tumor cells and label them as targets of innate immune cell attack.
• Innate immunity cells include neutrophils, monocytes, macrophages, dendritic cells, and NK cells.
• the cationic antimicrobial peptides include, for example, Lasioglossins such as Lasioglossin I, Lasioglossin II or Lasioglossin III, LTX-315, crotamine consisting of both L- and D- amino acids.
• Lasioglossins such as Lasioglossin I, Lasioglossin II or Lasioglossin III, LTX-315, crotamine consisting of both L- and D- amino acids.
• the positively charged part of these peptides interacts with negatively charged cancer cells. This interaction is followed by insertion of hydrophobic part of cationic antimicrobial peptides into cytoplasmic membrane creating a hole or an opening in the membrane, resulting in cell death.
• Lasioglossins are pentadecapeptides isolated from bee venom. Their primary sequences are: H-Val-Asn-Trp-Lys-Lys-Val-Leu-Gly-Lys-lle-lle-Lys-Val-Ala-Lys-NH(2) (LL-I), H-Val-Asn- Trp-Lys-Lys-lle-Leu-Gly-Lys-ile-ile-Lys-Val-Ala-Lys-NH(2) (LL-II) and H-Val-Asn-Trp-Lys-Lys- lle-Leu-Gly-Lys-lle-lle-Lys-Val-Val-Lys-NH(2) (LL-III).
• Ruxotemitide is an oncolytic peptide of a following formula:
• the present invention provides a pharmaceutical combination comprising resiquimod, polyinosinic:polycytidylic acid in the form of free acid or in salt form, lipoteichoic acid in the form of free acid or in salt form, mannan-BAM, an anti-CD40 antibody, and at least one cationic antimicrobial peptide.
• the present invention provides a pharmaceutical combination comprising resiquimod, polyinosinic:polycytidylic acid in the form of free acid or in salt form, lipoteichoic acid in the form of free acid or in salt form, mannan-BAM, an anti-CD40 antibody, and at least one cationic antimicrobial peptide selected from Lasioglossin I, Lasioglossin II, Lasioglossin III, or LTX-315, or crotamine consisting of amino acids both in L- and D- form.
• the present invention further provides a method of treatment of cancer, in particular treatment of solid tumors and their metastases, most preferably treatment of pancreatic adenocarcinoma, comprising a step of administering a pharmaceutical combination of active substances for immunotherapy comprising at least one TLR ligand, at least one compound that labels tumor cells as the target of immune cell attack, at least one anti-CD40 antibody, and further at least one compound that suppresses the anti-apoptotic mechanisms of tumor cells to a human or animal subject in need of such treatment.
• a pharmaceutical combination of active substances for immunotherapy comprising at least one TLR ligand, at least one compound that labels tumor cells as the target of immune cell attack, at least one anti-CD40 antibody, and further at least one compound that suppresses the anti-apoptotic mechanisms of tumor cells to a human or animal subject in need of such treatment.
• the step of administration of the pharmaceutical combination includes intratumoral administration of a mixture of active substances for immunotherapy comprising at least one TLR ligand, at least one compound that labels tumor cells as the target of immune cell attack, and at least one anti-CD40 antibody; and simultaneously with the intratumoral administration of the said mixture or prior to the intratumoral administration of the said mixture or subsequently to the intratumoral administration of the said mixture, administration of at least one cationic antimicrobial peptide intratumorally or systemically.
• a mixture of active substances for immunotherapy comprising at least one TLR ligand, at least one compound that labels tumor cells as the target of immune cell attack, and at least one anti-CD40 antibody
• the invention provides a method of treatment of cancer, in particular treatment of solid tumors, most preferably treatment of pancreatic adenocarcinoma, comprising a step of administering a pharmaceutical combination of active substances for immunotherapy comprising resiquimod, polyinosinic:polycytidylic acid, lipoteichoic acid, mannan-BAM, an anti-CD40 antibody, and to a human or animal subject in need of such treatment.
• the step of administration of the pharmaceutical combination includes intratumoral administration of a mixture of active substances comprising resiquimod, polyinosinic:polycytidylic acid, lipoteichoic acid, mannan-BAM, an anti-CD40 antibody, and simultaneously with the intratumoral administration of the said mixture or prior to the intratumoral administration of the said mixture or subsequently to the intratumoral administration of the said mixture, administration of Lasioglossin III, or LTX-315, or crotamine intratumorally or systemically.
• a mixture of active substances comprising resiquimod, polyinosinic:polycytidylic acid, lipoteichoic acid, mannan-BAM, an anti-CD40 antibody
• the substances used in the present invention are commercially available with the exception of mannan-BAM. Although the individual substances or their sub-combinations are already known in the art, it has now surprisingly been found that the combination of these types of substances, i.e. treatment based on the intervention of the corresponding mechanisms has a synergistic effect, causing a tumor to stop growing or even shrink and increase survival even in large and advanced solid tumors.
• the effect of the pharmaceutical combination according to the invention is tested on the example of pancreatic adenocarcinoma, which is a tumor resistant to conventional anti-tumor therapies.
• Mannan-BAM is a representative of ligands that stimulate phagocytosis in our therapy.
• the preparation of mannan-BAM is described below.
• mannan is anchored to the surface of tumor cells. It is recognized by mannan binding lectin so called MBL, which leads to activation of the complement cascade by the lectin pathway.
• MBL mannan binding lectin
• T umor cells labeled in this way are recognized by both phagocytes (neutrophils, macrophages, dendritic cells) and NK cells. Tumor cells are attacked, killed, phagocytosed, and professional antigen-presenting cells (especially dendritic cells and macrophages) subsequently present antigens to naive T cells in the lymph nodes, thereby also involving acquired immunity.
• Fig. 1 shows the effect of combining MBTA immunotherapy with application of Lasioglossin III, LTX-315, and crotamine, respectively in the treatment of mice with pancreatic adenocarcinoma
• Fig. 1A and Fig. 1 B show reduction of tumor growth
• Fig. 1C shows prolongation of survival of mice.
• Tissue culture media their supplements, mannan from Saccharomyces cerevisiae, lipoteichoic acid (LTA) from Bacillus subtilis, and sodium salt of polyinosinic: polycytidyl acid (poly(l: C)) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Resiquimod (R-848) was supplied by Tocris Bioscience (Bristol, UK). Biocompatible Anchor for cell Membrane (BAM, Mw 4000) was obtained from NOF EUROPE (Grobbendonk, Belgium).
• Anti-CD40 monoclonal antibody (rat lgG2a, clone PGK4.5/PGK45) was supplied by BioXCell (West Lebanon, NH, USA). Lasioglossin III was synthesized in IOCHB (Prague, Czech Republic), LTX-315 was synthesized by Schafer-N (Copenhagen, Denmark), and crotamine was delivered by Latoxan (Portes-les-Valence, France).
• the mouse pancreatic adenocarcinoma line Panc02 was donated by Prof. Lars Ivo Parteck (Greifswald, Germany). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and antibiotics (PAA, Pasching, Austria). The cells were cultured at 37 °C in an atmosphere saturated with water vapor and containing 5% carbon dioxide. SPF C57BL/6 mice (females) were obtained from Charles River Laboratories (Sulzfeld, Germany). Mice (weight 18 to 20 g) were housed in a barrier facility in a specific pathogen-free environment with free access to sterile food and water; the photoperiod was 12/12.
• DMEM Dulbecco’s modified Eagle medium
• PAA heat-inactivated fetal bovine serum and antibiotics
• mannan-BAM The synthesis of mannan-BAM was performed as previously described in article from Janotova et al. (Janotova T, Jalovecka M, Auerova M et al. (2014) The use of anchored agonists of phagocytic receptors for cancer immunotherapy: B16-F10 murine melanoma model. PLoS ONE 9: e85222.doi:10.1371/journal. pone.0085222).
• Subcutaneous transplantation Mice were injected subcutaneously with 4 x 10 5 Panc02 cells in 0.1 ml DM EM without additives into the pre-shaved right and left groin.
• MBTA immunotherapy During therapy, 50 pl of the therapeutic mixture consisting of 0.5 mg R-848, HCI form + 0.5 mg poly(l:C) + 0.5 mg LTA + 0.4 mg anti-CD40/ml 0.2mM mannan-BAM in PBS was administered intratumorally in the right tumors on days 0, 1 , 2, 8, 9, 10, 16, 17, 18, 24, 25, and 26.
• the combination of R-848, poly(l:C), LTA, mannan-BAM and anti-CD40 antibody is hereinafter referred to as ‘MBTA’.
• cationic antimicrobial peptides 50 pl of LTX-315 (7 mg/ml PBS), or Lasioglossin III (7 mg/ml PBS) or crotamine (20 pg/ml PBS) was administered intratumorally in the left tumors on days 0, 1 , 2, 8, 9, 10, 16, 17, 18, 24, 25, and 26.
• Tumor sizes were measured every other day using a digital calliper.
• a preparation for therapy based on combination of immunotherapy with the cytotoxic effect of cationic antimicrobial peptides has been tested for the treatment of pancreatic adenocarcinoma, which is considered to be one of the worst oncological diseases.
• Mice with two advanced tumors (bilateral model Panc02) were treated by intratumoral administration of an immunotherapeutic composition (MBTA) to one (right, primary) tumor simultaneously with intratumoral administration of cationic antimicrobial peptides to distal (left) tumor mimicking advanced metastasis.
• MBTA immunotherapeutic composition
• mice were inoculated subcutaneously with 4 x 10 5 mouse pancreatic adenocarcinoma Panc02 cells in 0.1 ml DMEM in the shaved area of the right and left groin. Twelve days after tumor cells transplantation, mice were randomized into groups of six and appropriate therapies were initiated immediately.
• MBTA mixture (PBS in control) was applied intratumorally (50 l MBTA mixture: 0.5 mg R-848 + 0.5 mg poly(l:C) + 0.5 mg LTA + 0.4 mg anti-CD40/ml 0.2 mM mannan-BAM in PBS) in 3 pulses (days 0, 1 , 2... 8, 9, 10... 16, 17, 18... 24, 25, 26) to right tumors.
• LTX-315 7 mg/ml PBS
• Lasioglossin III 7 mg/ml PBS
• crotamine 20 pg/ml PBS
• Fig. 1A, B The combination of MBTA and cationic antimicrobial peptides showed synergy in both reducing tumor growth (Fig. 1A, B) and prolonging mouse survival (Fig. 1C).
• Fig. 1C (A, B) Growth of tumor mass of right/left tumors.
• the pharmaceutical combination for use in treatment of cancer according to the present invention is used for treatment of cancer, more specifically for treatment of solid tumors such as pancreatic adenocarcinoma is.

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