TR2021017263A2 - A METHOD FOR ISOLATION OF MR1T CELLS AND PRODUCTION OF TRANSGENIC CAR-MR1T CELL - Google Patents
A METHOD FOR ISOLATION OF MR1T CELLS AND PRODUCTION OF TRANSGENIC CAR-MR1T CELLInfo
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Abstract
Mevcut buluş, MR1 (Major histocompatibility complex class I-related) T-hücre izolasyonu için bir yöntem ile ilgilidir. Buluşa ait yöntem; boş MR1 tetramerleri ile solid kanser hücre lizatlarının 20 ila 45 dakika süreyle inkübe edilmesi, solid kanser hücreleri tarafından sunulan MR1 antijenleri ile dolmuş olan dolu MR1 tetramerlerinin elde edilmesi, sağlıklı bir insan kanından izole edilen periferik kan mononükleer hücre (PBMC) örneğinin sunulması, dolu MR1 tetramerleri ile PBMC örneğinin inkübe edilmesi ve MR1 tetramerlerine bağlanan MR1T hücrelerinin izole edilmesi ve transgenik CAR-MR1T-EGFRt hücre üretim adımlarını ihtiva etmektedir.The present invention relates to a method for MR1 (Major histocompatibility complex class I-related) T-cell isolation. The method of the invention; Incubating solid cancer cell lysates with empty MR1 tetramers for 20 to 45 minutes, obtaining filled MR1 tetramers filled with MR1 antigens presented by solid cancer cells, presenting a peripheral blood mononuclear cell (PBMC) sample isolated from a healthy human blood, filled MR1 tetramers and incubating a PBMC sample and isolating MR1T cells bound to MR1 tetramers and generating transgenic CAR-MR1T-EGFRt cells.
Description
TARIFNAME MR1T HÜCRELERININ IZOLASYONU VE TRANSGENIK CAR-MR1T HÜCRE ÜRETIMI IÇIN BIR YÖNTEM BULUSUN ILGILI OLDUGU TEKNIK ALAN Mevcut bulus, MR1 (Major histocompatibility complex class l-related) T-hücre (MR1 Restricted T-hücre) izolasyonu için bir yöntem ile ilgilidir. Mevcut bulusa ait yöntem kapsaminda; bos MR1 tetramerleri solid tümör hücre Iizatlariyla ko-kültürlenmekte, solid kanser hücrelerinin sundugu MR1 iliskili tümör spesifik antijenler ile dolu olan MR1 tetramerleri saglikli bir insan kanindan izole edilen periferik kan mononükleer hücre (PBMC) ile inkübe edilmekte ve MR1 tetramerlerine baglanmis olan MR1T hücreleri izole edilmektedir. DESCRIPTION ISOLATION OF MR1T CELLS AND PRODUCTION OF TRANSGENIC CAR-MR1T CELL A METHOD FOR TECHNICAL FIELD OF THE INVENTION The present invention is MR1 (Major histocompatibility complex class l-related) T-cell (MR1 Restricted) It relates to a method for the isolation of T-cells. Within the scope of the method of the present invention; empty MR1 tetramers are co-cultured with solid tumor cell lines, MR1 tetramers packed with MR1-associated tumor-specific antigens are derived from healthy human blood. The isolated peripheral blood is incubated with mononuclear cell (PBMC) and MR1 MR1T cells bound to tetramers are isolated.
TEKNIGIN BILINEN DURUMU MR1T hücreleri, MR1 (Major histocompatibility complex class I-related) proteini yardimiyla antijenleri taniyip onlarla savasan, geleneksel olmayan T-hücreleridir. Geleneksel olmayan T- hücreleri olarak nitelendirilmelerinin sebebi, klasik T-hücrelerin aksine MHC/HLA (Major Histocompatibility Complex/Human Leukocyte Antigen) olmadan MR1 proteini tarafindan sunulan antijenleri tanimalaridir (Gherardi'n, N. A., vd. (2018). The di'verse family of MR1- MR1, birçok hücre tipinin yüzeyinde çok düsük seviyelerde eksprese edilen polimorfik olmayan MHC-l benzeri bir proteindir. Bakteriyel enfeksiyonun ardindan, antijen sunan hücreler (APC) antijen baglanmasina bagli gelisen protein stabilizasyonu nedeniyle MR1'in yüzey ekspresyonunu upregüle eder. MR1 birçok türde yüksek oranda korunmustur, örnegin insan ve fare MR1 proteinleri protein düzeyinde >%90 sekans homolojisi paylasmaktadir (Lepore, M., vol. (2017). Functionally diverse human T cells recognfze non-microbi'al antigens presented by MR1. eLi'fe, 6, 624476). KNOWN STATE OF THE TECHNIQUE MR1T cells with the help of MR1 (Major histocompatibility complex class I-related) protein are non-traditional T-cells that recognize and fight antigens. Unconventional T- The reason why they are called as MHC/HLA (Major) by MR1 protein without Histocompatibility Complex/Human Leukocyte Antigen) recognition of the presented antigens (Gherardi'n, N. A., et al. (2018). The di'verse family of MR1- MR1 is non-polymorphic, expressed at very low levels on the surface of many cell types. It is an MHC-1-like protein. After bacterial infection, antigen presenting cells (APC) Because of the protein stabilization due to antigen binding, the surface of MR1 upregulates its expression. MR1 is highly conserved in many species, for example human and mouse MR1 proteins share >90% sequence homology at the protein level (Lepore, M., vol. (2017). Functionally diverse human T cells recognfze non-microbi'al antigens presented by MR1. eLi'fe, 6, 624476).
Kanser immünoterapide halihazirda kullanilan en etkin yöntemlerden biri, transgenik kimerik antijen reseptörü (CAR, Chimeric antigen receptors) eksprese eden otolog ve klasik T-hücre (CAR-T) uygulamasidir. Fakat allojenik CAR-T hücre terapisinde kullanilan geleneksel T- hücreleri yüzeylerinde halihazirda T-hücre reseptörü eksprese ettiginden, antijenleri MHC Sinif 1 ve 2 araciligiyla tanimaktadir. Ancak söz konusu MHC proteini polimorfik (insandan insana degisen) oldugu için doku uyumsuzluguna sebep olabilmektedir. Öte yandan MR1T hücreleri, antijenleri MR1 proteini araciligi ile tanimalari, bu proteinin ise insandan insana degismemesi sebebiyle GVHD (Doku Uyumsuzlugu) olusturmayacagi öngörülmektedir. . Bu sebeple, MR1T hücreleri kanser immünoterapide genel kullanim için oldukça uygundur. Birçok çalisma, çesitli MR1T hücrelerinin kanseri elimine etmede basarili oldugunu ortaya koymustur (Wang, Z., vd. (2020). MR1 -restri'cted T cells.' the new dawn of cancer fmmunotherapy. Blosclence Reports, 40( 1 1); Mori, L., & De leero, G. (2020). 'Bohemlan Rhapsodyfof MR1T cells. Nature lmmunology, 21(2), 108-110). bir tümör iliskili antijene spesifik olarak baglanabilen bir T-hücre reseptörünü eksprese eden T-hücrenin izole edilmesine yönelik bir yöntemi açiklamaktadir. Yöntem T-hücrelerin preparasyonunun saglanmasi, preparasyonun MR1 proteinini eksprese eden kanser hücreleri ile temas ettirilmesi ve adi geçen kanser hücrelerine spesifik olarak reaktif olan bir T-hücrenin izole edilmesi adimlarini ihtiva etmektedir. monomorphi'c MHC class l related protein MR1" isimli yayin kapsaminda, MR1 araciligiyla çogu insan kanser türünü taniyabilen ve öldürebilen bir MR1 TCR klonu olan MC.7.GS izole edilmistir. Söz konusu MC.7.GS klonunun, bakteri metabolomlarinin MAIT hücreleri tarafindan taninmasi için kullanilan TCR profillerinden farkli olarak kanser hücrelerini tanidigi belirtilmektedir (Crowther, M. D., v.d. (2020). Genome-wi'de CRlSPR-CasQ screenlng reveals ublquitous T cell cancer targeti'ng via the monomorphlc MHC class l-related protein MR1. One of the most effective methods currently used in cancer immunotherapy is the transgenic chimeric. Autologous and classical T-cells expressing antigen receptors (CAR, Chimeric antigen receptors) (CAR-T) application. However, conventional T-cell therapy used in allogeneic CAR-T cell therapy. Since cells already express T-cell receptor on their surface, antigens are MHC Class It is recognized through 1 and 2. However, the MHC protein in question is polymorphic (human to human). It can cause tissue incompatibility because it changes). On the other hand, MR1T cells They recognize antigens through the MR1 protein, and this protein does not change from person to person. It is predicted that it will not cause GVHD (Tissue Incompatibility) due to . For this reason, the MR1T cells are very suitable for general use in cancer immunotherapy. Many studies, various demonstrated that MR1T cells were successful in eliminating cancer (Wang, Z., et al. (2020). MR1 -restricted T cells.' the new dawn of cancer fmmunotherapy. Blosclence Reports, 40( 1 1); Mori, L., & De leero, G. (2020). 'Bohemlan Rhapsodyfof MR1T cells. nature Immunology, 21(2), 108-110 ). expressing a T-cell receptor that can bind specifically to a tumor-associated antigen It describes a method for isolating T-cell. Method of T-cells cancer cells expressing the MR1 protein of the preparation of a T-cell that is specifically reactive to said cancer cells. It includes the steps of isolation. monomorphi'c MHC class l related protein MR1", via MR1 MC.7.GS isolate, an MR1 TCR clone that can recognize and kill most types of human cancer has been made. The bacterial metabolomes of the MC.7.GS clone in question were detected by MAIT cells. It recognizes cancer cells, unlike the TCR profiles used for the recognition of cancer cells. (Crowther, M. D., et al. (2020). CRlSPR-CasQ screenlng reveals in Genome-wi ublquitous T cell cancer targeti'ng via the monomorphlc MHC class l-related protein MR1.
Nature lmmunology, 21(2), 178-185). Nature Immunology, 21(2), 178-185).
Yukarida örnekleri verilen, teknigin bilinen durumuna ait MR1T hücresi izolasyonu yöntemleri, hücrelerin uzun süreli ko-kültür ile aktivasyonunu ihtiva etmektedir. Kanser hücreleri tarafindan aktif hale getirilen T-hücreler akis sitometri (flow-cytometry) yöntemi ile aktiflik belirteçlerine göre izole edilmekte, ardindan bu hücrelerin MR1T hücresi oldugunu teyit etmek için kanser hücreleri ile tekrar ko-kültür gerçeklestirilerek hücrenin aktivite durumu kontrol edilmektedir. Iki kez aktivasyona maruz birakilan T hücrelerinde yorulma (exhaustion) görülebilmektedir. MR1T cell isolation methods of the state of the art, examples of which are given above, It involves the activation of cells by prolonged co-culture. by cancer cells Activated T-cells are analyzed for activity markers by flow-cytometry. cancer cells are isolated according to MR1T cells. The activity status of the cell is checked by performing co-culture with the cells again. Two Exhaustion can be seen in T cells that are exposed to activation once.
Bunun sonucunda ise kanser immunoterapisi yeterliliginin azalmasi durumu ortaya çikmaktadir. As a result, a decrease in the adequacy of cancer immunotherapy has emerged. is coming out.
MR1T hücresi izolasyonunda karsilasilan problemlerden bir digeri de, aktif hale getirilen MR1T hücreleri tarafindan ortama interlökin-2 (IL-2) salgilanmasidir. Salgilanan IL-2 sitokini yakininda bulunan fakat kanser immünoterapi aktivitesi olmayan diger hücrelerin de aktif hale gelmesine sebep olmaktadir. Bunun nedeni ise kullanilan izolasyon markerlarinin (örnegin CDS, CD69, CD137, CD150) MR1T spesifik olmamasidir. Kontamine riskinin yüksek olmasi nedeniyle izole edilen aktif hücrelerin MR1T hücresi olmama ihtimali bulunmaktadir. Another problem encountered in MR1T cell isolation is the activated MR1T. is the secretion of interleukin-2 (IL-2) into the environment by the cells. Secreted IL-2 cytokine Activation of other cells that are nearby but do not have cancer immunotherapy activity. causing it to come. The reason for this is that the isolation markers used (for example, CDS, CD69, CD137, CD150) are non-MR1T specific. High risk of contamination Therefore, there is a possibility that the isolated active cells are not MR1T cells.
Bu bilgiler isiginda görülmektedir ki, son yillarda kanser immünoterapisi alaninda CAR-T hücrelerine ek vei'veya alternatif olarak “MR1T hücre” yaklasimi öne çikmaktadir. Ancak MR1 T hücrelerinin izolasyonu için kullanilan yöntemler, aktivasyon adimi sebebiyle elde edilen MR1T hücrelerinde yorulmaya yol açabilmektedir ve elde edilen hücrelerin MR1T hücresi oldugu kesin olarak tespit edilememektedir. Dolayisiyla ilgili teknik alanda, MR1T hücrelerinin aktive edilmeden izole edilmesine yönelik bir ihtiyaç bulunmaktadir. Sunulacak yeni yöntemler, özellikle kanser immünoterapisi alaninda bir ilerleme saglayacaktir. Mevcut bulus bu amaçla, MR1T hücrelerinin izolasyonu için, hücrelerin aktive edilmeden izolasyonuna imkan veren yeni bir yöntem saglamaktadir. In the light of this information, it is seen that CAR-T has been used in the field of cancer immunotherapy in recent years. In addition to cells or alternatively, “MR1T cell” approach stands out. But the MR1 T The methods used for the isolation of the cells are the MR1T obtained due to the activation step. It can cause fatigue in cells and the cells obtained are MR1T cells. cannot be determined precisely. Therefore, in the relevant technical field, the activation of MR1T cells There is a need for isolation without being isolated. New methods to be introduced, especially in the field of cancer immunotherapy will provide progress. For this purpose, the present invention For the isolation of MR1T cells, a new method allows isolation of cells without being activated. provides a method.
BULUSUN KISA AÇIKLAMASI Mevcut bulus bir yönüyle, MR1T hücresi izolasyonu için asagidaki adimlari ihtiva eden bir yöntem saglamaktadir: - bos MR1 tetramerleri ile solid kanser hücre Iizatlarinin inkübe edilmesi, - solid kanser hücrelerinin sundugu MR1 iliskili tümör spesifik antijenler ile dolmus olan MR1 tetramerlerinin elde edilmesi, - saglikli bir insan kanindan izole edilen periferik kan mononükleer hücre (PBMC) örneginin sunulmasi, - dolu MR1 tetramerleri ile PBMC örneginin 20 ila 45 dakika süreyle ko-kültür edilmesi, - MR1 tetramerlerine baglanmis olan MR1T hücrelerinin izole edilmesi. BRIEF DESCRIPTION OF THE INVENTION In one aspect, the present invention is a method for MR1T cell isolation comprising the following steps. The method provides: - incubation of solid cancer cell strains with empty MR1 tetramers, - filled with MR1-related tumor-specific antigens presented by solid cancer cells Obtaining MR1 tetramers, - peripheral blood mononuclear cell (PBMC) isolated from healthy human blood presentation of the sample, - co-culturing the PBMC sample with filled MR1 tetramers for 20 to 45 minutes, - Isolation of MR1T cells bound to MR1 tetramers.
Mevcut bulusa göre olan yöntem kapsaminda tümör iliskili antijenlerle dolu MR1 tetramerleri ile PBMC örneginin ko-kültür edilmesi islemi tercihen 30 dakika süreyle gerçeklestirilmektedir. MR1 tetramers filled with tumor-associated antigens in the method of the present invention co-culturing of the PBMC sample is preferably carried out for 30 minutes.
Mevcut bulusa göre olan yöntem kapsaminda söz konusu bos MR1 tetramerleri ile solid kanser hücre Iizatlarinin inkübe edilmesi islemi tercihen 4 ila 37°C arasinda gerçeklestirilmektedir. Solid cancer with said empty MR1 tetramers within the scope of the method according to the present invention The incubation of cell strains is preferably carried out between 4 and 37°C.
Mevcut bulusa ait yöntemde kullanilan bos MR1 tetramerleri tercihen floresan boya ile isaretlidir. Söz konusu floresan boya APC veya PE floresan boya olabilmektedir. Empty MR1 tetramers used in the method of the present invention are preferably coated with fluorescent dye. is marked. Said fluorescent dye may be APC or PE fluorescent dye.
Tercihen, mevcut bulusa ait yöntemde yer alan izolasyon islemi FACS sort etme yöntemi ile gerçeklestirilmektedir. Preferably, the isolation process in the method of the present invention is by FACS sorting method. is carried out.
Mevcut bulusa ait yöntemde kullanilan solid kanser hücre lizati tercihen A549, MM909.11, ihtiva eden gruptan seçilen bir hücre hattinin Iizatidir. The solid cancer cell lysate used in the method of the present invention is preferably A549, MM909.11, is a strain of a cell line selected from the group consisting of
Mevcut bulusa göre olan yöntem ayrica izole edilen MR1T hücrelerinin anti-CD3/anti-CD28 mikrobitleriyle veya PHA ile aktive edilmesi adimini ihtiva edebilmektedir. Tercihen, aktive edilen MR1T hücreleri lL-2, lL-7, lL-15, IL-12 ve lL-18 sitokinlerini ihtiva eden bir gruptan seçilen bir sitokin ile çogaltilmaktadir. The method according to the present invention also contains anti-CD3/anti-CD28 cells of isolated MR1T cells. microbites or PHA activation. Preferably, activated MR1T cells from the group containing the cytokines IL-2, IL-7, IL-15, IL-12 and IL-18 amplified by a selected cytokine.
Mevcut bulusa göre olan yöntem ayrica tercihen izole edilen MR1T hücrelerine solid tümör- spesifik CAR (kimerik antijen reseptörü) geninin entegre edilmesi yoluyla CAR-MR1T-EGFRt hücrelerinin elde edilmesi adimini ihtiva etmektedir. Söz konusu solid tümör tercihen mesane kanseri, meme kanseri, rahim agzi kanseri, endometrium kanseri, böbrek kanseri, dudak ve agiz kanseri, karaciger kanseri, melanom, mezotelyoma küçük hücreli olmayan akciger kanseri, melanom olmayan cilt kanseri, yumurtalik kanseri, pankreas kanseri, prostat kanseri, sarkom, küçük hücreli akciger kanseri, tiroid kanseri, glioblastoma, nöroblastoma ve kolorektal kanseri ihtiva eden bir gruptan seçilmektedir. The method according to the present invention is also preferentially to isolated MR1T cells from solid tumor cells. CAR-MR1T-EGFRt by integrating the specific CAR (chimeric antigen receptor) gene comprising the step of obtaining cells. The solid tumor in question is preferably the bladder. cancer, breast cancer, cervical cancer, endometrial cancer, kidney cancer, lip and oral cancer, liver cancer, melanoma, mesothelioma non-small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, thyroid cancer, glioblastoma, neuroblastoma, and colorectal selected from a group including cancer.
Mevcut bulusa ait yöntemde yer alan CAR-MR1T hücrelerinin elde edilmesi adimininda yer alan söz konusu entegre edilme islemi tercihen Ientiviral aktarim yoluyla gerçeklestirilmektedir. The step of obtaining CAR-MR1T cells in the method of the present invention is included. said integration is preferably carried out by Ientiviral transmission.
Mevcut bulusa göre olan yöntem ayrica elde edilen CAR-MR1 T-EGFRt hücrelerinin EGFR- spesifik monoklonal antikor ile isaretlenmesi adimini ihtiva edebilmektedir. The method according to the present invention furthermore EGFR- Labeling with a specific monoclonal antibody.
Mevcut bulus bir diger yönüyle, mevcut bulusa göre olan yöntemle elde edilen bir CAR-MR1T- EGFRt hücresi saglamaktadir. In another aspect of the present invention, a CAR-MR1T- EGFRt cell provides.
Mevcut bulus diger bir yönüyle, kanser immünoterapisinde kullanilmak üzere bulusa göre olan CAR-MRiT-EGFRt hücresini sunmaktadir. In another aspect of the present invention, it is a method according to the invention for use in cancer immunotherapy. presents the CAR-MRiT-EGFRt cell.
Mevcut bulusa göre olan yöntem ayrica tercihen, izole edilen MR1T hücrelerine SV4O Büyük T Antijeni (TAg) ve insan telomeraz (hTERT) genlerinin entegre edilmesi adimini ihtiva etmektedir. Söz konusu entegre edilme islemi tercihen Ientiviral aktarim yoluyla gerçeklestirilmektedir. The method according to the present invention is also preferably applied to isolated MR1T cells with SV4O Large It includes the step of integrating the T Antigen (TAg) and human telomerase (hTERT) genes. is doing. Said integration is preferably via Ientiviral transmission. is carried out.
Mevcut bulus bir diger yönüyle, mevcut bulusa göre olan yöntemle elde edilen bir SV40 TAg- hTERT-MRiT hücresi sunmaktadir. In another aspect of the present invention, an SV40 TAg- presents the hTERT-MRiT cell.
Mevcut bulus diger bir yönüyle, kanser immünoterapisinde kullanilmak üzere bulusa göre olan SV40 TAg-hTERT-MR1T hücresini saglamaktadir. In another aspect of the present invention, it is a method according to the invention for use in cancer immunotherapy. SV40 provides the TAg-hTERT-MR1T cell.
SEKILLERIN KISA AÇIKLAMASI Sekil 1, izole edilen MR1T hücrelerine Ientiviral aktarim yoluyla CAR geni entegre edilmesiyle CAR-MRtT-EGFRt hücre üretimini göstermektedir. BRIEF DESCRIPTION OF THE FIGURES Figure 1, by integrating the CAR gene into isolated MR1T cells by Ientiviral delivery. CAR-MRtT-EGFRt shows cell production.
BULUSUN AYRINTILI AÇIKLAMASI Mevcut bulus ile, iki defa aktive edilmemis dolayisiyla anti-tümör etkisi yüksek MR1 T hücrelerin izole edilmesi; baska bir deyisle MR1T hücrelerin “exhaustion” sürecine ugramadan izole edilmesi için bir yöntem sunulmasi amaçlanmaktadir. DETAILED DESCRIPTION OF THE INVENTION With the present invention, MR1 T cells with high anti-tumor effect have not been activated twice. to isolate; In other words, MR1T cells are isolated without undergoing the "exhaustion" process. It is intended to present a method for
MR1T hücreleri, polimorfik olmayan MHC-l benzeri bir protein olan MR1 proteini tarafindan sunulan antijenleri taniyip onlarla savasan, geleneksel olmayan T hücreleri olmalari bakimindan son yillarda dikkat çekmektedir. Ancak bu hücrelerin T-hücreleri arasindaki insidansi oldukça azdir (%0.1) ve yüksek verimlilikle izole edilebilmeleri önem tasimaktadir. MR1T cells are secreted by the MR1 protein, a non-polymorphic MHC-1-like protein. non-traditional T cells that recognize and fight presented antigens has attracted attention in recent years. However, between these cells and T-cells The incidence is very low (0.1%) and it is important that they can be isolated with high efficiency.
Halihazirda MR1T hücrelerinin izolasyonu için kullanilan yöntemler, yorulmaya (exhaustion) yol açabilmekte ve elde edilen hücrelerin MR1T hücre oldugunun yüksek bir dogrulukla saptanmasina izin vermemektedir. Bunlara ek olarak, teknigin bilinen durumuna ait yöntemlerde aktive olmus MR1T hücrelerinin klonlamasi alt klonlama yöntemi (Subcloning) ile 96-kuyucuklu plakalarda oyuk basina 1-3 hücre yerlestirilerek yapilabilmektedir. Bu da, MR1T hücrelerinin klinik uygulamalar için gereken sayida çogaltilmasini zorlastirmaktadir. Bu durum mevcut MR1T hücrelerinin izolasyon ve çogaltilmasinda zaman kaybina ve yüksek maliyetlere sebep olmaktadir. The methods currently used for the isolation of MR1T cells are subject to exhaustion. and with high accuracy that the cells obtained are MR1T cells. does not allow detection. In addition to these, the state of the art Cloning of activated MR1T cells in the methods was performed by subcloning method. This can be done by placing 1-3 cells per well in 96-well plates. This is the MR1T makes it difficult to reproduce the cells in the number required for clinical applications. This situation Loss of time and high costs in isolation and propagation of existing MR1T cells. causes.
Dolayisiyla, MR1T hücrelerinin etkili ve verimli bir sekilde izole edilmesi için yeni yöntemlerin gelistirilmesi kanser immünoterapisi alaninda büyük önem tasimaktadir. Mevcut bulus sahipleri, iki kere aktivasyona maruz birakilan T hücrelerinde yorulma (exhaustion) ve kanser immunoterapisi yeterliliginin azalmasi durumuna çözüm olarak hücreleri aktive etmeden izole edebilecek bir yöntem gelistirmislerdir. Therefore, new methods for isolating MR1T cells effectively and efficiently are needed. development is of great importance in the field of cancer immunotherapy. present invention owners, exhaustion and cancer in T cells exposed to twice activation Isolation without activating the cells as a solution to the decrease in immunotherapy adequacy. They developed a method that could
Buna göre mevcut bulus bir yönüyle, MR1T hücresi izolasyonu için yeni bir yöntem saglamaktadir. Söz konusu yöntem asagidaki adimlari ihtiva etmektedir: - bos MR1 tetramerleri ile solid kanser hücre Iizatlarinin inkübe edilmesi, - solid kanser hücrelerinin sundugu MR1 iliskili tümör spesifik antijenler ile dolmus olan MR1 tetramerlerinin elde edilmesi, - saglikli bir insan kanindan izole edilen periferik kan mononükleer hücre (PBMC) örneginin sunulmasi, - dolu MR1 tetramerleri ile PBMC örneginin 20 ila 45 dakika süreyle ko-kültür edilmesi, - MR1 tetramerlerine baglanmis olan MR1T hücrelerinin izole edilmesi. Accordingly, in one aspect of the present invention, a new method for MR1T cell isolation it provides. This method includes the following steps: - incubation of solid cancer cell strains with empty MR1 tetramers, - filled with MR1-related tumor-specific antigens presented by solid cancer cells Obtaining MR1 tetramers, - peripheral blood mononuclear cell (PBMC) isolated from healthy human blood presentation of the sample, - co-culturing the PBMC sample with filled MR1 tetramers for 20 to 45 minutes, - Isolation of MR1T cells bound to MR1 tetramers.
Görülecegi üzere mevcut bulusa ait yöntem, solid tümör kanser hücre Iizatlariyla inkübe edilmis bos MR1 tetramerlerinin kanser hücre Iizatlarinda bulunan MR1T hücre spesifik antijenleri ile doldurulmasi ve insan kanindan izole edilen PBMC ile ko-kültür edilmesi prensibine dayanmaktadir. Peptid-MHC (pMHC) multimerleri, antijene özgü T hücrelerinin saptanmasi ve izolasyonu için "altin standart" haline gelmistir. Ayni kökenli pMHC ile TCR etkilesimi kisa ömürlü olmasina ragmen, bu etkilesim, tek bir molekül üzerine birden fazla pMHC dahil edilmesinin sagladigi "avidite etkisi" ile stabilize edilebilir. Elde edilen pMHC tetramerleri, antijene özgü T hücrelerinin dogrudan ex vi'vo olarak saptanmasina ve fenotiplenmesine izin vermek için periferal kan mononükleer hücreleri (PBMC) içindeki ayni kökenli T-hücrelerine kararli bir sekilde baglanabilir (Lepore, M., vd. (2017). Functionally diverse human Tcel'ls recogni'ze non-microbi'ai' antigens presented by MR1. eLi'fe, 6, e24476; Dolton, G., vd. (2018). Optimized Peptide-MHC Multimer Protocols for Detecti'on and Iso/ati'on of Autoi'mmune T-Celi's. Frontiers i'n immunology, 9, 1378). As can be seen, the method of the present invention is incubated with solid tumor cancer cell lines. MR1T cell specificity found in cancer cell lines of empty MR1 tetramers antigens and co-culturing with PBMC isolated from human blood. is based on the principle. Peptide-MHC (pMHC) multimers of antigen-specific T cells It has become the "gold standard" for detection and isolation. TCR with cognate pMHC Although the interaction is short-lived, this interaction can be applied to more than one molecule on a single molecule. It can be stabilized by the "avidity effect" provided by the inclusion of pMHC. The resulting pMHC tetramers allow direct ex vivo detection of antigen-specific T cells and in peripheral blood mononuclear cells (PBMC) to allow phenotyping can bind stably to T-cells of origin (Lepore, M., et al. (2017). diverse human Tcel'ls recogni'ze non-microbi'ai' antigens presented by MR1. eLi'fe, 6, e24476; Dolton, G., et al. (2018). Optimized Peptide-MHC Multimer Protocols for Detecti'on and Iso/ati'on of Autoi'mmune T-Celi's. Frontiers in immunology, 9, 1378).
Mevcut bulusa göre olan yöntem, MR1 iliskili tümör spesifik antijenler ile dolu MR1 tetramerleri ile PBMC örneginin kisa süreli bir inkübasyonunu ihtiva etmesi bakimindan avantajlidir. 20 ila 45 dakikalik ko-kültür süresi içerisinde hücrelerin aktive olmasi mümkün olmadigindan, diger hücrelerde aktivasyon ve kontaminasyon riski olusmamakta, bu sayede yalnizca MR1T hücresi izolasyonu gerçeklestirilmis olmaktadir. Söz konusu inkübasyon süresi örnegin 20 konusu dolu MR1 tetramerleri ile PBMC örneginin ko-kültür edilmesi islemi 30 dakika sürdürülmektedir. The method according to the present invention uses MR1 tetramers filled with MR1-associated tumor-specific antigens. advantageous in that it contains a short-term incubation of the PBMC sample with 20 to Since it is not possible for cells to be activated within the 45-minute co-culture period, other there is no risk of activation and contamination in cells, so only MR1T cell isolation is performed. The said incubation period is for example 20 Co-culturing the PBMC sample with the subject filled MR1 tetramers was 30 minutes. is being continued.
Tercihen, söz konusu bos MR1 tetramerleri ile solid kanser hücre Iizatlarinin inkübe edilmesi islemi 4 ila 37°C arasinda gerçeklestirilmektedir. Bos MR1 tetramerleri ile solid kanser hücre Mevcut bulusa ait yöntemde kullanilan bos MR1 tetramerleri tercihen floresan boya ile isaretlidir. Söz konusu floresan boya APC veya PE floresan boya olabilmektedir. Preferably, incubation of solid cancer cell strains with said empty MR1 tetramers. The process is carried out between 4 and 37°C. Solid cancer cell with Bos MR1 tetramers Empty MR1 tetramers used in the method of the present invention are preferably coated with fluorescent dye. is marked. Said fluorescent dye may be APC or PE fluorescent dye.
Mevcut bulusa ait yöntemin son adiminda yer alan izolasyon islemi, tercihen FACS sort etme yöntemi ile gerçeklestirilmektedir. The isolation process involved in the last step of the method of the present invention, preferably FACS sorting carried out by the method.
Mevcut bulusa ait yöntemde kullanilan solid kanser hücre Iizati tercihen A549, MM909.11, ihtiva eden gruptan seçilen bir hücre hattinin Iizatidir. The solid cancer cell strain used in the method of the present invention is preferably A549, MM909.11, is a strain of a cell line selected from the group consisting of
Mevcut bulusa ait yöntem ayrica tercihen, izole edilen MR1T hücrelerinin anti-CD3/anti-CD28 mikrobitleriyle veya PHA ile aktive edilmesi adimini ihtiva etmektedir. Mevcut bulusa ait yöntem ayrica tercihen, aktive edilen MR1T hücrelerinin IL-2, IL-7, IL-15, IL-12 ve IL-18 sitokinlerini ihtiva eden gruptan seçilen bir sitokin ile çogaltilmasi adimini ihtiva etmektedir. The method of the present invention is also preferably anti-CD3/anti-CD28 of isolated MR1T cells. Activation with microbites or PHA. Method of the present invention also preferentially activate the IL-2, IL-7, IL-15, IL-12 and IL-18 cytokines of activated MR1T cells. containing the step of amplification with a cytokine selected from the group consisting of.
Kimerik antijen reseptörleri (CAR'Iar), tümör reddine aracilik etmek için T-hücrelerini yeniden yönlendiren ve yeniden programlayan sentetik reseptörlerdir. Bugüne kadar kullanilan en basarili CAR'Iar, kemorefrakter veya nükseden B-hücresi maligniteleri olan hastalarda tam remisyon olasiligi sunan CD19'u hedefleyenlerdir. CAR'Iar tipik olarak bir hastanin T- hücrelerine y-retroviral vektörleri veya diger rastgele entegre vektörler kullanilarak dönüstürülür. Bu da klonal çogaltma , onkojenik transformasyon, transgen ekspresyonu ve transkripsiyonel susturma ile sonuçlanabilir. Chimeric antigen receptors (CARs) regenerate T-cells to mediate tumor rejection. They are synthetic receptors that direct and reprogram. Most used to date in patients with successful CARs, chemorefractories, or recurrent B-cell malignancies. are those targeting CD19 that offer the possibility of remission. CARs typically include a patient's T- cells using γ-retroviral vectors or other randomly integrated vectors. is converted. This includes clonal amplification, oncogenic transformation, transgene expression, and may result in transcriptional silencing.
MR1T hücrelerinin kanser dokusu üzerinde infiltre edilmesini saglayan antijenlerin biyokimyasal özellikleri henüz bilinmediginden özellikle solid tümörlerde MR1T hücrelerinin MR1-antijen kompleksine gösterecegi anti tümör etkinligin tümör spesifik olup olmadigi bilinmemektedir. MR1T hücreleri anti kanser etkinligi gösterebilir, fakat MR1T antijenleri kanser türüne spesifik olmayabilir . Hedeflenen kanser türüne karsi spesifiteyi artimak amaciyla mevcut bulus ile, izole edilen MR1T hücrelerinin spesifitesi CAR geni ekleyerek artirilmaktadir. Böylelikle solid tümörlere spesifik CAR-MR1T-EGFRt hücreleri üretilerek; mevcut CAR-T uygulamalarina ek olarak yeni, GVHD (doku uyumsuzlugu) olusturmayan, solid tümörlere karsi daha etkin bir kanser immünoterapi uygulama yönteminin sunulmasi hedeflenmektedir. Antigens that allow MR1T cells to infiltrate on cancer tissue Since the biochemical properties are not yet known, especially in solid tumors, MR1T cells Whether its anti-tumor activity to the MR1-antigen complex is tumor specific. unknown. MR1T cells may show anticancer activity, but MR1T antigens It may not be specific to the type of cancer. Increasing specificity against targeted cancer type With the present invention, the specificity of isolated MR1T cells is by adding the CAR gene. is being increased. Thus, by producing CAR-MR1T-EGFRt cells specific to solid tumors; new, non-GVHD (tissue mismatch) solid, in addition to existing CAR-T applications presenting a more effective cancer immunotherapy method against tumors is targeted.
Dolayisiyla mevcut bulusa ait yöntem, tercihen ayrica izole edilen MR1T hücrelerine solid tümör-spesifik CAR (kimerik antijen reseptörü) geninin entegre edilmesi yoluyla CAR-MR1T- EGFRt hücrelerinin elde edilmesi adimini ihtiva etmektedir. Bu yolla elde edilen CAR-MR1T- EGFRt hücrelerinin seçili tümör tipine daha hizli ulasmasi ve etkili yanit olusturmasi beklenmektedir. Bahsi geçen solid tümör; mesane kanseri, meme kanseri, rahim agzi kanseri, endometrium kanseri, böbrek kanseri, dudak ve agiz kanseri, karaciger kanseri, melanom, mezotelyoma küçük hücreli olmayan akciger kanseri, melanom olmayan cilt kanseri, yumurtalik kanseri, pankreas kanseri, prostat kanseri, sarkom, küçük hücreli akciger kanseri, tiroid kanseri, glioblastoma, nöroblastoma ve kolorektal kanseri ihtiva eden bir gruptan seçilmektedir. Therefore, the method of the present invention is preferably applied to separately isolated MR1T cells. CAR-MR1T- by integrating the tumor-specific CAR (chimeric antigen receptor) gene Includes the step of obtaining EGFRt cells. CAR-MR1T- obtained in this way Faster access of EGFRt cells to the selected tumor type and effective response expected. The aforementioned solid tumor; bladder cancer, breast cancer, cervix cancer, endometrial cancer, kidney cancer, lip and mouth cancer, liver cancer, melanoma, melanoma non-small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, from a group that includes thyroid cancer, glioblastoma, neuroblastoma, and colorectal cancer. is selected.
Yukarida açiklandigi gibi, kati tümörlerin ürettigi tümör iliskili antijenleri (tumor associated antigen, TAA) hedefleyen CAR'Iar teknikte siklikla kullanilmakta ve yüksek spesifite ile basari orani saglamaktadir. Dolayisiyla mevcut bulusa ait yöntemde kullanilan söz konusu CAR geni TAA spesifik CAR geni tasarimlaridir. As described above, tumor-associated antigens (tumor associated antigens) produced by solid tumors antigen, TAA) targeting CARs are frequently used in the art and have been successfully used with high specificity. provides the rate. Thus, the said CAR gene used in the method of the present invention They are TAA specific CAR gene designs.
Bulusa ait yöntemde, solid tümör-spesifik CAR geni izole edilen MR1T hücrelerine tercihen Bulusa ait yöntemle elde edilen CAR-MRiT-EGFRt hücreleri tercihen ayrica EGFR-spesifik monoklonal antikor ile isaretlenmektedir. Söz konusu antikor, bulusa ait CAR-MR1T-EGFRt hücrelerinin izolasyonuna imkan saglamasi bakimindan avantajlidir. In the method of the invention, the solid tumor-specific CAR gene is preferentially to isolated MR1T cells. CAR-MRiT-EGFRt cells obtained by the method of the invention are preferably also EGFR-specific labeled with a monoclonal antibody. Said antibody is the CAR-MR1T-EGFRt of the invention. It is advantageous in terms of allowing the isolation of cells.
Mevcut bulus diger bir yönüyle, bulusa ait yöntemle elde edilen CAR-MR1T-EGFRt hücrelerini saglamaktadir. In another aspect of the present invention, CAR-MR1T-EGFRt cells obtained by the method of the invention. it provides.
Mevcut bulus diger bir yönüyle, kanser immünoterapisinde kullanilmak üzere bulusa göre olan CAR-MRiT-EGFRt hücresini sunmaktadir. In another aspect of the present invention, it is a method according to the invention for use in cancer immunotherapy. presents the CAR-MRiT-EGFRt cell.
Hücresel yaslanmanin telomerlerin kisalmasindan kaynaklandigi bilinmektedir. Insan ve sigir hücrelerinde telomer kisalmasi ve replikatif yaslanma, telomerazin ters transkriptaz bileseninin (TERT) ektopik ekspresyonu ile önlenebilmektedir. Uzun ömürlü memelilerden gelen hücrelerin neoplastik dönüsüme karsi direnç sergiledigi mekanizmalar tam olarak açikliga kavusturulmamis olsa da, bir aday mekanizma bu türlerin hücrelerinde neoplastik transformasyon için replikatif yaslanmanin önlenmesinin gerekliligidir. Bu kavram, normal insan hücrelerini tam bir tümörijenik duruma dönüstürmek için gereken genetik modifikasyonlara iliskin deneylerin sonuçlariyla desteklenmektedir. Normal hücrelerin, SV4O büyükT antijeni, onkojenik RasG12V ve hTERT kodlayan üç retrovirüs tarafindan art arda transdüksiyonu, tamamen tümörijenik olan hücrelerin üretildigini göstermistir. hTERT olmadan sadece SV4O TAgve Ras kodlayan retrovirüslerle transdüksiyon yapildiginda, hücreler tümör üretememistir. Dolayisiyla insan hücrelerinin tümörijenik dönüsümü için hTERT'nin gerekli olduguna dair kanitlara ragmen, hTERT'nin ektopik ekspresyonunun, onkoproteinlerle is birligi yapmadan tek basina hücrelerin normal özelliklerini degistirmedigi gözlenmistir. Bu durum ilk olarak hücre kültüründe gösterilmistir. hTERT eksprese eden hücreler normal hücre döngüsü kontrol noktalarini korumakta ve kaiyotipik instabilite göstermemektedirler. Bu tür hücrelerin görünüste normal davranisi, “telomerizasyon” teriminin ortaya çikmasina neden olmustur. It is known that cellular aging is caused by the shortening of telomeres. man and cattle telomere shortening and replicative aging in cells, telomerase reverse transcriptase component (TERT) can be prevented by its ectopic expression. from long-lived mammals The mechanisms by which cells resist neoplastic transformation have not been fully elucidated. Although not established, a candidate mechanism is neoplastic in cells of this species. It is necessary to prevent replicative aging for transformation. This concept is normal the genetics needed to transform human cells into a fully tumorigenic state This is supported by the results of experiments on modifications. Normal cells, SV4O succession of three retroviruses encoding the bigT antigen, oncogenic RasG12V, and hTERT. transduction showed that cells that were completely tumorigenic were produced. without hTERT When transduced only with retroviruses encoding SV4O TAg and Ras, cells become tumor cells. failed to produce. Thus, hTERT is required for tumorigenic transformation of human cells. Although there is evidence that ectopic expression of hTERT is associated with oncoproteins It was observed that it did not change the normal properties of cells alone. This situation is the first demonstrated in cell culture. hTERT-expressing cells have normal cell cycle they maintain control points and do not show chiaotypic instability. Such cells Its seemingly normal behavior gave rise to the term “telomerization”.
Ayrica, telomerize hücrelerin bir konakçi hayvana transplantasyon sonrasinda da tamamen normal özellikleri korudugu gösterilmistir (Darimont, C., vd. (2002). SV40 TAg Tantigen and telomerase are required to obtain immortalized human adult bone cells without loss of the differentiateo' phenotype. Cell growth & differentiation .' the molecular biology journal of the Cooperation of hTERT, SV40 TAg T antigen and oncogenic Ras in tumorigenesis: a cell transplantation model using bovine adrenocortical cells. Neoplasia (New York, N. Y.), 4(6), 493-500). In addition, telomerized cells are also completely completely absent after transplantation into a host animal. It has been shown to preserve normal features (Darimont, C., et al. (2002). SV40 TAg Tantigen and telomerase are required to obtain immortalized human adult bone cells without loss of the differentiateo' phenotype. Cell growth & differentiation .' the molecular biology journal of the Cooperation of hTERT, SV40 TAg T antigen and oncogenic Ras in tumorigenesis: a cell transplantation model using bovine adrenocortical cells. Neoplasia (New York, N.Y.), 4(6), 493-500).
Izole edilen MR1T hücreleri primer hücresi olup, kisa süreli yasam döngüsüne sahiptir. Mevcut bulus kapsaminda izole edilen MR1T hücrelerinin sinirsiz sayida çogaltilmasi, kanser tedavisinde kullanimi konusunda avantaj saglayacaktir. Ayrica zaman alici ve masrafli izolasyon süreçleri minimize edilecektir. Dolayisiyla mevcut bulusa ait yöntem kapsaminda söz konusu hücrelere tercihen telomeraz geni (hTERT) ve SV40 TAg (Simian virüs 40, büyük T antijeni) antijen geni entegre edilmektedir. Böylece sinirsiz sayida çogaltilabilecek transgenik, universal, kullanima hazir (Off-the-Shelf) MR1T hücrelerinin sunulmasi hedeflenmektedir. Söz konusu entegre etme islemi tercihen lentiviral aktarim yoluyla gerçeklestirilmektedir. Isolated MR1T cells are primary cells and have a short life cycle. Available Unlimited proliferation of MR1T cells isolated within the scope of the invention, cancer will provide an advantage in its use in the treatment of Also time consuming and costly isolation processes will be minimized. Therefore, within the scope of the method of the present invention, telomerase gene (hTERT) and SV40 TAg (Simian virus 40, large T) preferentially to the cells of interest. antigen) antigen gene is integrated. Thus, an unlimited number of transgenic, universal, ready-to-use (Off-the-Shelf) MR1T cells. Promise The said integration process is preferably carried out by lentiviral transmission.
Mevcut bulus diger bir yönüyle, bulusa ait yöntemle elde edilen SV40 TAg-hTERT-MR1T hücrelerini saglamaktadir. Another aspect of the present invention is the SV40 TAg-hTERT-MR1T obtained by the method of the invention. nourishes its cells.
Mevcut bulus diger bir yönüyle, kanser immünoterapisinde kullanilmak üzere bulusa göre olan SV40 TAg-hTERT-MR1T hücresini sunmaktadir. In another aspect of the present invention, it is a method according to the invention for use in cancer immunotherapy. SV40 presents the TAg-hTERT-MR1T cell.
Mevcut bulusa göre olan MR1 T hücre izolasyonu yöntemi, asagidaki Örnekler`de detayli olarak açiklanmaktad ir. ÖRNEKLER 1. MR1 T Hücre izolasyonu Kanser immünoterapi uygulamalarinda kullanilmak üzere, saglikli bir insan kanindan MR1T hücrelerini izole edebilmek için gelistirilen yeni yöntem kapsaminda; öncelikle bos MR1 tetramerleri (APC veya PE floresan boya isaretli) solid kanser hücre (A549, MM909.11, PBS+%1 insan serum albumin solüsyonu içerisinde homojenizatör cihazinda Iizat hale dönüstürülmesiyle elde edildi . Bu inkübasyon ile, kanserli hücre Iizatlarindaki MR1 antijenleri bos MR1 tetramerlerinin içerisine dolduruldu. MR1 iliskili tümör spesifik antijenler ile dolu MR1 tetramerleri saglikli insan kanindan izole edilen allojenik periferik kan mononükleer hücre (PBMC, Peripheral blood mononuclear cell ) ile inkübe edildi. Böylelikle yalnizca MR1T hücreleri tetramerlere baglanmis oldu. APC veya PE floresan hedefli FACS sort etme islemiyle bu tetramerlere bagli MR1T hücreleri izole edildi. Daha sonra anti-CDS/anti-CD28 mikrobitleriyle veya PHA ile aktif hale getirilip IL-2, IL-7, IL-15 veya IL-12, IL-18 sitokinleri ile 7- 14 gün çogaltildi. 2. Elde Edilen MR1T Hücrelerine CAR Geni Eklenmesi Çalismalarin bu asamasinda, izole edilen MR1T hücrelerine solid tümör hedefli CAR (Chimeric antigen receptors) reseptör geni Ientiviral aktarim yoluyla entegre edilerek CAR-MR1T-EGFRt hücreleri üretildi. Saglikli bir insan kanindan izole edilen MR1T hücrelerine Ientiviral gen aktarim yoluyla solid tümör (mesane kanseri, meme kanseri, rahim agzi kanseri, endometrium kanseri, böbrek kanseri, dudak ve agiz kanseri, karaciger kanseri, melanom, mezotelyoma küçük hücreli olmayan akciger kanseri, melanom olmayan cilt kanseri, , yumurtalik kanseri, pankreas kanseri, prostat kanseri, sarkom, küçük hücreli akciger kanseri, tiroid kanseri, glioblastoma, nöroblastoma veya kolorektal kanseri) iliskili antijenlere spesifik CAR geni entegre edildi (Sekil 1). The MR1 T cell isolation method according to the present invention is detailed in the Examples below. is explained. EXAMPLES 1. MR1 T Cell isolation MR1T from healthy human blood for use in cancer immunotherapy applications Within the scope of the new method developed to isolate cells; firstly empty MR1 tetramers (APC or PE fluorescent dye labeled) solid cancer cell (A549, MM909.11, Isolate in the homogenizer device in PBS+1% human serum albumin solution. obtained by conversion. With this incubation, MR1 antigens in cancer cell strains The bos was packed into MR1 tetramers. MR1 packed with MR1-associated tumor-specific antigens Allogeneic peripheral blood mononuclear cell tetramers isolated from healthy human blood (PBMC, Peripheral blood mononuclear cell ). Thus, only the MR1T cells were bound to tetramers. With APC or PE fluorescent-targeted FACS sorting MR1T cells bound to these tetramers were isolated. Then anti-CDS/anti-CD28 Microbites or PHA activated with IL-2, IL-7, IL-15 or IL-12, IL-18 cytokines 7- 14 days increased. 2. Insertion of the CAR Gene into the Obtained MR1T Cells At this stage of the studies, solid tumor-targeted CAR (Chimeric antigen receptors) receptor gene CAR-MR1T-EGFRt by integrating via intraviral delivery cells were produced. Intiviral gene into MR1T cells isolated from healthy human blood solid tumor by transmission (bladder cancer, breast cancer, cervical cancer, endometrial cancer) cancer, kidney cancer, lip and mouth cancer, liver cancer, melanoma, mesothelioma non-small cell lung cancer, non-melanoma skin cancer, , ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, thyroid cancer, CAR gene specific for antigens associated with glioblastoma, neuroblastoma, or colorectal cancer integrated (Figure 1).
Buna göre, TAA spesifik CAR'i (TAA-CD kodlayan Ientiviral vektör, sentezlendi. Lentivirüs üretim için gerekli olan zarf pCMV-VSV-G plazmidi ve psPAX2 plazmidi temin edildi. Genetik modifikasyon amaçli kullanilacak CAR kodlayici plazmit ile coli' DH5a susuna entegre edilip plazmitler çogaltildi. izole edilmis zarf, paketleme ve CAR- EGFRt plazmitleri, FuGene transfeksiyon reaktifi ile muamele edildi ve daha sonra konakçi hücreler olan HEK293T kullanilarak Ientivirüs üretimi gerçeklestirildi. Paketlenmis rekombinant toplandi. Üretilen CAR-EGFRt Ientivirüsler, konak hücre proteinlerinden saflastirilmasi ve virüs konsantrasyonunun artirilmasi (20X-100X) amaciyla, üretici talimatlarina uygun olarak TFF (Tangential flow filtration) filtrasyon ve konsantrasyon isleminden geçirildi. Lentivirüs titre testinin ve sterilite, saflik ve replikatif kompetent Lentivirüs (RCL) dahil diger kalite kontrol testlerinin tamamlanmasindan sonra virüsler -80°C'de saklandi. Accordingly, Ientiviral encoding TAA-specific CAR (TAA-CD) vector, synthesized. Envelope pCMV-VSV-G plasmid and psPAX2 required for lentivirus production plasmid was obtained. With the CAR-encoding plasmid to be used for genetic modification coli' was integrated into the DH5a strain and the plasmids were amplified. insulated envelope, packaging and CAR- EGFRt plasmids were treated with FuGene transfection reagent and then host Ientivirus production was performed using HEK293T cells. Packaged recombinant gathered. Produced CAR-EGFRt Ientiviruses were purified from host cell proteins and the virus TFF in accordance with the manufacturer's instructions to increase the concentration of TFF (20X-100X). (Tangential flow filtration) filtration and concentration process. lentivirus titrate test and other quality control, including sterility, purity, and replicative competent Lentivirus (RCL). After completion of the tests, the viruses were stored at -80°C.
Saglikli üç insan donörden alinan kan örneklerinden periferik mononükleer hücrelerin (PBMC) izolasyonu için, söz konusu örnekler Ficoll ile birlestirildi ve density-gradient santrifüj yöntemi ile PBMC'Ier izole edildi. MR1T hücreleri TAA yüklü MR1 tetramerleri kullanilarak izole edildi. Peripheral mononuclear cells (PBMC) from blood samples from three healthy human donors For isolation, these samples were combined with Ficoll and density-gradient centrifugation method. and PBMCs were isolated. MR1T cells were isolated using TAA-loaded MR1 tetramers.
Anti-CD3/anti-CD28 mikroboncuklari (Dynabead) veya tümör Iizati ile ko-inkübe edilen MR1+ kanser hücre hatlari ile ilk MR1T-hücresi aktivasyonu yapildi ve lentivirüs transdüksiyon islemi, Vectofusin 1 (10 ug/mL, Miltenyi), protamin sülfat (40 ug/mL, Sigma) ile olusturulan virüslerle gerçeklestirildi. Hücreler, T-hücre besi yerinde (50IU/ml IL-2, 10 ng/ml IL-7, 5 ng/ml EGFR-spesifik monoklonal antikor, olusturulan CAR-MR1T-EGFRt hücrelerinin isaretlenmesi ve izolasyonunda kullanildi. Söz konus antikor ile ayrica gerekli görüldügünde CAR-MR1T- EGFRt hücrelerinin programli hücre ölümüne yönlendirilmesi saglanmaktadir. Kesilmis EGFRt isaretlenmesi için florokrom bagli anti-EGFR (örnegin A kullanilarak flow cytometry yöntemi ile analizi gerçeklestirildi. CAR-MR1T-EGFRt hücrelerinin izolasyonu için, Biotin bagli anti-EGFR antikorlari ile isaretlenmeyi takiben Streptavidin-bead kullanilarak izolasyon gerçeklestirildi. Hastaya tranzfüze edilen CAR-MR1T-EGFRt hücrelerinin, gerekli görüldügünde ortamdan kaldirilmasi amaciyla EGFR-spesifik monoklonal antikor yoluyla hücrelerin programli hücre ölümü saglandi. 3. Elde Edilen MR1T Hücrelerine Telomeraz Gem' Eklenmesi Mevcut bulusa ait yöntemle izole edilen allojenik MR1T hücreleri ölümsüzlestirilerek, sinirsiz çogalabilen MR1T hücre suslari üretildi. Izole edilen allojenik MR1T hücrelerine Ientiviral aktarim yöntemi ile Büyük T antijen (SV40, Large T antigen - Simian virus 40; UniProtKB - P entegre edildi. Bu yolla elde edilen SV40 TAg-hTERT-MR1T hücrelerine, kanser hücre özelligi (ölümsüzlük, immortality) kazandirilmis oldu. Gamma irradyasyona ( tabi tutulan inaktive edilmis MR1T hücreleri, kanser immünoterapi uygulamalarinda kullanilabilmektedir. MR1+ co-incubated with anti-CD3/anti-CD28 microbeads (Dynabead) or tumor strain First MR1T-cell activation was performed with cancer cell lines and lentivirus transduction treatment, Vectofusin 1 (10 ug/mL, Miltenyi), protamine sulfate (40 ug/mL, Sigma) performed with viruses. Cells in T-cell medium (50IU/ml IL-2, 10 ng/ml IL-7, 5 ng/ml EGFR-specific monoclonal antibody, labeling of generated CAR-MR1T-EGFRt cells and used in isolation. With the said antibody, CAR-MR1T- EGFRt cells are directed to programmed cell death. Cut EGFRt fluorochrome-linked anti-EGFR (for example, flow using A) Analysis was carried out by cytometry method. For isolation of CAR-MR1T-EGFRt cells, Using Streptavidin-bead following labeling with biotin-bound anti-EGFR antibodies isolation was performed. CAR-MR1T-EGFRt cells transfused into the patient via EGFR-specific monoclonal antibody for removal when seen. programmed cell death of the cells was achieved. 3. Addition of Telomerase Gem to Obtained MR1T Cells Allogeneic MR1T cells isolated by the method of the present invention were immortalized and Proliferable MR1T cell lines were produced. Intiviral to isolated allogeneic MR1T cells Large T antigen (SV40, Large T antigen - Simian virus 40; UniProtKB - by transfer method) P is integrated. get it this way SV40 TAg-hTERT-MR1T cells, cancer cell feature (immortality, immortality) has been earned. Gamma irradiated (inactivated MR1T cells can be used in cancer immunotherapy applications.
Yukaridaki bilgiler isiginda görülmektedir ki mevcut bulus ile sunulan yöntem; kanser immünoterapisi alaninda kullanimi bulunan MR1T hücrelerin pratik ve etkili sekilde izole edilebilmesine, bu hücrelerin solid kanser-spesifik hale getirilmesine ve sinirsiz çogalabilen MR1T hücrelerinin elde edilmesine imkan saglamasi bakimlarindan avantajlidir. In the light of the above information, it is seen that the method presented with the present invention; cancer Isolation of MR1T cells, which are used in the field of immunotherapy, in a practical and effective way. can be produced, these cells can be made solid cancer-specific and It is advantageous in terms of enabling the obtaining of MR1T cells.
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