LT6677B - Method of production of cytokine induced/activated killer cells from peripherial blood, bone marrow or apheresis product and use thereof - Google Patents
Method of production of cytokine induced/activated killer cells from peripherial blood, bone marrow or apheresis product and use thereof Download PDFInfo
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Abstract
Description
IŠRADIMO SRITISFIELD OF INVENTION
Išradimas skirtas medicinos sričiai, konkrečiai - citokinais indukuotų kilerinių ląstelių išgavimui iš periferinio kraujo arba kaulų čiulpų arba aferezes produkto, jų aktyvavimui, dauginimui ir panaudojimui imunoterapijoje, siekiant sunaikinti arba sumažinti onkologinį procesą ar jo metastazavimą, taip pat naikinti metastazes, užkertant kelią naujų navikinių darinių susiformavimui, pasitelkiant kilerines ląsteles ir jų produkuojamus imunologiškai aktyvius baltymus.The present invention relates to the field of medicine, in particular to the cytokine-induced production of killer cells from peripheral blood or bone marrow or apheresis product, their activation, proliferation and use in immunotherapy to destroy or reduce the oncological process or its metastasis and to prevent new tumor derivatives. formation using killer cells and the immunologically active proteins they produce.
TECHNIKOS LYGISTECHNICAL LEVEL
Prieš daugiau nei du dešimtmečius, įrodžius, kad imuninė sistema gali atpažinti ir eliminuoti vėžines ląsteles, didelis dėmesys skirtas imuninių ląstelių tyrimams, mėginant išsiaiškinti kokį poveikį jos turi vėžio genezei. Dabar jau yra žinoma, kad su vėžiu kovoja tiek įgimtas, tiek įgytas imunitetas [Teng MWL, Swann JB, Koebel CM, Schreiber RD, Smyth MJ. Immune-mediated dormancy: An equilibrium with cancer. Journal of Leukocyte Biology 2008; 84: 988-93]. Nepaisant didelės kovojančių ląstelių įvairovės, vėžys, pasitelkdamas savo gynybinius mechanizmus, išvengia sunaikinimo [Dunn GP, Old LJ, and Schreiber RD. The three Es of cancer immunoediting. Annual Review of Immunology 2004a; 22: 329-60].More than two decades ago, when the immune system was shown to be able to recognize and eliminate cancer cells, a great deal of attention was devoted to research on immune cells to try to determine their effect on cancer genesis. It is now known that both innate and acquired immunity fight cancer [Teng MWL, Swann JB, Koebel CM, Schreiber RD, Smyth MJ. Immune-mediated dormancy: An equilibrium with cancer. Journal of Leukocyte Biology 2008; 84: 988-93]. Despite its wide variety of fighting cells, cancer, through its defense mechanisms, avoids destruction [Dunn GP, Old LJ, and Schreiber RD. The three Es of cancer immunoediting. Annual Review of Immunology 2004a; 22: 329-60].
Dėl imuninės sistemos spaudimo, vėžys, norėdamas išlikti, įgyja savybių, mažinančių jo imunogeniškumą, ir (arba) slopina imuninės sistemos komponentus [Chen DS and Mellman I. Oncology meets immunology: The cancer-immunity cycle. Immunity 2013; 39: 1-10]. Pastebėjus, kad geresnę prognozę turi pacientai, kuriems dėl infekcijos buvo sužadinta imuninė sistema, pradėta taikyti imunoterapija [Cann SAH, van Netten JP, van Netten C. Dr William Coley and tumour regression: A place in history or in the future. Postgraduate Medical Journal 2003; 79: 672-80], kurios tikslas yra aktyvinti imunines ląsteles ar tikslingai jas nukreipti j vėžinių ląstelių sunaikinimą.Under pressure from the immune system, cancer, in order to survive, acquires properties that reduce its immunogenicity and / or suppress immune system components [Chen DS and Mellman I. Oncology meets immunology: The cancer-immunity cycle. Immunity 2013; 39: 1-10]. Patients who have been triggered by immunosuppression due to infection have been shown to have a better prognosis [Cann SAH, van Netten JP, van Netten C. Dr. William Coley and tumor regression: A place in history or in the future. Postgraduate Medical Journal 2003; 79: 672-80], the purpose of which is to activate or purposefully direct immune cells towards the destruction of cancer cells.
Imunoterapija turi didelį pranašumą prieš tradicinius gydymo metodus, kadangi yra didesnio specifiškumo ir nesukelia sveikų ląstelių žūties [Eggermont LJ, Paulis LE, Tel J, Figdor CG. Towards efficient cancer immunotherapy: Advances in developing artificial antigen-presenting cells. Trends in Biotechnology 2014; 32 (9): 456-65]. Vienas iš ląstelinės imunoterapijos komponentų yra natūralūs kileriai (NK). Tai ląstelinio imuniteto ląstelės, kurių funkcija yra tiek tiesiogiai, tiek per prisijungusias signalines molekules (antikūnai) atpažinti pakitusias savas ląsteles ir jas sunaikinti. Ši savybė gali būti pritaikoma kovoje su vėžiniais susirgimais. Kadangi vėžinė ląstelė yra ta pati nuosava ląstelė, tik su pasikeitusiais MHC I komplekso antigenais, NK ląstelės geba jas atskirti, identifikuoti ir sunaikinti [Kuby Immunology 8th edition 2019; ISBN-10: 1-4641-8978-1], Pačios NK ląstelės nėra priskiriamos T ląstelių grupei. Natūralūs citotoksiškumo receptoriai tiesiogiai gali dalyvauti apoptozės proceso paleidime po to, kai prisijungia prie taip vadinamo FAS ligando, kuris parodo, kad ląstelė pakitusi (infekuota virusu ar vėžinė). MHC priklausomi receptoriai dalyvauja apoptozės proceso paleidime. NK lątelė pati susireguliuoja, priklausomai nuo esamų ant ląstelės-taikinio signalų kiekio ir stiprumo, kaip jai elgtis: ar žudyti, ar ignoruoti ląstelę-taikinį [Terunuma H, Deng X, Dewan Z, Fujimoto S, Yamamoto N. Potential role of NK cells in the induction of immune responses: implications for NK cell-based immunotherapy for cancers and viral infections. Int Rev Immunol. 2008;27(3):93110]. Todėl NK ląstelės elgsenoje yra svarbios aktyvacinės ir inhibitorinės molekulės, tokios kaip: Ly49, NCR, CD16 (aktyvacinės), ir inhibitorinės: KIR, CD94/NGK2, ILT arba LIR, Ly49 ir kt. [lannello A, Debbeche O, Samarani S, Ahmad A. Antiviral NK cell responses in HIV infection: I. NK cell receptor genes as determinants of HIV resistance and progression to AIDS. J Leukoc Biol. 2008 Jul;84(1): 1 -26. doi: 10.1189/jlb.0907650. Epub 2008 Apr 3],Immunotherapy has a major advantage over conventional methods of treatment in that it is more specific and does not cause healthy cell death [Eggermont LJ, Paul LE, Tel J, Figdor CG. Towards efficient cancer immunotherapy: Advances in developing artificial antigen-presenting cells. Trends in Biotechnology 2014; 32 (9): 456-65]. One of the components of cellular immunotherapy is the natural killer (NK). These are cellular immune cells whose function is to recognize and destroy abnormal cells either directly or via attached signaling molecules (antibodies). This feature can be applied in the fight against cancer. Because the cancer cell is the same native cell, but with altered antigens of the MHC I complex, NK cells are able to distinguish, identify, and destroy them [Kuby Immunology 8th edition 2019; ISBN-10: 1-4641-8978-1], NK cells themselves are not assigned to the T cell family. Natural receptors for cytotoxicity may be directly involved in triggering the apoptosis process after binding to a so-called FAS ligand, which indicates that the cell is altered (infected with a virus or cancerous). MHC-dependent receptors are involved in triggering the apoptosis process. The NK cell adjusts itself depending on the amount of cell-to-target signaling available and how it behaves: whether to kill or ignore the cell-target [Terunuma H, Deng X, Dewan Z, Fujimoto S, Yamamoto N. Potential role of NK cells in the induction of immune responses: implications for NK cell-based immunotherapy for cancers and viral infections. Int Rev Immunol. 2008; 27 (3): 93110]. Therefore, activating and inhibitory molecules such as: Ly49, NCR, CD16 (activating), and inhibitory: KIR, CD94 / NGK2, ILT or LIR, Ly49 and others are important in NK cell behavior. [lannello A, Debbeche O, Samarani S, Ahmad A. Antiviral NK cell responses in HIV infection: I. NK cell receptor genes as determinants of HIV resistance and progression to AIDS. J Leukoc Biol. 2008 Jul; 84 (1): 1 -26. doi: 10.1189 / jlb.0907650. Epub 2008 Apr 3],
Funkciškai NK ląstelės gali lizuoti savo taikinį per citolitinių granulių išskyrimą. Savo citoplazmoje NK ląstelės turi granules, kuriose yra uždarytos įvairios proteazės ir perforinai, žinomi kaip granzimai. Šios medžiagos paleistos į išorę gali lizuoti šalia esančias ląsteles arba sukelti šalia esančių ląstelių apoptozę. Ližės ir apoptozės sunaikinimo būdai taip pat yra svarbūs imunologiniu požiūriu, toliau priklausomai nuo vykstančio proceso pasijungia kitos imuninės sistemos ląstelės, tokios kaip neutrofilai, makrofagai [Smyth MJ, Hayakawa Y, Takeda K, Yagita H. New aspects of natural-killer-cell surveillance and therapy of cancer. Nat Rev Cancer. 2002 Nov;2(11):850-61]. Kitas būdas - atpažinti taikinį yra per prisijungusius antikūnus, ši savybė svarbi taikant NK ląsteles kartu su monokloniniais antikūnais, jų terapinis poveikis tokiu atveju gali tik sustiprėti. Prisijungusį antikūną NK ląstelės atpažįsta per CD16 receptorių, kurį pačios ir ekspresuoja. Šis receptorius, susijungęs su antikūno Fc galu, aktyvuoja NK ląsteles ir jos pradeda ližės procesą atpalaiduodamos granzimus. Taip pat NK ląstelės yra jautrios citokinams, tokios molekulės kaip 1L-12/15/18/2 ir CCL5 aktyvuoja NK ląsteles in vivo ir in vitro, taip pat interferonai - tiek gama, tiek alfa - aktyvuoja NK ląsteles priešvirusiniam atsakui. NK ląsteles pačios išskiria interferonus ir TNF alfa molekulę, kuri yra svarbi priešvėžinio imuniteto reguliacijoje. NK ląstelės pasižymi ir tikslia savas-svetimas reguliacija per MHC I atpažinimo mechanizmus. Jeigu ląstelė aptikta NK, ląstelės sekretuoja mažesnius nei įprasta MHC I kiekius, NK ląstelės tokias ląsteles sunaikina. Manoma, kad ši funkcija evoliucionavo iš būtinybės kompensuoti T limfocitų funkciją, kuriai sutrikus ar išsekus, nesant NK ląstelių, organizmas gali žūti, todėl NK ląstelės yra labai svarbi įgimto imuniteto dalis, užtikrinanti tvarkingą visų imuninių grandžių reguliaciją ir darbą. Jos yra neatsiejama imuninės sistemos dalis, taip pat jos pasižymi labai gera savybe, jog esant taikiniui ar pereinant į gynybinę funkciją jos nesekretuoja didelių kiekių biologiškai aktyvių citokinų, todėl taikant jas didelėmis terapinėmis dozėmis, tiek kaip padauginus natyvias ląsteles, tiek modifikuotas genetiškai, nebus sukeliamas citokinų atpalaidavimo sindromas, kas būdinga taikant T limfocitų terapijas ar CAR-T ląsteles. Todėl NK ląstelės yra labai patogus įrankis, kuriant genetiškai modifikuotų ląstelių gydymo metodus [vadovėlis: DOI 10.1007/sl 1523-017-0489-2]. NK ląstelės dažnai neturi tiksliai išreikšto antigeno receptoriaus ir yra dalis įgimto imuniteto, tai reiškia, kad jos gali labai greitai reaguoti į antigeną, be išankstinio apsimokymo prieš jį. Moksliniai duomenys rodo, jog ši savybė yra labai svarbi vėžio išgyvenamumo ir sunaikinimo balanse. Jos gali greitai aptikti pakitusias ląsteles ir jas sunaikinti be išankstinio žinojimo kas jose pakito. Nes, pavyzdžiui, T ląstelėms, kad atpažintų ir reaguotų į taikinį, yra būtina prieš tai būti su juo susidūrus [Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL, Yokoyama WM, Ugolini S. Innate or adaptive immunity? The example of natural killer cells. Science. 2011 Jan 7;331 (6013):44-9. doi: 10.1126/science. 1198687], Tuo atveju jeigu vėžinė ląstelė nesukelia uždegimo, tai T ląstelės jos neatpažins ir eigoje vystysis vėžinis susirgimas, NK ląstelės šioje grandyje gali pradėti elgtis kaip imunosupresoriai, nes nėra aktyvacijos signalo, tylint T ir kitoms imuninės sistemos ląstelėms (makrofagai, dendritinės, neutrofilai), tačiau pačios NK ląstelės turi ne tik bendrus receptorius citokinams ir įvarioms signalinėms molekulėms, užkuriančioms arba supresuojančioms imuninę funkcija, jos turi alternatyvius receptorius, kurie yra specifiniai vėžinių ląstelių ližei (NKG2D, NKp44, NKp46, NKp30 ir DNAM). Būtent per juos N K ląstelės geba tiesiogiai dalyvauti vėžinio proceso kontrolėje. Jos taip pat sąveikauja su dendritinėmis ląstelėmis, formuodamos kompleksinį imuninį atsaką [Vadovėlis: doi:10.5772/intechopen.78804, Terunuma H, Deng X, Dewan Z, Fujimoto S, Yamamoto N. Potential role of NK cells in the induction of immune responses: implications for NK cell-based immunotherapy for cancers and viral infections. Int Rev Immunol. 2008,27(3):93-110. doi: 10.1080/08830180801911743]. Šią funkciją labai svarbu paminėti, turint omenyje mūsų išradime minimą produktą kaip dendritinėmis ląstelėmis praimuotos NK ląstelės. Šios ląstelės yra praėjusios apmokymo etapus in vitro ir gali būti taip, jog jų suleidimas pacientui bus naudingas ūmia reakcija į jo vėžines ląsteles, tačiau šiuo atveju visada išlieka nepataikymo tikimybė, todėl praimuotos NK ląstelės nėra išskiriamos į atskirą produktą, geriausiai yra taikyti mišrią technologiją, tuo metu šansai paveikti naviką tiesiog yra padidinami.Functionally, NK cells can lyse their target via secretion of cytolytic beads. In their cytoplasm, NK cells contain granules that contain various proteases and perforins known as granzymes. These substances, when released externally, can lyse adjacent cells or induce apoptosis of adjacent cells. The methods of killing lithium and apoptosis are also important from an immunological point of view, further downstream of other immune system cells, such as neutrophils, macrophages [Smyth MJ, Hayakawa Y, Takeda K, Yagita H. New aspects of natural-killer-cell surveillance and therapy of cancer. Nat Rev Cancer. 2002 Nov; 2 (11): 850-61]. Alternatively, target recognition is via binding antibodies, an important feature of NK cells in combination with monoclonal antibodies, which may only exacerbate their therapeutic effect. The bound antibody recognizes NK cells via the CD16 receptor, which it expresses itself. This receptor, which binds to the Fc end of the antibody, activates NK cells and initiates the lytic process by releasing granzymes. NK cells are also sensitive to cytokines, molecules such as 1L-12/15/18/2 and CCL5 activate NK cells in vivo and in vitro, and interferons, both gamma and alpha, activate NK cells for antiviral response. NK cells themselves secrete interferons and the TNF alpha molecule, which is important for the regulation of antitumor immunity. NK cells also exhibit precise self-foreign regulation via MHC I recognition mechanisms. If a cell is detected in NK, the cells secrete less than normal amounts of MHC I, NK cells destroy such cells. This function is thought to have evolved from the need to compensate for the function of T lymphocytes, which can be disrupted or depleted in the absence of NK cells, making NK cells an essential part of the innate immune system that ensures the proper regulation and function of all immune circuits. They are an integral part of the immune system and are very good at secreting large amounts of biologically active cytokines at the target or defense function and, therefore, at high therapeutic doses, both as native cells and genetically modified, will not induce cytokine release syndrome characterized by T-lymphocyte therapies or CAR-T cells. Therefore, NK cells are a very handy tool for developing methods for the treatment of genetically modified cells [textbook: DOI 10.1007 / sl 1523-017-0489-2]. NK cells often lack the highly expressed antigen receptor and are part of the innate immune system, meaning that they can react very quickly to the antigen without prior training. Scientific evidence indicates that this property is very important in the balance of cancer survival and destruction. They can quickly detect abnormal cells and destroy them without prior knowledge of what has changed. Because, for example, for T cells to recognize and respond to a target, it is necessary to be exposed to it in advance [Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL, Yokoyama WM, Ugolini S. Innate or adaptive immunity? The example of natural killer cells. Science. 2011 Jan 7; 331 (6013): 44-9. doi: 10.1126 / science. 1198687], If the cancer cell does not cause inflammation then the T cells will not recognize it and in the course of the development of the cancer, NK cells in this chain may act as immunosuppressants in the absence of activation signal silencing T and other immune system cells (macrophages ), but NK cells themselves have not only common receptors for cytokines and various signaling molecules that stimulate or suppress immune function, but also have alternative receptors specific for cancer cell lysis (NKG2D, NKp44, NKp46, NKp30 and DNAM). It is through these that N K cells are able to participate directly in the control of the cancer process. They also interact with dendritic cells to form a complex immune response [Textbook: doi: 10.5772 / intechopen.78804, Terunuma H, Deng X, Dewan Z, Fujimoto S, Yamamoto N. Potential role of NK cells in the induction of immune responses: implications for NK cell-based immunotherapy for cancers and viral infections. Int Rev Immunol. 2008.27 (3): 93-110. doi: 10.1080 / 08830180801911743]. This function is very important to mention in view of the product of the present invention as NK cells primed with dendritic cells. These cells have undergone training steps in vitro and may be injected into the patient to benefit from an acute reaction to his cancerous cells, but in this case there is always a risk of maladministration, so that primed NK cells are not isolated into a single product; at that time, the odds of affecting the tumor are simply increased.
Nepaisant didelės pažangos moksle, naujų vaistų sukūrime ir naviko biologijos pažinime, vėžys vis dar išlieka viena iš pagrindinių mirties priežasčių ne tik tarp besivystančių, bet ir išsivysčiusiose šalyse. Šiuo metu svarbiausi vėžio gydymo metodai yra trys: chirurgija (naviko pašalinimas), chemoterapija (vaistų vartojimas) ir radioterapija (spindulinis gydymas). Kiekvienas būdas turi apribojimų ar pašalinių poveikių, pvz.: chirurginis gydymas gali būti taikomas, tik šalinant kietą, apčiuopiamą naviką, o problemų kyla dėl nepašalintų pradinio naviko liekanų ar metastazių. Chemoterapija yra grįsta sparčiai besidalijančių ląstelių naikinimu įvairiais citostatikais, tačiau organizme greitai proliferuoja ne tik vėžinės ląstelės, bet ir plaukų folikulų, žarnyno epitelio, kaulų čiulpų ląstelės ir kt. [Aly HAA. Cancer therapy and vaccination. Journal of Immunological Methods 2012; 382: 1-23]. Imuninės sistemos ląstelės, pvz.: makrofagai, gali slopinti chemoterapijos efektyvumą priklausomai nuo naviko rūšies. Vienas pirmųjų tyrimų, siekiant išsiaiškinti makrofagų įtaką chemoterapijai, parodė, kad vėžio gydymui skiriamas preparatas doksorubicinas pelėse, susargdintose leukemija ar limfoma, sustiprina priešvėžinį makrofagų poveikį. Kitas pavyzdys yra B ląstelės, kurios netiesiogiai skatina ortotopinės epitelinio audinio karcinomos progresavimą. Chemoterapija, neinhibuojant B ląstelių, yra neveiksminga. Tik kartu su anti-CD20 antikūnais, vaistas paklitakselis yra veiksmingas prieš šios rūšies vėžį [Coffelt SB and de Visser KE. Immune-mediated mechanisms influencing the efficacy of anticancer therapies. Trends in Immunology 2015; 36 (4): 198-216],Despite great advances in science, in the development of new drugs and in the knowledge of tumor biology, cancer remains one of the leading causes of death, not only in developing countries but also in developed countries. Currently, the most important treatment methods for cancer are three: surgery (removal of the tumor), chemotherapy (medication), and radiotherapy (radiation therapy). Each method has limitations or side effects, such as: surgery can only be applied to remove a solid, palpable tumor, and problems arise from unresolved initial tumor remnants or metastases. Chemotherapy is based on the destruction of rapidly dividing cells by various cytostatics, but not only cancer cells, but also hair follicles, intestinal epithelium, bone marrow cells, etc. proliferate rapidly in the body. [Aly HAA. Cancer therapy and vaccination. Journal of Immunological Methods 2012; 382: 1-23]. Immune cells, such as macrophages, may inhibit the effectiveness of chemotherapy, depending on the type of tumor. One of the first studies to elucidate the effect of macrophages on chemotherapy showed that doxorubicin, a treatment for cancer, enhances the anti-cancer effect of macrophages in mice with leukemia or lymphoma. Another example is B cells, which indirectly promote the progression of orthotopic epithelial tissue carcinoma. Chemotherapy without inhibiting B cells is ineffective. Only in combination with anti-CD20 antibodies, paclitaxel is effective against this type of cancer [Coffelt SB and de Visser KE. Immune-mediated mechanisms influencing the efficacy of anticancer therapies. Trends in Immunology 2015; 36 (4): 198-216],
Norint pasiekti geresnių rezultatų, yra taikomas kombinuotas kelių metodų gydymas. Gydymas tradiciniais chemoterapijos metodais yra kenksmingas, kadangi yra pažeidžiamos ir sveikos ląstelės, todėl tikimasi didelių pasiekimų, pradėjus plačiai taikyti imunoterapiją, specifiškai nukreiptą prieš vėžines ląsteles. Imunoterapija vėžio gydymo metodas, kuomet imuninės sistemos komponentai yra aktyviai arba pasyviai suaktyvinami ir nukreipiami piktybinių ląstelių sunaikinimui. Dėl didesnio specifiškumo yra išvengiama pašalinių poveikių, kuriuos sukelia įprasti gydymo metodai [Eggermont LJ, Paulis LE, Tel J, Figdor CG. Towards efficient cancer immunotherapy: Advances in developing artificial antigen-presenting cells. Trends in Biotechnology 2014; 32 (9): 456-65],A combination of several methods is used to achieve better results. Conventional chemotherapy treatment is detrimental because of the vulnerability of healthy cells, and major advances are expected in the widespread use of immunotherapy specifically targeting cancer cells. Immunotherapy is a method of treating cancer by activating or passively activating components of the immune system and targeting the destruction of malignant cells. Higher specificity avoids side effects caused by conventional treatments [Eggermont LJ, Paul LE, Tel J, Figdor CG. Towards efficient cancer immunotherapy: Advances in developing artificial antigen-presenting cells. Trends in Biotechnology 2014; 32 (9): 456-65],
Pirmosios imunoterapijos užuomazgos siekia XIX a. pabaigą, kai W. Coley pastebėjo, kad pacientas, pasiekęs visišką naviko remisiją, prieš tai persirgo ūmia streptokokų sukelta infekcija. Vėliau, sukūręs vakciną iš negyvų Streptococcus pyogenes ir Serratia marcescens bakterijų, pradėjo gydyti neoperuotinu vėžiu (dažniausiai sarkoma) sergančius žmones. Vakcinos paskirtis buvo sužadinti imuninę sistemą [Cann SAH, van Netten JP, van Netten C. Dr William Coley and tumour regression: A place in history or in the future. Postgraduate Medical Journal 2003; 79: 672-80; Parish CR. Cancer immunotherapy: the past, the present and the future. Immunology and Cell Biology 2003; 81: 106-13]. Maždaug tuo pat metu R. Pearl pranešė, kad tuberkuliozė turi priešvėžinį poveikį, todėl sugalvota vėžio gydymui panaudoti vakciną, taikomą tuberkuliozės gydymui - BCG (baccile Calmette-Guėrin). BCG vakcina šiuo metu yra vienintelė bakterinės kilmės vakcina, taikoma imunoterapijoje neinvazinio šlapimo pūslės vėžio gydyme [Mayer G. Immunology chapter one: innate (non-specific) immunity. Microbiology and Immunology On-Line Textbook. University of South Carolina School of Medicine. http://pathmicro.med.sc.edu/ghaffar/innate.htm [cituota 2015 05 01].; Cann SAH, van Netten JP, van Netten C. Dr William Coley and tumour regression: A place in history or in the future. Postgraduate Medical Journal 2003; 79: 672-80]. Plaučių vėžio imunoterapijoje BCG yra taikoma tik kaip adjuvantas imuninės sistemos stimuliavimui [Raez LE, Fein S, Podack ER. Lung Cancer Immunotherapy. Clinical Medicine & Research 2005; 3 (4): 221-8],The first beginnings of immunotherapy date back to the 19th century. late in the year when Coley noticed that a patient who had complete tumor remission had previously had an acute streptococcal infection. Later, after developing a vaccine against dead Streptococcus pyogenes and Serratia marcescens bacteria, he began to treat people with non-operable cancer (mostly sarcoma). The purpose of the vaccine was to stimulate the immune system [Cann SAH, van Netten JP, van Netten C. Dr William Coley and tumor regression: A place in history or in the future. Postgraduate Medical Journal 2003; 79: 672-80; Parish CR. Cancer immunotherapy: the past, the present and the future. Immunology and Cell Biology 2003; 81: 106-13]. At about the same time, Pearl reported that tuberculosis had an anticancer effect, so he came up with a vaccine for the treatment of tuberculosis called BCG (baccile Calmette-Guerrin). The BCG vaccine is currently the only vaccine of bacterial origin used in immunotherapy for the treatment of non-invasive bladder cancer [Mayer G. Immunology chapter one: innate (non-specific) immunity. Microbiology and Immunology On-Line Textbook. University of South Carolina School of Medicine. http://pathmicro.med.sc.edu/ghaffar/innate.htm [cited 2015-01-01]; Cann SAH, van Netten JP, van Netten C. Dr. William Coley and tumor regression: A place in history or in the future. Postgraduate Medical Journal 2003; 79: 672-80]. In lung cancer immunotherapy, BCG is applied only as an adjuvant to stimulate the immune system [Raez LE, Fein S, Podack ER. Lung Cancer Immunotherapy. Clinical Medicine & Research 2005; 3 (4): 221-8],
Antigenų, susijusių su naviku (TAA), atradimas paskatino specifinės imunoterapijos vystymą [Parish CR. Cancer immunotherapy: the past, the present and the future. Immunology and Cell Biology 2003; 81: 106-13; Wayteck L, Breckpot K, Demeester J, De Smedt SC, Raemdonck K. A personalized view on cancer immunotherapy. Cancer Letters 2014; 352: 113-25], kurios tikslas yra sužadinti imuninės sistemos atsaką į naviką [Bluestone JA, Small EJ. The future of cancer treatment: Will it include immunotherapy? Cancer Cell 2012; 22: 7-8], žinant, kad efektyvus imuninis atsakas neišsivysto dėl sumažėjusio imuninių ląstelių atpažinimo, silpno naviko imunogeniškumo ir imunosupresinės naviko aplinkos [Kumar S and Mason M. Principles of cancer treatment by immunotherapy. Surgery 2012; 30 (4): 198-202],The discovery of tumor-associated antigens (TAAs) has led to the development of specific immunotherapy [Parish CR. Cancer immunotherapy: the past, the present and the future. Immunology and Cell Biology 2003; 81: 106-13; Wayteck L, Breckpot K, Demeester J, De Smedt SC, Raemdonck K. A personalized view on cancer immunotherapy. Cancer Letters 2014; 352: 113-25], the purpose of which is to induce an immune system response to a tumor [Bluestone JA, Small EJ. The future of cancer treatment: Will it include immunotherapy? Cancer Cell 2012; 22: 7-8], knowing that an effective immune response does not develop due to reduced immune cell recognition, weak tumor immunogenicity, and immunosuppressive tumor environment [Kumar S and Mason M. Principles of cancer treatment by immunotherapy. Surgery 2012; 30 (4): 198-202],
Pagal veikimą naviko imunoterapija yra skirstoma į pasyviąją ir aktyviąją. Pasyvioji imunoterapija apima visas imunologiškai aktyvias medžiagas, imunomoduliuojančias ląsteles ir nepriklauso nuo paties paciento imuninės sistemos funkcionavimo, kadangi jam yra pateikiamos ląstelės, pasižyminčios priešvėžiniu poveikiu [Aly HAA. Cancer therapy and vaccination. Journal of Immunological Methods 2012; 382: 1-23], Toliau pasyviąją imunoterapiją galima smulkiau suskirstyti į terapiją monokloniniais antikūnais, stimuliuojančiais citokinais ir adoptyviomis T ląstelėmis [Wayteck L, Breckpot K, Demeester J, De Smedt SC, Raemdonck K. A personalized view on cancer immunotherapy. Cancer Letters 2014; 352. 113-25]. Pagal kito šaltinio skirstymą prie pasyviosios imunoterapijos dar yra priskiriama kaulų čiulpų transplantacija [Zavala VA and Kalergis AM. New clinical advances in immunotherapy for the treatment of solid tumours. Immunology 2015],By function, tumor immunotherapy is divided into passive and active. Passive immunotherapy encompasses all immunologically active substances, immunomodulatory cells, and is independent of the functioning of the patient's own immune system as it is provided with cells having anticancer activity [Aly HAA. Cancer therapy and vaccination. Journal of Immunological Methods 2012; 382: 1-23], Passive immunotherapy can be further subdivided into therapy with monoclonal antibodies, cytokine-stimulating and adoptive T cells [Wayteck L, Breckpot K, Demeester J, De Smedt SC, Raemdonck K. A personalized view on cancer immunotherapy. Cancer Letters 2014; 352. 113-25]. According to another source, passive immunotherapy includes bone marrow transplantation [Zavala VA and Kalergis AM. New clinical advances in immunotherapy for the treatment of solid tumors. Immunology 2015],
Aktyviajai imunoterapijai yra būtina tinkamai funkcionuojanti paciento imuninė sistema, kadangi gydymo metu kovai su vėžinėmis ląstelėmis yra sužadinami recipiento imuniniai mechanizmai. Aktyviajai imunoterapijai yra priskiriamos įvairios vakcinos: BCG, peptidinės, DNR, dendritinių ląstelių ir kt, kurios turėtų būti kiek galima mažiau toksiškos organizmui, galėtų sukelti priešvėžinį imuninį atsaką ne tik prieš pirminį naviką, bet ir prieš metastazes, kurių poveikyje susidarytų imuninė atmintis, apsauganti nuo ligos atsinaujinimo [Moingeon P. Cancer vaccines. Vaccine 2001; 19: 1305-26.; Yannelli JR and Wroblewski JM. On the road to a tumor cell vaccine: 20 years of cellular immunotherapy. Vaccine 2004; 23: 97-113].Active immunotherapy requires the patient's immune system to function properly, as the recipient's immune mechanisms are activated to fight the cancer cells during treatment. Active immunotherapy includes a variety of vaccines: BCG, peptide, DNA, dendritic cells, etc., which should be minimally toxic to the body, could trigger an antitumor immune response, not only against the primary tumor, but also against metastases that would trigger immune memory. from relapse [Moingeon P. Cancer vaccines. Vaccine 2001; 19: 1305-26; Yannelli JR and Wroblewski JM. On the road to tumor cell vaccine: 20 years of cellular immunotherapy. Vaccine 2004; 23: 97-113].
TRUMPAS IŠRADIMO APRAŠYMASBRIEF DESCRIPTION OF THE INVENTION
Citokinais indukuotų ląstelių dauginimas yra globali problema, šios ląstelės labai priklauso nuo donoro, jo esamos būklės, taikytų ankstesnių gydymo schemų ir kitų neapibrėžtų / nežinomų parametrų. Mūsų sukurtas ląstelių dauginimo metodas yra išskirtinis tuo, kad gaunamas ganėtinai stabilus ląstelių skaičius preparate su dideliu NK ląstelių kiekiu jame. Taip pat ląstelių gamybai naudojamas unikalus mišinys aktyvuojančių molekulių, išlaikomas specifinis aktyvacijos periodas ir proliferacijos periodas, skirtas ląstelių kryptingam dalinimuisi ir brendimui.Cytokine-induced cell proliferation is a global problem and is highly dependent on the donor, its current status, previous treatment regimens, and other uncertain / unknown parameters. The cell proliferation method we have developed is unique in that it produces a fairly stable number of cells in a preparation with a high concentration of NK cells. It also uses a unique mixture of activating molecules for cell production, maintaining a specific activation period and a proliferation period for targeted cell division and maturation.
Periferinis kraujas arba kraujo aferezatas arba kaulų čiulpų aspiratas yra separuojamas, išskiriant tik mononuklearinių ląstelių frakciją, ši frakcija yra išsėjama ląstelių auginimo terpėje ant specialaus bioreaktoriaus arba flakono, kuriame auginama iki 17-20 parų. Auginant ląsteles reaktoriuje, matuojamas gliukozės suvartojimas, tai atspindi ląstelių augimo aktyvumą ir parodo laiką kada jau galima stabdyti gamybą ir rinkti ląsteles šaldymui ir tolesniam naudojimui. Terpė nupilama, surenkamos ląstelės, jos yra skaičiuojamos ir šaldomos dozėmis po 0,5-5,0 mlrd. ląstelių šaldymo terpėje su autologiniu paciento serumu. Atliekamas sterilumo tyrimas ir kiti privalomi tyrimai.Peripheral blood or blood apheresis or bone marrow aspirate is separated by isolating only the mononuclear cell fraction, which is seeded in cell culture medium in a special bioreactor or vial for up to 17-20 days. When growing cells in a reactor, glucose consumption is measured, which reflects cell growth activity and shows the time at which production can be stopped and cells harvested for refrigeration and subsequent use. The medium is drained, cells are harvested, counted and frozen at doses of 0.5 to 5.0 billion. cells in refrigerated media with autologous patient serum. A sterility test and other required tests shall be carried out.
IŠSAMUS IŠRADIMO APRAŠYMASDETAILED DESCRIPTION OF THE INVENTION
I. Mononuklearinių ląstelių išskyrimas iš kraujo, aferezato, kaulų čiulpų aspiratoI. Isolation of mononuclear cells from blood, apheresis, bone marrow aspirate
Kraujas skiedžiamas hidroetilkrakmolo tirpalu, skirtu infuzijoms kaip plazmos pakaitalas, skiedžiama santykiu 1:1. Sumaišytas tirpalas paliekamas nusistovėti kambario temperatūroje 20-40 min., per tiek laiko paprastai išsiskiria du sluoksniai, apatinis, turtingas eritrocitais, ir viršutinis, kuriame yra eritrocitų, plazmos bei branduolinių kraujo elementų. Viršutinis sluoksnis yra surenkamas į centrifuginius mėgintuvėlius ir centrifuguojamas prie 1000-1500 G 5-15 min. kambario temperatūroje, po centrifugavimo supernatantas yra nupilamas, o nuosėdos suspenduojamos druskos tirpale arba likusiame viršutiniame kraujo / hidroksietilkrakmolo tirpalo mišinyje. Paprastai, esant 50-300 ml kraujo, centrifugatas yra skiedžiamas iki 40 ml baigtinio tūrio. Jeigu kraujo yra daugiau, atitinkamai proporcingai yra skiedžiamas ir centrifugatas. Tuomet j 15 arba 50 ml mėgintuvėlius yra pripilama fikolo arba perkolo tirpalo, kurio tankis yra nuo 1,0 iki 1,12 g/ml. Tirpalo pilama ne daugiau kaip 1/3 mėgintuvėlio tūrio. Ant šio tirpalo pipete iš lėto, palenkus mėgintuvėlį, kuo gulsčiau atsargiai pilamas skiestas ląstelių centrifugatas iki mėgintuvėlio viršaus. Mėgintuvėliai centrifuguojami prie 1100-1500 G 10-20 min. kambario temperatūroje, stabdis nenaudojamas. Po centrifugavimo mėgintuvėliuose matomos trys frakcijos: apačioje - eritrocitų frakcija, virš eritrocitų - mononuklearų sluoksnis, plaukiojantis aukščiau fikolo skaidraus sluoksnio, virš mononuklearų skluoksnio - trombocitai kartu su praskiesta hidroksietilkrakmolu plazma. Pipete atsargiai yra surenkamas mononuklearų sluoksnis ir supilamas j naujus mėgintuvėlius, kuriuose yra įpilta druskos tirpalo arba ląstelių auginimo terpės. Vyksta ląstelių plovimo nuo gradiento medžiagos (fikolo arba perkolo) etapas. Ląstelės gerai išmaišomos ir centrifuguojamos prie 500-850 G 10-20 min. kambario temperatūroje. Stabdis nenaudojamas. Procedūra gali būti kartojama kelis kartus. Po plovimo ląstelės suskaičiuojamos ir išsėjamos j ląstelių auginimo reaktorių (tūris 1L) arba flakonus (175 cm2 ploto), tokiu santykiu: 20-100 milijonų ląstelių ir 100 ml indukuojamosios terpės, skirtos NK ląstelėms, T limfocitams arba dendritinėms ląstelėms auginti su papildomais priedais, kurie gali būti anti CD3 ir (arba) antiCD28 ir (arba) antiCD127 ir (arba) antiCD335 molekulė, bei interferonais, tokie kaip interferonas alfa ir (arba) interferonas gamma, įskaitant visus jų pogrupius. Bioreaktorius / flakonai yra inkubuojami ląstelių kultūroms skirtuose inkubatoriuose 37 °C temperatūroje ir 5-15 % CO2 aplinkoje 12-24 valandas. Po inkubacijos, jeigu auginama bioreaktoriuje, pridedama proliferacinės terpės, kurioje yra citokinai, tokie kaip IL-2 ir (arba) IL-15 ir (arba) IL-18. Jeigu dirbama su flakonais, proliferaciją skatinantys citokinai dedami tiesiai į terpę, papildant ją auginimo terpe 25 % nuo viso terpės tūrio. Ląstelės auginamos iki 17-20 parų, stebimas gliukozės ir (arba) laktato kiekis. Auginant flakonuose, terpė keičiama nauja, papildant ją tiek aktyvacinėmis, tiek proliferaciją aktyvuojančiomis molekulėmis. Terpės keičiamas kiekis nusprendžiamas pagal metabolitų suvartojimą / atsiradimą (gliukozė / laktatas). Auginant flakonuose, ląstelės resuspenduojamos pipete (išvengiant didelių ląstelių agregatų). Auginant bioreaktoriuje, matuojami tik metabolitai, ir vizualiai stebimas ląstelių augimas ant reaktoriaus pagrindo. Kuomet gliukozės ar laktato koncentracijos pasiekia kritines ribas (12-13±2 mmol), auginimas yra stabdomas ir ląstelės yra renkamos bei šaldomos. Gaminant praimuotas NK ląsteles, 5-7 auginimo dienoje į ląstelių kultūrą įpilamas 1-10 min. Autologinių / alogeninių su specifiniu antigenu brandintų denritinių ląstelių. Toliau auginimas nesikeičia. Ląstelės šaldomos koncentracijoje nuo 0,5-5,0 mlrd. Ląstelių / 25 ml terpės skirtos šaldymui. Šaldymo terpė sudaryta iš 10 % DMSO (dimetilsulfoksidas) autologinio paciento serumo arba plazmos. DMSO niekada nėra įmaišomas kaip grynas tirpalas į ląstelių suspensiją, jis maišomas tik praskiestas su serumu. Tirpalai, maišant su DMSO kaista, todėl visi tirpalai atšaldomi, prieš pradedant ląstelių šaldymo procedūrą. Su šaldymo terpe sumaišytos ląstelės yra išpilstomos j šaldymo mėgintuvėlius, kurie dedami j šaldymo dėžutę arba šaldymo įrenginį ir šaldomi pagal standartinį protokolą -1 °C/min., patalpinant juos j -80 °C temperatūrą porai valandų. Užšaldytas preparatas yra saugomas iki jo sunaudojimo -80 °C arba -196 °C temperatūroje.The blood is diluted 1: 1 with hydroethyl starch solution for infusion as a plasma substitute. The reconstituted solution is left to stand at room temperature for 20-40 minutes, during which time two layers, the lower, rich in red blood cells and the upper containing erythrocytes, plasma and nuclear blood cells, are usually released. The supernatant is collected in centrifuge tubes and centrifuged at 1000-1500 G for 5-15 min. at room temperature, the supernatant is discarded after centrifugation, and the precipitate is suspended in saline or the remaining supernatant of blood / hydroxyethyl starch solution. Typically, the supernatant is diluted to a final volume of 40 ml with 50 to 300 ml of blood. If there is more blood, the centrifuge is diluted accordingly. Then 15 or 50 ml tubes are filled with a solution of ficol or percol in a density of 1.0 to 1.12 g / ml. Not more than 1/3 of the tube volume is added. Pipette this solution slowly, tilting the tube gently, diluting the diluted cell supernatant as slowly as possible to the top of the tube. Centrifuge tubes at 1100-1500 G for 10-20 min. at room temperature, the brake is not used. After centrifugation, three fractions are visible in the tubes: the lower part of the erythrocyte fraction, the top of the erythrocytes a mononuclear layer floating above the phycol clear layer, and the mononuclear layer platelets together with diluted hydroxyethyl starch plasma. Carefully pipette the mononuclear layer and transfer to new tubes containing saline or cell culture medium. Gradient cell washing step (ficol or percol) is underway. Cells are well mixed and centrifuged at 500-850 G for 10-20 min. at room temperature. The brake is not used. The procedure may be repeated several times. After washing, cells are counted and plated in a cell growth reactor (1L volume) or vials (175 cm 2 area) at a ratio of 20-100 million cells and 100 ml induction medium for growth of NK cells, T lymphocytes or dendritic cells with additional additives. , which may be an anti-CD3 and / or antiCD28 and / or antiCD127 and / or antiCD335 molecule, and interferons such as interferon alpha and / or interferon gamma, including any subsets thereof. The bioreactor / vials are incubated in cell culture incubators at 37 ° C and 5-15% CO 2 for 12-24 hours. After incubation, if grown in a bioreactor, a proliferative medium containing cytokines such as IL-2 and / or IL-15 and / or IL-18 is added. In the case of vials, proliferation-promoting cytokines are added directly to the medium, supplemented with culture medium at 25% of the total volume of the medium. Cells are cultured for up to 17-20 days with glucose and / or lactate levels monitored. When growing in vials, the medium is changed by addition of both activating and proliferating molecules. The amount of medium to be changed is determined by the metabolite intake / appearance (glucose / lactate). When growing in vials, cells are resuspended in a pipette (avoiding large cell aggregates). When growing in a bioreactor, only metabolites are measured and cell growth on the reactor base is visually observed. When glucose or lactate concentrations reach critical limits (12-13 ± 2 mmol), growth is stopped and cells are harvested and frozen. For the production of primed NK cells, 1-10 min is added to the cell culture on culture day 5-7. Autologous / allogeneic antigen-matured denitrocytes. Further cultivation does not change. Cells are frozen at concentrations ranging from 0.5 to 5.0 billion. Cell / 25 ml medium for freezing. Refrigeration medium consisted of 10% DMSO (dimethylsulfoxide) autologous patient serum or plasma. DMSO is never mixed as a pure solution in cell suspension, it is only diluted with serum. The solutions heat up when mixed with DMSO, so all solutions are cooled before starting the cell freezing procedure. Cells mixed with the refrigerant medium are dispensed into freezer tubes, which are placed in a freezer box or freezer and frozen according to the standard protocol at -1 ° C / min, stored at -80 ° C for a couple of hours. Frozen product is stored at -80 ° C or -196 ° C until use.
Iš specialiai atskirai surinkto mėginio yra atliekami būtini ląstelių kokybinių parametrų tyrimai (bakterinės, grybelinės infekcijos, paviršiaus žymenys (CD3/16/56/8/4/69 gali būti pildoma pagal mokslinį progresą), gyvybingumo analizė, endotoksinų ir mikoplazmos tyrimas).From a specially collected sample, the necessary cell qualitative parameters tests (bacterial, fungal infections, surface markers (CD3 / 16/56/8/4/69 may be completed according to scientific progress), viability analysis, endotoxin and mycoplasma assay) are performed.
II. CIK atšildymas ir suleidimasII. CIK thawing and injection
Užšaldytas mėgintuvėlis / maišelis ištraukiamas iš šaldymo vietos ir nedelsiant yra patalpinamas į indą su šiltu +37 °C temperatūros vandeniu. Atšildoma iki kol ledas visiškai ištirps ir pripilamas toks pats tūris fiziologinio skysčio arba autologinio serumo, jeigu yra vietos maišelyje, jeigu maišelis yra 25 ml, tai lašinama tiesiai iš maišelio į veną, likus 5-10 % tūrio j maišelį steriliai pripilama dar 20 ml fiziologinio skysčio, preparatas toliau sulašinamas kol maišelyje neliks skysčio. Lašinama intraveniškai 5 ml/min greičiu. Stebima paciento būsena, pasireiškus galvos skausmams ar pykinimui, lašinimimo greitis sumažinamas iki 2 ml/min. Neigiamos reakcijos į CIK preparato taikymą pasireiškia retai, dažniausiai tai būna aukšta temperatūra, galvos skausmas, kas praeina be papildomo vaistinio įsikišimo per 30-45 minutes. Esant labai aukštai temperatūrai, reikia naudoti acetaminofeną (500 mg tab. peros) iškarto po lašinimo, jeigu tai pirmas kartas, jeigu antras kartas tai 30 minučių iki lašinimo. Lašinama per sistemą su tinkleliu ir ne plonesne nei 23 G adata.The frozen tube / bag is removed from the freezer and immediately placed in a container with warm water at + 37 ° C. Thaw until the ice has completely dissolved and fill with the same volume of physiological fluid or autologous serum, if space is present in the pouch, if the pouch is 25 ml, drip directly into the pouch, and 5 to 10% of the pouch is sterile filled with liquid, the product should continue to be added until no fluid is left in the bag. Intravenous infusion at a rate of 5 ml / min. Patient monitoring for headaches or nausea reduces the drip rate to 2 ml / min. Adverse reactions to CIK administration are rare, usually in the form of high temperature, headache, and resolved without additional drug intervention within 30-45 minutes. At very high temperatures, acetaminophen (500 mg tab. Per day) should be used immediately after dosing for the first time and 30 minutes before dosing for the second time. It is administered through a grid system with a minimum of 23G needle.
CIK taikymo indikacijos ir gydymo pavyzdžiaiIndications for CIK application and treatment examples
Indikacijos procedūrai atlikti:Indications for performing the procedure:
Karcinomų, sarkomų ir kraujo vėžių (skystųjų navikų) 1-os, 2-os, 3-ios, 4-os stadijų onkologiniai susirgimai.Carcinomas, sarcomas and blood cancers (liquid tumors) stage 1, 2, 3, 4 oncology.
Ligoniams taikyto gydymo CIK ląstelėmis pavyzdžiai ir jų rezultatai:Examples and results of CIK treatment in patients:
Ligonė A.A. IV stadijos metastazuojanti kolorektalinė karcinoma, taikomas gydymas chemoterapija, tirozinkinazės inhibitoriais, paciento išgyvenamumo prognozė - 2-3 mėn. Taikytas gydymas autologinėmis CIK ląstelėmis nuo 2018 metų. Atlikti 7 CIK kursai po 1,0 mlrd. Ląstelių i.v. CIK taikoma tarpuose tarp chemoterapijų. Pacientas yra stabilioje būsenoje, liga neprogresuoja, dingo ascitas, būklė gera.Hospital A.A. Stage IV metastatic colorectal carcinoma, chemotherapy, tyrosine kinase inhibitors, survival time 2-3 months. Treatment with autologous CIK cells has been applied since 2018. 7 CIK courses of $ 1.0 billion were completed. The cells i.v. CIC is applied at intervals between chemotherapies. The patient is in a stable state, the disease is not progressing, the ascites have disappeared and the condition is good.
Ligonė R.P., 2014 m. diagnozuotas krūties vėžys, taikytas chirurginis gydymas, chemoterapija, 2017 metais navikas metastazavo j plaučius ir kepenis, 2018 į smegenis, prognozė bloga, navikai operuoti kur chirurgiškai įmanoma, 2018 metais pradėtas taikyti imunoterapinis gydymas CIK preparatu. Suleista iki 2019 metų balandžio mėnesio 8 CIK kursai po 1,0 mlrd. ląstelių i.v. Šiai dienai pacientė stabili, liga neprogresuoja, vėžio žymenys “tyli, būklė gera.Hospital R.P., 2014. diagnosed breast cancer, applied surgical treatment, chemotherapy, in 2017 tumor metastasized to lungs and liver, 2018 to brain, prognosis is poor, tumors operated where surgically possible, in 2018 immunotherapy treatment with CIK was started. Issued by April 2019, 8 CIK courses at $ 1.0 billion each. of cells i.v. Today, the patient is stable, the disease does not progress, the signs of cancer are 'silent, and the condition is good.
Ligonė R.R., 2011 metais diagnozuota sarkoma, plitimas labai greitas, auglys išplito į kaulus, kepenis, plaučius, gydymas chemoterapija, radioterapija ligą sulaikė, 2017 prasidėjo aktyvus ligos progresas. Nuo 2018 metų taikyta CIK terapija. 8 kursai po 1,0 mlrd. ląstelių i.v., ligos progresavimas apstojo, metastazės plaučiuose kalcifikavosi, progresavimas visur liovėsi, išskyrus dubens kaulo vieną metastazę, kitos metastazės pradėjo nykti, stebimi daugybiniai uždegiminiai procesai metastazėse. 2019 metų balandžio mėnesį liga laikoma stabilia, paciento būklė gera.R.R., diagnosed with sarcoma in 2011, very rapid spread, tumor spread to bone, liver, lung, chemotherapy treatment, radiation therapy suppressed disease, 2017 began active disease progression. Since 2018, CIK therapy has been applied. 8 courses at $ 1.0 billion each. cells i.v., disease progression stopped, lung metastases calcified, progression ceased except for pelvic bone one metastasis, other metastases began to subside, multiple inflammatory processes in metastases are observed. In April 2019, the disease was considered stable and the patient was in good health.
Ligonis V.S., 2018 metais diagnozuota skrandžio karcinoma G3, IV stadija, metastazės pilvaplėvėje. Taikyta operacinis gydymas ir chemoterapija, tarpuose CIK imunoterapija. Atlikti 9 CIK kursai po 1,0-1,5 mlrd. ląstelių i.v. Pacientas 2019 metų balandžio mėnesiui yra stabilus, metastazių plitimas ir naujų atsiradimas nestebimas, pilvaplėves metastazės nyksta.Patient V.S., 2018 diagnosed with gastric carcinoma G3, stage IV, peritoneal metastasis. Surgical and chemotherapy was applied, including CIK immunotherapy. Completed 9 CIK courses of $ 1.0-1.5 billion each. of cells i.v. The patient is stable by April 2019, no spread of metastases and no new onset, no abdominal metastases.
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