WO2020003009A1 - Vaccin autologue et procédé de préparation du vaccin et surveillance du patient cancéreux pendant et après la vaccination - Google Patents

Vaccin autologue et procédé de préparation du vaccin et surveillance du patient cancéreux pendant et après la vaccination Download PDF

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WO2020003009A1
WO2020003009A1 PCT/IB2019/050752 IB2019050752W WO2020003009A1 WO 2020003009 A1 WO2020003009 A1 WO 2020003009A1 IB 2019050752 W IB2019050752 W IB 2019050752W WO 2020003009 A1 WO2020003009 A1 WO 2020003009A1
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cells
ctcs
autologous
vaccine
patient
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Vladimír BOBEK
Katarína KOLOŠTOVÁ
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Cellpeutics Sp. Z O.O.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages

Definitions

  • the invention relates to a personalized immunotherapeutic anti-tumor vaccine using the immunization property of sporadic cells and their parts, methods of its production and the timing of its production and its administration to the patient based on monitoring of the patient, whereby monitoring relies on the real – time sporadic cell assessment.
  • the vaccine can be used to induce a personalized anti-tumor immune response in autologous vaccination, which causes an immunogenic reaction in the organism against the currently present tumor cells.
  • a tumor cell comes from innate cells of the organism to which tolerance is established. However, tumor cells produce specific tumor antigens that are not present in other types of innate cells of the organism.
  • the immune system uses both the main components of immunity: the innate (non-specific) immune system (macrophages, NK cells, granulocytes, complement) as well as the adaptive immune system (specific T and B lymphocytes).
  • DCs Dendritic cells represent a connection between both the systems.
  • DCs were originally described by Ralph Steinman and Zanvil Cohn in 1973 (5,6). DCs play an important role in mediating the innate immune response and invoking the adaptive immune response. DCs, frequently referred to as a “natural adjuvant” have been recognized to be the most efficient antigen presenting cells (APCs), being able to activate the cells of the naive and memory immune response. DCs have an excellent capacity to acquire and process the antigen and to present it to T cells. Subsequently, they express high level of co-stimulating or co-inhibiting molecules that determine the degree of immune activation or anergy (7).
  • APCs antigen presenting cells
  • DCs first absorb antigens through phagocytosis, pinocytosis and endocytosis with the use of Fc receptors, integrins (avp3 or ⁇ v ⁇ 5), lectin receptors of type C (CLR, including the mannose receptor and DEC205), apoptotic cell receptors and uptake receptors.
  • Antigens are processed into the form of peptides with the use of an endogenous pathway for presentation on MHC molecules of class I to CD8 + T cells, or are processed through an exogenous pathway for presentation on HLA molecules of class II to CD4+ T cells.
  • DCs can also process antigens by cross presentation by connection of the cytosol pathway (8) and vacuolar pathway (9).
  • the antigens are transferred into the cytoplasm, where they are processed by the proteasome before binding to newly created molecules of MHC class; this process may also involve participation of the endoplasmic reticulum.
  • the vacuolar pathway is less marked, but it is expected to be present in endocytic compartments because the pathway is resistant to proteasome inhibitors, but sensitive to inhibitors of lysosomal proteolysis and is dependent on cathepsin S. It has been newly found out that signaling through TLRs (Toll like receptor) influences phagosome maturation and also induces accumulation of molecules in phagosomes for optimal cross-presentation on HLA (10).
  • DCs migrate into the secondary lymphatic tissue as lymph nodes (capturing antigens from the skin and organs), the spleen (capturing antigens from blood), or Peyer's patches (capturing antigens from the intestine lumen).
  • DC infiltration correlates with intratumoral T-cell infiltration and at the same time with a better prognosis in patients with melanoma (12). It has been proved in a mouse model that strategically localized DCs dwell in lymph nodes and can induce the T-cell response much sooner and independently of migratory DC populations (13). These DCs are present in the lymphatic endothelial sinus, where antigens soluble in lymph are captured, and they initiate the immune response without delay. It has been recently proved that the subpopulation that most activates the immune response are DCs expressing the AXL, SIGLEC1 genes - called AS DCs (14).
  • T-lymphocytes To induce efficient immune response, these specific tumor antigens must especially be presented to naive T-lymphocytes. This task is fulfilled by cells called APCs. Their most efficient representatives are mature dendritic cells (mDCs). T-lymphocytes require the antigen to be processed by the APCs: absorbed, broken, bound to molecules of the major histocompatibility complex (MHC) and presented on its surface.
  • MHC major histocompatibility complex
  • DCs are the most important cells among the antigen presenting cells. They express 50 times more MHC molecules than macrophages and on their surface they have a great quantity of costimulatory molecules. This is a heterogeneous group of cells that differ not only with their anatomic location, but also with their surface characteristics and functions. They are dispersed in most tissues. DCs can also absorb bigger fragments of cells carrying various proteins, including those that carry tumor specific antigens, and even whole dead or dying cells. Genes for tumor antigens can be introduced into DCs by means of recombinant viruses or a plasmid. Advantages of DCs activated this way are high concentration of the antigen, its natural conformation and continuous production until the death of these cells.
  • DCs arguedly occupy one of leading positions in the development of anti-tumor vaccines.
  • clinical trials are currently being conducted with activated DCs in patients with solid tumors (melanoma, prostate carcinoma, uterine cervix carcinoma etc.) (1).
  • tumor vaccines most frequently use specific tumor antigens that come from human or animal primary tumors to activate the immune system. These are e.g. vaccines from DCs for the treatment of malignant melanoma (as the antigen e.g. survivin, telomerase, MART-1, MAGE-3 is used), renal tumors (again survivin, TERT), prostate carcinoma antigen (PSA), colorectal carcinoma antigen (CEA) etc. (2).
  • antigens for a particular histological tumor type are used that is well specified and described.
  • whole tumor cells coming from primary tumors or from uniform tumor lines are used for the preparation of DC vaccines (3,3a).
  • Immunotherapy with the use of DCs based on vaccination principles is an approved procedure to use the potential of the patient's innate immune system to remove tumor cells of metastasizing carcinomas, e.g. in the case of hormone refractory prostate carcinoma.
  • many types of DC vaccines have been tested and in some cases their administration was associated with a clinical result, there is a number of possible components, especially antigens for the generation of DC vaccines, and relatively functional vaccination schedules.
  • Vaccines whose administration will be integrated with local activation of the immune response especially appear to be significant in future.
  • Another problem is that traditional monitoring of the immune response by means of analyzing some activated cell subgroups (e.g. -CD8+IFN ⁇ +) does not correspond to the clinical result of therapeutic vaccines.
  • activated cell subgroups e.g. -CD8+IFN ⁇ +
  • enhanced knowledge of the biology of DC and the key mechanisms that participate in generating the immune response have provided valuable tools for the improvement of DC-based vaccination substances and established them as a potential treatment strategy for patients with cancer.
  • Circulating tumor cells represent a direct interconnection of the primary tumor and metastases as these cells are released from the primary tumor to the bloodstream, where they subsequently circulate and “seed” remote tissues with respect to the primary tumor, or they re-seed the tumor they originally moved away from.
  • the theories of seeding (concerning remote tissues) and self-seeding (re-seeding of the original tumor) have so far provided a sufficient amount of evidence that this is a process programmed on the level of tumor cells and tumors, but at the same time closely related to the functionality of the immune system. In a simplified manner, this could be described as when the immune system does not work, tumor cells proliferate.
  • CTCs are essential for the occurrence of distant metastases and their detection is considered as a manifestation of aggressiveness of tumors and their ability of metastasize into remote organs.
  • Current findings indicate that metastatic dissemination may often be an early manifestation of tumor progression and not only a manifestation of an advanced tumor disease.
  • Properties of CTCs are directly related to dynamic modifications of the stage of the disease. In one patient, the mutation profiles of CTCs before and after the treatment only share few sequential variants (5).
  • antigens of CTCs can be assumed to be unique and to differ from the primary tumor both on the level of gene expression and of the mutational profile of DNA (4,5).
  • CTCs are the only tumor tissue that is accessible relatively non-invasively and repeatedly.
  • the examination comprising a CTC test is also called fluid biopsy, just as it provides the possibility of using CTCs as markers of a tumor disease.
  • CTCs can be used as a prognostic, predictive or diagnostic marker has been verified in several hundreds of clinical trials. To put it simply, CTCs can be used to monitor the course of a tumor disease, especially in situations when the response to the type of the administered treatment is of interest.
  • Generating DCs for clinical trials comprises cultivation of DCs from monocytes or CD34+ haematopoietic precursors and possibly enrichment of the circulating blood DC subgroups.
  • the generated DC subgroups differ from each other; however, in all cases, activation of the immune response has been confirmed preclinically as well as clinically.
  • DC vaccines used in clinical trials usually mature for 24-48 hours and the production of cytokines is evaluated just within this time period (18). However, most of the cytokines produced in the course of DC maturation are expressed during 24 hours. Then, their production is re-started after encountering T cells and the CD40L receptors. Protocols where DCs are matured for up to 24 hours may be ideal to maintain the capability of producing cytokines after an injection in vivo.
  • DCs are usually activated by incubation with peptides, proteins, RNA or autologous/allogeneic tumor cells (19). Peptides are directly presented by MHC molecules on the surface of DCs while proteins and tumor cells require processing to peptides before binding to MHC molecules.
  • DCs Several studies have confirmed the ability of DC cells to induce a strong specific T cell response after mRNA activation (20-23).
  • Transfection of DCs with mRNA can be achieved with the use of e.g. a cationic lipid (i.e. DOT AP N-( 2,3-dioleoyloxy-1-propyl) trimethylammonium methyl sulfate) or by electroporation (24-28).
  • a cationic lipid i.e. DOT AP N-( 2,3-dioleoyloxy-1-propyl) trimethylammonium methyl sulfate
  • electroporation 24-28.
  • lymph nodes Migration into lymph nodes (LN) is critical for the induction of the immune response.
  • DC vaccines have been administered intradermally, subcutaneously, intravenously, intranodally or intratumorally, the optimum administration mode has not been determined yet.
  • Intratumoral administration of DC vaccines showed retention in the puncture site with a small quantity of DCs in the lymph nodes (29,30).
  • More recent strategies involve administration of DC vaccines using more than one administration mode, i.e. intradermally, intravenously and intranodally.
  • DC vaccines can contain mature DCs (phenotypically mature as documented by upregulation of costimulation molecules), (32). They can also produce IL-12, which is a key factor for inducing efficiency of the vaccine. However, it is not known how much IL-12 is actually sufficient and whether DCs will still produce IL-12 after the injection or in case of encountering T cells. Since there is a causal relationship between the production of IL-12 and the efficiency of the vaccine, it is essential to know whether IL-12 is actually produced after a DC injection (32).
  • circulating tumor cells may be a prognostic, predictive, diagnostic as well as a therapeutic marker.
  • the invention relates to the use of circulating tumor cells (CTCs), either separately or in combination with other tumor cells to produce vaccines from dendritic cells (DCs).
  • CTCs circulating tumor cells
  • DCs dendritic cells
  • the immune reaction against the tumor process can be surprisingly induced with the use of the so-called CTCs isolated from the patient in a time period when his/her health condition is satisfactory (i.e. no sign of tumor progression is present: the tumor is not growing, metastases are not detected, protein biomarkers are normal).
  • the only thing that reflects ongoing activation of tumor processes during this period is a growing number of CTCs in a drawn sample of the patient's peripheral blood.
  • CTCs in the patient's blood in the remission period has already been described, but according to the present invention, these cells can be used for immunization in the time period when the patient is in the state of remission according to the clinical terminology.
  • the newly evolved tumor cells shed in the bloodstream are actually used to find “relative” tumor cells in the bloodstream and elsewhere according to the invention, in addition before any progression can be detected with the use of prior-art methods.
  • biological material isolated from the patient is exclusively used, in the case of tumor cells in the form that is incapable of proliferation, so there is no additional risk for the patient.
  • CTCs can be used to detect an incoming relapse of the disease and to start to direct the immune system against something that de facto does not exist to a greater extent yet - i.e. the invention related to personalized therapeutically preventive vaccines.
  • CTCs appearing in the patient's blood in the remission period can be assumed to have been present in the patient's body in the period of the previous conducted therapy, which means that they are uniquely and individually specifically modified by therapeutic interventions, which also contributes to specificity of their antigenic portfolio as will be shown further below.
  • tumor cells are a heterogeneous population of cells, are not generically stable and in the course of an oncological disease more new and antigenically different tumor cells are continuously produced.
  • various variants of tumor cells are produced and spread in the course of the disease while these cells are different both from the original cells of the primary tumor and from each other.
  • Newly produced tumor cells are also different in case an experimental animal is inoculated with identical cells of the same cell line for the purpose of induction of homogeneous models of tumor growth.
  • the production of changed tumor cells is further induced by the ongoing therapy, both on the level of chemotherapy and on the level of biological therapy.
  • the invention also provides a sufficiently fast preparation method of an autologous anti-tumor vaccine based on activated dendritic cells to enable completion of the preparation and application of the vaccine in the period when the newly formed tumor cells are not subject to the next genetic change yet.
  • DC vaccines can be advantageously prepared repeatedly in short interval thanks to an available source of antigens.
  • the invention successfully solves the essential problem of current vaccines based on DCs, most frequently produced from cells of a cell culture, e.g. commercial cell lines, or from cells of the primary tumor as these vaccines cannot activate the immune response to newly formed, antigenically different tumor cells that circulate in the bloodstream and that can create or have created remote metastases in spite of the ongoing therapy.
  • all patients with the same primary tumor type receive the same standard add-on (adjuvant) treatment after its removal at present.
  • the relatively low number of CTCs in peripheral blood approximately equal to an antigen presenting cell and a CTC will meet in the blood, inducing an immune response of the organism.
  • CTCs are considerably differentiated so an antigen presenting cell of the immune system frequently does not recognize a CTC and does not identify it as a potential threat.
  • the aim is to achieve ideally a long-term and individually specific response to immunization by means of the currently produced vaccine.
  • the effect of the vaccine, or efficiency of the immunization process can be then (repeatedly) monitored during the time period following the administration of the vaccine.
  • the direct effect is measurable by an increase of CD8+IFN ⁇ +, however, repeated tests have not confirmed correlation between these cells and the real clinical impact.
  • the efficiency of the vaccine can be monitored indirectly by means of the CTC level in peripheral blood.
  • a long-term response will not mean complete eradication of CTCs and tumor cells e.g. in metastatic foci, the aim is to minimize their number, providing a sustainable model of tumor tissue growth shown in Fig. 3, also called “stable disease”.
  • CTCs Molecular testing of the characteristics of primary tumors and CTCs shows that besides antigens of the primary tumor, CTCs also express antigens that can be considered as new signs of aggressiveness of the tumor disease. Thus, CTCs are used as a source of new targeted antigens, i.e. antigens that treatment can be directed against.
  • the first embodiment of the invention is an individual autologous vaccine based on viable autologous dendritic cells for induction of an immune response against circulating tumor cells to invoke a personalized anti-tumor immune response on autologous vaccination.
  • the vaccine induces an immunogenic reaction against the currently circulating and other tumor cells.
  • the vaccine contains as the active component the patient's innate antigens, these innate antigens being antigens coming from autologous CTCs, currently isolated from the patient's peripheral blood.
  • Antigen in the sense of biological material usable for application of DCs will hereinafter refer to material derived from tumor cells in a form selected from the group: apoptotic tumor cells, lysed or killed tumor cells; isolated parts of tumor cells selected from RNA, especially mRNA, DNA and proteins; and from cells of released vesicles, which may be macrovesicles and microvesicles, e.g. exosomes formed spontaneously from CTCs in higher numbers than is common with other cells, or lyophilized parts of CTCs.
  • one form is used, e.g. lysate, but simultaneous use of two or more forms of antigens from tumor cells is not excluded either.
  • Viable intact tumor cells are not suitable as the antigen due to risks for the patient.
  • the above-described antigen is processed by dendritic cells in a natural way and presented in a form suitable for the patient's immune system.
  • anti-tumor vaccines For the anti-tumor vaccines to be efficient, they must be prepared individually for every patient with the use of the patient's innate immunization antigens.
  • autologous innate herein means that the material is collected individually from every single patient for his own vaccine production.
  • isolated means that the material is freed of at least some other materials it usually occurs in its natural environment with.
  • currently isolated means that the material is collected from the patient at such a point of time that the prepared vaccine can correspond to the current time development of the tumor disease of the patient, e.g. according to an individual time schedule adapted to the current state, with the aim to personalize the preparation of the vaccine.
  • Such a suitable point of time can be determined by monitoring with the use of standardized diagnostic methods as ultrasound, magnetic resonance (MRI), positron emission tomography-computer tomography (PET-CT) and/or with the use of standard tumor markers (PSA, CA125, CAE) and/or in a preferred embodiment even substantially earlier than with the use of the well-known diagnostic methods, by means of a marker according to the present invention, which is the presence and/or number and/or characteristics of CTCs in the patient's peripheral blood.
  • diagnostic methods as ultrasound, magnetic resonance (MRI), positron emission tomography-computer tomography (PET-CT) and/or with the use of standard tumor markers (PSA, CA125, CAE) and/or in a preferred embodiment even substantially earlier than with the use of the well-known diagnostic methods, by means of a marker according to the present invention, which is the presence and/or number and/or characteristics of CTCs in the patient's peripheral blood.
  • CTCs refer to cells being released from the primary or secondary tumor to the bloodstream. Determination of CTCs can be carried out within fluid biopsy, referring here to collection of peripheral blood and its analysis for CTCs, fragments of circulating free DNA (cfDNA) from tumor cells, exosomes etc., wherein the result of the examination can be specification of the ongoing oncological disease, especially its development dynamics.
  • fluid biopsy referring here to collection of peripheral blood and its analysis for CTCs, fragments of circulating free DNA (cfDNA) from tumor cells, exosomes etc.
  • CTCs present in the blood should be a marker that can differentiate patients that need further additional treatment after passing a certain line (stage) of treatment (e.g. adjuvant). Therefore, personalization of oncological care is mentioned in the context of CTCs and fluid biopsy, namely on the basis of characteristics of CTCs.
  • CTCs Current collection of CTCs to obtain antigens is suitably carried out during a relapse stage detectable on the level of the above-mentioned standard diagnostic methods and/or on the level of an increased number of CTCs and/or CTC clusters (i.e. clusters of especially CTCs that can be detected in the blood grouped with cells of the immune system and/or erythrocytes and/or thrombocytes).
  • the strategy of using CTCs for the production of personalized vaccines is based on the possibility of monitoring the course of standard treatment of patients with the use of CTCs.
  • CTCs can be isolated and used for the production of a vaccine that will be unique for this time period (i.e. currently in the real time) and will induce an immune response especially against antigens present in CTCs.
  • CTCs can be advantageously obtained from a non-adherent fraction (supernatant) of the monocyte fraction of cells that is formed during the preparation of dendritic cells as will be described below.
  • the supernatant can be used as a supplementary/alternative source of CTCs and/ or as a source of lymphocytes that can be stored at -20 °C for later used under standardized conditions according to protocols comprising freezing of cells.
  • the currently isolated CTCs are CTCs isolated from the supernatant from the non-adherent monocyte fraction of peripheral blood cells in a culture flask after 1.5 - 3h incubation in a culture medium.
  • CTCs are preferably obtained from the said supernatant by filtration as described below.
  • CTCs can be preferably obtained by blood filtration on a separation membrane with the pore size of 5 - 12 ⁇ m, more preferably with the use of capillary forces and most preferably as viable CTCs that will allow both cultivation to obtain a higher number of CTCs as will be described in more detail below, and obtaining undamaged RNA from CTCs.
  • the goal of the lysis of tumor cells is to obtain antigens in the form of e.g. proteins and RNA and at the same time the possibility to use lysis buffers that are suitable for samples with a small number of cells (1 to 1000 cells) and do not require further purification steps.
  • a typical individual tumor cell comprises relatively few transcripts of most genes. Sequencing data indicate that in murine embryonal stem cells there are approximately 505,000 mRNA molecules, in murine embryonal fibroblasts there are approximately 22,000 mRNA molecules (51,52). It is obvious that within the analysis of individual cells, every loss during the extraction caused by washing can bring about a significant and even a complete loss of some transcripts (53,54).
  • RNA accessible and maintains its integrity without inhibition of the downstream enzymatic reactions also in the course of freezing and defrosting.
  • methods 3a - 3c described below, were used.
  • CTCs are getting from the position of a marker that describes the current state to the position of an effector that determines what antigens the immune response will be directed against.
  • CTCs are an aggressive group of cells that initiation of the growth of metastatic tumor foci can be ascribed to. Based on the hypothesis that metastases would not appear without CTCs, the object of the fight against a tumor disease should not only be the primary tumor, secondary tumor, but also CTCs according to the authors of the invention.
  • CTCs compared to other tumor cells used in the preparation process of vaccines is, besides the above-mentioned low collection requirements, also the time profile of their occurrence.
  • first CTCs appear in blood at least in a situation when the primary tumor achieves the size of 1 mm 3 and a reappearance of CTCs is associated with a worse prognosis and a lack of response to the administered therapy (see Fig. 1) (6).
  • the therapeutic use of CTCs according to the invention brings a substantial advantage as compared to other types of tumor cells, especially disseminated tumor cells used for vaccination so far.
  • DTCs disseminated tumor cells
  • a body fluid refers to any fluid filling any bodily cavity naturally (as e.g. bone marrow, cerebrospinal fluid) and/or due to an advanced tumor disease (as pleural, pericardial and synovial exudate, ascites, cephalic fluid), or a fluid introduced into a bodily cavity (peritoneal wash, uterine wash).
  • DTCs can only be isolated in the final stage of an oncological disease, e.g. after several months (their presence is an adverse prognostic indicator for the patient), which makes them unsuitable for monitoring of the course of therapy.
  • DTCs provide a sufficient quantity for immunization in late stages of a disease, but on the other hand, this will complicate the fight of activated cells of the immune system against the created excess of tumor cells.
  • CTCs can be advantageously continuously isolated in a situation when they can be used to monitor the progress of the disease or the treatment process. If an increasing number of CTCs is detected, the same cells are advantageously used as a source of immunization antigens as long as the disease has not progressed into a stage when an excessive presence of tumor cells is life threatening. So the present invention also makes it possible to determine the most suitable moment of transition from monitoring to therapy.
  • DTCs are usable in other preferred embodiments of the invention as an auxiliary source of antigens for activation of dendritic cells as an adjuvant (add-on) therapy supplementing the CTC therapy.
  • DTCs can be obtained by filtration of collected body fluid similarly to CTCs on a separating membrane with the pore size of 5 - 12 ⁇ m, more preferably with the use of capillary forces and most preferably as viable cells, and can be used for activation of DCs in the same forms as CTCs.
  • Antigens from primary tumor cells isolated from the patient's primary tumor can also be used in an adjuvant manner in a vaccine according to the invention.
  • Primary tumor cells are especially applicable in cases when due to the size of the primary tumor and an advanced stage of the disease, during an indicated surgical removal of the primary tumor, not only a sufficient quantity of biological material for the diagnostics, but also a sufficient quantity of tumor tissue for isolation of tumor cells of the primary tumor can be obtained.
  • antigens can be in the form mentioned above for antigens from CTCs again.
  • Tumor cells used in a vaccine according to the present invention are preferably cells that are enriched to increase their quantity/concentration, preferably by filtration based on the cell size and/or proliferated by means of short-term 3-5-day and/or long-term 5-21-day cultivation in vitro, preferably in a culture medium without the addition of serum and/or with addition of the patient's autologous serum and/or one or more other usual growth factors. It has been found out that long-term cultivation is advantageous especially for preservation of CTCs e.g. in a frozen state and obtaining a higher number of antigens. Short-term cultivation is advantageous for immediately following vaccination for quick obtaining of minimally genetically altered cells. Cells from both the types of cultivation can be used for the activation of DCs, which can partly simulate variability of cells in the course of time.
  • Concentration of cells refers to separation of CTCs from peripheral blood (or DTCs from a body fluid), preferably by filtration based on the size of cells, again preferably on a separating membrane with the pore size of 5 - 12 ⁇ m, and more preferably with the use of capillary forces, when the concentration of CTCs/DTCs in the obtained preparation is increased at least with the factor of 10 6 .
  • This way, a few CTCs/DTCs up to hundreds of CTCs/DTCs can be obtained.
  • Tumor cells can be preferably cultivated in vitro, which lead to proliferation, which is in some cases (e.g. the number of CTCs per 8 ml of blood is lower than 100) necessary to achieve a sufficient quantity of tumor cells for immunization. Viable cells are necessary for this.
  • a sufficient quantity of CTCs means an empirically determined quantity of cells that can invoke an immune response. The determination can be done by a skilled person with the use of routine procedures. A sufficient quantity is preferably on the order of hundreds of CTCs or DTCs. Cultivation is not necessary in case in vitro RNA amplification is used as described below. Cultivation may be also be advantageous to e.g. purify CTCs of white blood cells, as a control point for counting, documentation etc.
  • Cultivation for CTCs/DTCs is advantageously carried out under standardized cultivation conditions (37 °C, 5 % CO 2 ) in a culture growth medium as e.g. CellGRO, Xvivo, RPMI, DMEM, MEM.
  • a culture growth medium as e.g. CellGRO, Xvivo, RPMI, DMEM, MEM.
  • a serum- free medium is suitable in a situation when it is desirable to maximally restrict the influence of external and non-autologous factors as e.g. cytokines from fetal bovine serum leading to activation of an immune response.
  • Cultivation in a medium with an addition of autologous serum leads to minimization of external influences on immunization of the patient's DCs.
  • Cultivation conditions can be determined by a skilled person without the need of experimenting or with simple tests.
  • RNA molecules e.g. mRNA isolated from the CTC can be non-specifically amplified, preferably by in vitro propagation of tumor cells or in vitro RNA amplification) and subsequent immunization of dendritic cells by means of these transcripts.
  • DCs prepared this way are capable of presenting tumor antigens in the lymph nodes and of activating antigen-specific T lymphocytes in the patient's body.
  • another embodiment of the invention is an autologous vaccine where antigens are presented by autologous in vitro matured DCs after activation by the above mentioned forms of antigens derived from CTCs while RNA can be non-specifically amplified, preferably by in vitro propagation of tumor cells or in vitro RNA amplification.
  • Dendritic cells can be obtained from monocytes.
  • Monocytes represent a type of leukocytes, agranulocytes that forms 3-8 % of peripheral blood leukocytes. They are produced in the bone marrow from a separate development line of primitive leukocytes (reticular cells). Precursor cells are called monoblasts, which monocytes are differentiated from through the promonocyte stage. They circulate in blood for about 8 hours and then they enter tissues, get even bigger and become macrophages. Monocytes are virtually non-functional in the peripheral blood so they are precursors of macrophages or also immature macrophages. The main function of circulation of monocytes in blood is to provide a supply of macrophages for the needs of all tissues in the body. Blood as the source of monocytes can be collected as peripheral blood or the patient is subjected to leukapheresis, which separates the monocyte fraction on the basis of the cell size.
  • Monocytes can also be isolated from blood using a density gradient isolation protocol, e.g. Histopaque 1077, when the entire PMBC (peripheral blood mononuclear cells) fraction of polymorphonuclear cells of peripheral blood is separated and subsequently, monocytes are isolated thanks to their adhesion to plastic surfaces.
  • the obtained PBMC fraction is, after possible washing steps e.g. with phosphate buffer (PBS), for this purpose incubated in a culture medium as e.g. CellGRO, Xvivo, RPMI, DMEM, MEM, suitably in a culture flask for approx. 1.5 - 3 h. This time period during which monocytes settle and adhere to the bottom of the culture flask is different in individual patients.
  • the rest of non-adhered cells is removed in the supernatant, which can still be used for the isolation of CTCs as described above.
  • the settled adhered monocytes can be differentiated by cultivation towards a cell structure that has characteristics of especially dendritic cells (DCs).
  • DCs dendritic cells
  • DCs are produced by in vitro cultivation of monocytes obtained by separation from the monocyte fraction of peripheral blood cells on a separation membrane with the pore size of 7 - 10 ⁇ m, more preferably with the use of capillary forces. This is preferred because separation on a separation membrane, especially with the use of the capillary force, is considerate to monocytes, and further for the high separation rate and low price.
  • Another possibility of obtaining a pure monocyte culture is elutriation, i.e. automatic separation of PBMCs, or their immunomagnetic separation based of positive selection of cells expressing the antigen CD14 (CD14+).
  • Another advantageous possibility of obtaining matured dendritic cells for the purposes of the invention, especially CD1+ CD19 cells is a closed automatic system for maturation and activation of DCs using the principle of immunomagnetic separation (ClinimacsTM, MiltenyiBiotec,http://www.miltenyibiotec.com/en/clinical-applications/clinimacs-system/clinimacs-instruments/clinimacs-prodigy.aspx).
  • a source of DCs can further be separate monocytes, CD34+ positive bone marrow cells and free differentiated DC cells of a myeloid (mDC) or plasmatocytoid (pDC) DC character.
  • mDC myeloid
  • pDC plasmatocytoid
  • Enhanced production of DCs can be induced by administering an injection of the growth factor Flt3.
  • Cultivation of monocytes is generally carried out to differentiate cells with the properties of DCs (i.e. a population of immature DCs) from monocytes during the cultivation.
  • DCs i.e. a population of immature DCs
  • Additions of various growth factors (especially IL-6, GM-CSF) to standard culture media as e.g. CellGRO, Xvivo, RPMI, DMEM, MEM) are used in the course of the cultivation of monocytes to DCs.
  • standard culture media as e.g. CellGRO, Xvivo, RPMI, DMEM, MEM
  • the cultivation time is preferably 2-6 days.
  • Immature DCs mature immediately after being subsequently in vitro exposed to an encounter with an antigenic stimulus. Maturation of DCs is essential for the presentation of antigens to immune cells.
  • Activation of DCs means their transition to a state when they are able to further present the antigen.
  • Active DCs present the HLA-antigen complex to T cells CD4 and/or CD8 to induce clonal amplification of T-cell response.
  • DCs are preferably activated in vitro by means of one or more above-mentioned forms of antigens derived from circulating tumor cells.
  • an autologous vaccine can additionally contain DCs activated in vitro by analogous materials derived from DTCs and/or primary tumor cells.
  • activation of DCs can be carried out with materials (including whole attenuated cells) derived from CTCs, DTCs, or primary tumor cells separately, or the said materials can be used for activation of DCs in any mixture.
  • Attenuated CTCs are CTCs that have been deprived of their ability to proliferate,but are still viable cells so that DCs can present new antigens obtained from CTCs to the immune system.
  • the attenuation is carried out e.g. with the use of UV-C or electromagnetic irradiation.
  • Apoptotic CTCs are cells obtained e.g. by incubation outside a CO 2 incubator with signs of apoptosis as e.g. presence of a fragmented cell nucleus or presence of caspase 3.
  • Dendritic cells are preferably produced by in vitro cultivation from a monocytary fraction of peripheral blood cells collected before the therapy or at the time of determination of the patient's diagnosis, especially before the start of chemotherapy, radiotherapy, biological or surgical treatment, to obtain cells that are not influenced by these interventions.
  • Another embodiment of the invention relates to the use of autologous CTCs, currently isolated from the patient's peripheral blood as a source of antigens for the production of an autologous vaccine for induction of the immune response against the currently present tumor cells of the patient.
  • CTCs the use may analogously comprise antigens of autologous DTCs, currently isolated from the patient's body fluids, and/or primary tumor cells, currently isolated from the patient's primary tumor.
  • the said cells as well as derived materials may be used separately or in any combinations.
  • a number of cells may be empirically determined which the number of cells determined at a certain time point is compared to and if the determined number of cells is higher than the said threshold number of cells, e.g. higher than 10 or higher than 100 cells, the preparation of a vaccine is started. Further, a percentage may be set and if the determined number of cells is e.g. 5% higher than the set number of cells or 5% higher than the number of cells determined at a prior time point, the preparation of a vaccine is started etc. This way, a significant difference may be defined, i.e. such a rate of exceeding of a determined threshold value when the production of a vaccine should preferably be started. A significant rate of exceeding the value of the quantity of CTCs is e.g.
  • the preparation of a vaccine can be started even if a single CTC is found (the number of CTCs is not a limiting factor for the start of the vaccine production process) by RNA transcription in vitro and subsequent immunization of dendritic cells by means of these transcripts.
  • the defined time intervals between collections can be the same or different and they can e.g. take from one week to one year.
  • another object of the invention is the use when samples of peripheral blood are additionally collected from a patient in determined time intervals wherein the presence of CTCs and/or their sensitivity to the autologous vaccine produced currently for an earlier time point is tested ex vivo, and the preparation of an autologous vaccine produced currently for a later time point is started if the number of isolated CTCs for the later time point is higher by a significant difference than their specified threshold number and/or the number of isolated CTCs for the earlier time point, always in relation to the same volume of blood.
  • samples of body fluids can be additionally collected from the patient in regular time intervals wherein the presence of DTCs and/or their sensitivity to the autologous vaccine produced currently for an earlier time point is tested ex vivo.
  • a vaccine according to the invention can be suitably administered before the start of chemotherapy, after the end or interruption of chemotherapy, alternatively also with another biological treatment, preferably in situations wherein the use treatment is immunotherapeutic one.
  • Another object of the invention is a preparation method of an autologous vaccine for induction of the immune response against tumor cells wherein the vaccine comprises as the active component the patient's innate antigens wherein the method comprises the following steps:
  • the DCs in step d) are preferably produced by in vitro cultivation from a monocyte fraction of peripheral blood cells collected before or at the time of determination of the patient's diagnosis, especially before the start of chemotherapy, radiotherapy, biological or surgical treatment, to obtain a sufficient quantity of cells not influenced by this therapy.
  • steps b) to d) above may additionally comprise one or more steps, however always in the order specified below, selected from the group:
  • the material obtained above from step d) is generally intended for immediate use so it can be directly used for vaccination, or it can just be supplemented e.g. with a buffer and/or the osmotic pressure can be adjusted e.g. with the use of NaCl, or possibly a preservative can be added.
  • the vaccine or antigen can be stored before the use at -20 to -80 °C.
  • the test for the capability of phagocytosis is preferably used to test sufficient maturation of DCs as only a functional, mature DCs are capable of phagocytizing antigens from its surroundings.
  • the used test which will accelerate the entire process of the degree of maturation of DCs is commercially available (pHrodoTM Green Zymosan BioparticlesTM Conjugate for Phagocytosis, ThermoFisherScientific) and is based on the principle of activation of fluorescence at a certain degree of acidity (pH) so the result is that if a cell phagocytizes proteins from its surroundings, its phagosomes become green (fluorescence microscopy).
  • the new pHrodo® Green conjugates which are sensitive to pH, provide faster and more precise results than other phagocytosis tests.
  • the pHrodo® Green conjugates are not fluorescent outside a cell at neutral pH, but they fluoresce brilliant green at acidic pH, e.g. in phagozomes.
  • Using this type of a phagocytosis test is advantageous compared to other more complicated and more expensive methods as flow cytometry and efficiently saves time.
  • tests focused on the activity of the endocytosis/pinocytosis process can be used.
  • Another possibility of maturation testing consists in analyzing gene expression of a selected group of genes that are typical for mature DCs.
  • An example is expression of e.g. CD80, CD83, CD86 and HLA-DR as shown in Table 2. This will bring a principal acceleration of the entire process of quality testing of the vaccine compared to the prior art based e.g. on analysis of proteins with the use of flow cytometry.
  • the gene expression test can be conducted with a substantially smaller quantity of mature DCs.
  • Mature DCs can be stored at -20 to -80 °C for several weeks (58).
  • Antigens are preferably presented to autologous in vitro matured DCs after activation as antigens from CTCs in the above-mentioned forms while RNA can be non-specifically amplified, preferably by in vitro propagation of tumor cells or in vitro RNA amplification.
  • the preparation method of a vaccine based on CTCs described above can additionally comprise steps during which DTCs obtained from the patient's body fluids are processed separately in an analogous manner.
  • the DTC-based vaccination material then acts together with the CTC-based vaccine as an adjuvant vaccine.
  • acting together means mixing of immunizing antigens (the above-mentioned forms of antigens coming from CTCs and DTCs) during the activation of immature DCs.
  • immunizing antigens the above-mentioned forms of antigens coming from CTCs and DTCs
  • the result of combining the CTC and DTC immunization is only one vaccine.
  • Application of separate CTC and DTC vaccines can also be considered so that their effects can overlap in a certain period.
  • the preparation method of an autologous vaccine may additionally comprise the following steps, separately from the preparation of the CTC vaccine
  • dendritic cells are preferably used that are produced by in vitro cultivation from a monocytary fraction of cells collected before or at the time of determination of the patient's diagnosis, especially before the start of chemotherapy, radiotherapy, biological or surgical treatment.
  • one or more supplementary steps may be carried out, however always in the order specified below, selected from the group:
  • the vaccine use as well as the methods according to the invention will further make it possible to reduce costs of anti-tumor vaccination because when CTCs are used for DC activation according to the invention, not only the costs of obtaining the primary tumor during the surgery, purification of the primary tumor cells etc are eliminated, but also to induce the same systemic immune reaction as with prior-art vaccines, a lower quantity of DCs will be necessary, which will however be more specifically immunized and provide a more specific and more efficient immune response.
  • a vaccine according to the invention can be generally applied e.g. subcutaneously, intranodally, intravenously. All the above-mentioned administration types will be tested on the level of a clinical trial.
  • the invention provides a novel sufficiently sensitive, efficient, acceptable and safe for the patient, easy to implement in practice and from the point of view of costs acceptable method of monitoring the patient in the course of a tumor disease or before its occurrence for identification of appearance of circulating tumor cells, i.e. an increased risk of occurrence of a tumor.
  • CTCs can be considered as a valid prognostic marker of a tumor disease.
  • the invention makes it possible for CTCs to become a predictive biomarker, i.e. one determining probability of efficiency of the scheduled therapy.
  • the invention further enables use of circulating tumor cells as a diagnostic biomarker, especially in situations when standard methods cannot be used to determine the origin of a primary tumor.
  • the novel method of monitoring with the use of CTCs may replace or supplement the traditional monitoring of the immune response to vaccines by means of analyzing some activated cell subgroups (e.g. -CD8+IFN ⁇ +), which does not correspond to the clinical result.
  • the novel method of monitoring is especially advantageous for determining the moment when to start further therapy, especially preferably when to start a therapy with one of the vaccines according to the invention described above and/or when to start preparation of a vaccine for induction of the immune response by means of some of the methods according to the invention described above.
  • the invention also comprises a monitoring method of the patient to determine whether the preparation of a current autologous vaccine for induction of the immune response against tumor cells should be started and/or for the occurrence, progress or recurrence of his/her tumor disease wherein the vaccine comprises as the active component antigens coming from cells of the individual patient or whether different diagnostics or therapy should be started wherein the method comprises the following steps:
  • step e) of preparation of a new current autologous vaccine is skipped.
  • the invention further provides the monitoring method described above wherein in points c) to e) instead of or additionally to the number of CTCs, at least one marker present on the CTCs and/or at least one marker associated with the tumor, used as standard for the particular type of tumor tissue is used for the monitoring of expression determination.
  • the size of the current sample of the patient's peripheral blood is at least 2 x 8 ml due to a low number of CTCs, more is preferred.
  • CTCs always means gene expression of specific genes associated with the tumor as shown here e.g. in Fig. 5.
  • the number of cells always refers to the number of cells related to the same volume of blood or body fluid.
  • a prior collection may be either the collection immediately preceding the current collection or one of earlier collections.
  • testing is preferably carried out by determining the gene expression of a suitable marker and/or testing for the presence of mutations, most preferably with the use of next generation sequencing (NGS).
  • NGS next generation sequencing
  • the monitoring method of the response to treatment also comprises testing of the mutation and expression profile of the primary tumor (viable and/or fixed), e.g. by continuously monitoring the blood for the presence of antigens specific for the primary tumor.
  • Monitoring gene expression using the qPCR method, mutational analysis or using the NGS method can be suitably used again.
  • the monitoring method of the response to treatment can advantageously also comprise testing of the concentration and mutation profile of free circulating DNA wherein at least one biomarker of the mutational origin is used that was originally detected in CTCs and/or in the primary tumor and/or in DTCs. It is true that testing the mutation profile of free circulating DNA is well-known in the art, but not the use of the ratio of the quantity of free DNA and mutated free DNA for quick monitoring of the cell response to vaccines. With respect to the number and/or character of CTC, free circulating DNA and free mutated DNA clearly appear to be the best test for monitoring of immunization.
  • the monitoring method of the response is preferably conducted with the same patient repeatedly, preferably in regular time intervals.
  • the time intervals are defined with respect to individual needs of the patients. So, a time schedule (personalized schedule) is advantageously innovatively created that is especially governed by the current state of the patient.
  • the state of the patient refers to current numbers and the character of CTCs present in peripheral blood in combination with a standard diagnostic procedure for individual types of tumor diseases.
  • the time intervals between collections can be the same or different and they can e.g. take from one week to one year.
  • material which is at least one element selected from the group of circulating tumor cells, disseminated tumor cells, primary tumor cells; forms of antigens derived from these cell types as mentioned above, can be stored for later use for immunization. Lyophilization can be preferably used for long-term storage.
  • the body fluid is preferably selected from the group of bone marrow; cerebrospinal fluid, pleural, pericardial and synovial exudate; ascites; cephalic fluid; and peritoneal wash.
  • Phagosomes are displayed by means of (pHRodo Green Zymosan A Bioparticles®) in lighter shades on a dark background (originally green fluorescence).
  • the scale represents 10 ⁇ m - shows shows the result of testing the phagocytizing capacity by displaying of active phagosomes in DCs by means of vital fluorescence staining
  • the process of obtaining the vaccine from sporadic cells can be schematically divided into the following stages, which will be further described in detail.
  • CTCs were isolated from the patient's own blood (i.e. autologously) through separation based on the size of tumor cells. This stage comprised the following steps:
  • CTCs circulating tumor cells
  • CTCs For the selection of CTCs from full blood (EDTA, 2 x 8 ml), a filtration method using capillarity on a membrane with the pore size of 7-10 ⁇ m was used. (Metacell s.r.o., Czech Republic). The captured cells were subsequently in vitro cultivated in a serumless medium RPMI for 3-5 days under standard conditions (37°C, 5% CO 2 ), the protocol is described in the references (45-47). The evaluation or presence of CTCs in the sample comprised a cytomorphological analysis of CTCs using vital fluorescence microscopy.
  • cytopathological criteria were evaluated as the cell size (tumor cells, i.e. also CTCs generally have a size of >15 ⁇ m), nucleus size (>10 ⁇ m) and the shape of the cell nucleus, irregularity of the nucleus membrane, ratio of the volume of the nucleus and cytoplasm, presence, appearance and number of nucleoli, presence of proliferation (detection of mitoses) or/and creation of 2D and 3D cell structures (48).
  • Fig. 3 shows viable CTCs captured on a separation membrane in the course of a separation protocol based on cell size (MetaCellTM)).
  • the nuclei originally colored light blue
  • cytoplasm is grey (originally green) showing the irregular shape of cells.
  • a cytomorphological analysis of in vitro cultivated fraction enriched with CTC confirmed the process of proliferation of isolated CTCs at individual control points (7, 14, 21 and 28 days).
  • Fig. 4 shows CTCs proliferating under in vitro conditions.
  • the nuclei originally colored light blue
  • cytoplasm is grey (originally green).
  • ascitic fluid was collected directly from the peritoneum to a test tube (50 ml, Corning) without anticoagulation agents and filtered through a membrane (MetaCellTM). After evaluation of the density of the ascitic fluid, the ascitic fluid may be diluted with RPMI or PBS before the entire filtration.
  • TA tumor-associated genes
  • the evaluation or the presence of CTCs in the sample also comprised a molecular analysis (gene expression testing).
  • gene expression testing For each of the types of separated CTCs specified below, the presence of CTCs in the enriched cell fraction on the culture membrane was also proved by means of a gene expression analysis for groups of specific tumor-associated (TA) genes.
  • TA tumor-associated
  • CTCs were detected on the basis of expressing of the tumor-associated genes KRT18, KRT19 (keratin 18/19), EpCAM (epithelial cell adhesion molecule), MUC1 (mucin 1), MGB (mammaglobine), HER2, ESR (estrogen receptor), PGR (progesterone receptor), CD45 and CD68 (leukocyte markers), CD24 and CD44 (stem cell markers) and ACTB (control gene).
  • RNA fractions isolated from the whole blood (the so-called leukocyte fraction), RNA isolated from the CTC enriched cell fraction immediately after the filtration and RNA isolated from the CTC enriched fraction after the filtration on the separation (culture) membrane (MetaCell®), (49,50) and short-term in vitro incubation 3-5 days).
  • the comparison indicates the rate of enrichment of the obtained cell fraction with cells with tumor characteristic compared to the leukocyte fraction (the leukocyte fraction was isolated from peripheral blood by lysis of erythrocytes).
  • the CTC examination using a gene expression analysis in combination with a cytomorphological analysis comprises information about the presence/absence of CTCs in peripheral blood and their characteristics (expression of tumor-associated genes).
  • the expression of genes associated with chemoresistance is also evaluated in relation to the administered treatment.
  • the table indicates that through a suitable combination of X - indicated genes the origin of tested tumor cells, in our case of CTCs, can be confirmed/refuted to confirm their presence in the tested sample. What is advantageous is that within one sample, several markers could be monitored at the same time (up to 30 genes from one CTC enriched fraction), which is advantageous compared to standardized immunohistochemical analyses when up to five antibodies can be used at the most.
  • CTCs captured on the filtration membrane from the peripheral blood sample are further specified. Collection of blood and isolation of CTCs are carried out in the interval of four weeks (shown as points on the full line). The intervals are adapted to the type of therapy that the patient is currently undergoing (personalization of collection intervals).
  • a vaccine is immediately prepared from the isolated CTCs (e.g. in week 12 in Fig. 2) and applied (indicated with a syringe). After the vaccination a decrease of CTCs is assumed, however, at the control collection in week 36, CTCs reappear, indicating a possible relapse.
  • Fig. 6 Monitoring of CTC changes in the course of an ongoing neoadjuvant therapy of breast cancer is shown in Fig. 6 (a so far unpublished article of the authors of the invention (57)).
  • the specifications of the tumor disease of the patient are shown in the table in the figure.
  • Fig. 6 shows
  • a neoadjuvant therapy of the FEC type (FEC Fluorouracil, Epirubicin, Cyclophosphamide) was indicated in a patient with breast cancer (histotype: triple-negative, TNBC - triple negative breast cancer).
  • FEC Fluorouracil, Epirubicin, Cyclophosphamide FEC Fluorouracil, Epirubicin, Cyclophosphamide
  • histotype triple-negative, TNBC - triple negative breast cancer
  • NGS Next Generation Squencing, GeneReaderTM, Oiagen
  • QiaAcr testing panel offers sequencing analysis of 1250 mutations in twelve most frequently mutated genes associated with the tumor process with the use of NGS, analysis of mutations in the gene KRAS, NRAS, ERBB2, ERBB3, KIT, ALK, PDGFR, EGFR, BRAF, PI3KCA, RAF2.
  • the results of the sequencing analyses are automatically evaluated by the QIA-Analyse software and interpreted by QIA- Interpreter (Qiagen - Ingenuity).
  • QIA- Interpreter Qiagen - Ingenuity.
  • the central database of mutations for comparing of sequencing data among laboratories makes it possible to minimize errors in the data analysis and to standardize it.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • This supernatant can also be further used as a source of possible T cells. After centrifugation and rewashing of the obtained cells, this cell fraction can be kept frozen (-20°C). Further, filtration of the supernatant (12 ml, or aliquot volumes) through a separation membrane can provide further CTCs, especially in patients with an advanced tumor disease. The separated CTCs are further cultivated in vitro as described in point i a.
  • Immature DCs were subsequently differentiated for 4 days (37 °C, 5% CO 2 ) in a growth medium RPMI 1640 (SigmaAldrich).
  • media of the Cellgrow (Cellgenix), Mo-DC Differentiation Medium (Miltenyi Biotech)) type can be used.
  • the growth medium was supplemented with interleukin IL-4 (2500 U/ml) and the growth factor GM-CSF (20 ng/ml) and the cells were cultivated for 4 days.
  • DC Poly(I:C) (VacciGradeTM InvivoGen) was added to the control population of the DCs in the concentration of 25 ⁇ g/ml.
  • cDNA was generated by means of a High capacity RNA to cDNA kit® (Themofisher), gene expression was tested according to the protocol with the use of a TaqmanFast Advanced Master Mix® (Thermofisher reaction chemistry) on a Cobas 4800 device (Roche).
  • the control genes for the tested fraction were ACTB, CD45, CD68.
  • the protocol for testing the quality of produced and especially for testing the gene expression level of activated DCs was created on the basis of published data of the group Castiello et al. (37).
  • CD83 molecule CD83
  • CD86 CD86
  • mRNA mRNA. Hs.171182
  • Hs01567026_m1 7.
  • HLA-DR molecule HLA-DRA
  • mRNA alpha chain Hs.520048 HLA-DR Hs00219575_m1
  • Intracellular adhesive molecule 1 Hs.643447 ICAM1 Hs00164932_m1
  • interferon alpha induced protein 27 (IFI27), transcript variant 2, mRNA. Hs.532634 IFI27 Hs01086373_g1
  • tumor necrosis factor alpha-induced protein 6 (TNFAIP6)
  • mRNA Hs.437322
  • Viability of cells was determined by staining with a ReadyProbes® Cell Viability Imaging Kit (Blue/Green), (Thermofisher).
  • ReadyProbes® Cell Viability Image Imaging Kit (Blue/Green) is a ready-to-use assay that enables quick and easy determination of viability of cells. Viability was the determined with the use of the stable reagent NucBlue® Live (Hoechst 33342) and the reagent NucGreen® Dead according to the manufacturer's protocol per 1 ml of a growth medium (e.g. RPMI 1640) by counting of living and dead cells. NucBlue® Live reacts with the nuclei of all cells and can be detected with a standard DAPI filter (blue fluorescent protein, BFP - blue).
  • NucGreen® Dead only reacts with the nuclei of cells with disrupted plasmatic membrane integrity and is detected with the use of a standard FITC filtration kit / (green fluorescent protein, GFP - green). This combination is suitable for determination of viability by means of fluorescence microscopy or flow cytometry.
  • RNAse free water was used for the production of the solution. 5 ⁇ l of the solution per cell is taken into account, i.e. the lysis of a greater number of cells was adapted proportionally (55).
  • CTCs were transferred on a separating (culture) membrane with the pore size of 7 - 10 ⁇ m (MetaCellTM) to the lysis solution (500 ⁇ l) in an Eppendorf tube (1.5 ml). Presence of not lysed cells to check proper execution of the lysis process was detected after removal of the membrane from the lysis solution. The membrane was stained using the accelerated May Grünwald staining protocol. After 15 minutes, no compact cells were present on the membrane. The concentration of RNA was not measured, the quantity of the lysis solution was adapted by the number of present CTCs documented in the course of the cytomorphological analysis.
  • the freezing and defrosting method is commonly used for the lysis of bacterial and mammal cells.
  • the technique comprises freezing of cell suspension in a bath or freezing device on dry ice/ethanol and then defrosting of the material at the room temperature or the temperature of 37 °C (at least 1 h).
  • This method of lysis causes that the cells are inflated and finally they get disintegrated due to the process of formation of ice crystals during freezing and their mixing during defrosting. For efficient lysis, more cycles are required, and the process may be relatively long. However, freezing/defrosting has proved to efficiently release proteins present in the cytoplasm of mammal cells.
  • CTCs were transferred on a separation (culture) membrane MetaCellTM into a homogenization test tube of type C (Miltenyi) and mechanically dissociated by means of a GentleMACSTM Dissociator (Miltenyi) using the installed software "m_spleen04".
  • the cell suspension was centrifuged at 1000 x g for 10 minutes. The pellet was resuspended in PBS, removed to a test tube of type M (Miltenyi) and lysed using a GentleMACSTM Dissociator with the use of the "protein_01" software.
  • the suspension was filtered through a 70 ⁇ m and 30 ⁇ m filter, then sonicated for 30 minutes or/and lysed with BSA lysis buffer.
  • a test for the presence of living tumor cells was introduced. If viable cells were still present, further lysis and freezing, defrosting steps were carried out until 0% viability was achieved.
  • concentration of proteins in the lysate can be determined with a standardized assay with bicinchoninic acid (BCA) (Pierce Biotechnology/Thermo Fisher Scientific, USA) according to the manufacturer's instructions.
  • BCA bicinchoninic acid
  • the phagocytary capacity was tested with the use of pHrodo® Green Zymosan A BioParticles® conjugates for phagocytosis (Thermofisher, USA): The cells were washed 1x with a solution for living cells and treated with a suspension of the pHrodoTM Green Zymosan A BioParticles® conjugate in a living cell imaging solution in the concentration of 1 mg/ml. The cells were incubated for 90 minutes at 37 °C. The nuclei were stained with NucBlue® Live Cell Stain.
  • Imaging of active phagosomes is possible thanks to fluorescence of pHrodo particles in phagosomes of DC cells cultivated from monocytes in an in vitro culture as shown in Fig. 7, where phagosomes are shown as light objects on a dark background.
  • the scale represents 10 ⁇ m.
  • the quantity of the cell lysate was related to the number of detected CTCs on the separation membrane (the number of isolated CTCs varied in the range of 100 - 150 cells). Samples with more numerous representation of CTCs were stored (-20°C) for further testing.
  • RNA isolated from the enriched fraction of CTCs was added in various concentrations 100, 250, 500, 750 and 1000 ng RNA) in H 2 O directly to the DC culture. The concentration and purity of RNA was measured with a spectrophotometer (NanoDrop).
  • the T cells were obtained from the fraction of not adhered polymorphonuclear cells, or by direct isolation with the use of immunomagnetic separation (anti CD4, anti CD8, Dynabeads, Invitrogen)
  • T cells autologous or/and non-autologous
  • activated DCs the DCs immunized with CTC antigens in various forms as described above.
  • the culture medium where common cultivation is conducted was gradually collected in time and frozen for later testing of the expression of cytokines, especially INF ⁇ , using the standardized ELISA assay.
  • the calibration curve of the INFy values then makes it possible to determine the optimum quantity of cells and the time required for immunization.
  • ACTB and CD45 were used as control genes.
  • the relative quantification method according to Livak et al was used (36).
  • the calculation bears on values measured by means of qPCR (so-called Quantification Cycles - Cq) and compared them both to the control genes in the pattern and among control groups of samples on the basis of different Cq values for the control gene and the gene of interest, i.e. target.
  • the invention provides personalized immunotherapeutic anti-tumor vaccines usable in oncology whose production and administration can be exactly timed based on monitoring of the patient, making sure that the anti-tumor immune response is focused directly against the tumor cells that are beginning to spread at the particular time point in the organism and could possibly produce metastases.

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Abstract

Un vaccin anti-tumoral immunothérapeutique personnalisé autologue utilise la propriété d'immunisation de cellules sporadiques et de leurs parties, des procédés pour sa production et la synchronisation de sa production et son administration au patient sur la base de la surveillance du patient, la surveillance reposant sur l'évaluation de cellules sporadiques en temps réel. Le vaccin peut être utilisé pour invoquer une réponse immunitaire anti-tumorale personnalisée en vaccination autologue, ce qui provoque une réaction immunogène dans l'organisme contre les cellules tumorales actuellement présentes.
PCT/IB2019/050752 2018-06-26 2019-01-30 Vaccin autologue et procédé de préparation du vaccin et surveillance du patient cancéreux pendant et après la vaccination WO2020003009A1 (fr)

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TWI798031B (zh) * 2021-03-25 2023-04-01 聯發科技股份有限公司 監測數據通道的省電方法及用戶設備
CN115718095A (zh) * 2022-03-28 2023-02-28 南京诺源医疗器械有限公司 一种循环肿瘤细胞自动扫描方法及装置
CN115718095B (zh) * 2022-03-28 2023-09-01 南京诺源医疗器械有限公司 一种循环肿瘤细胞自动扫描方法及装置

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