TWI403332B - A method to produce immune cells and induce immune effector cells - Google Patents
A method to produce immune cells and induce immune effector cells Download PDFInfo
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Abstract
Description
本發明係有關於一種產生免疫細胞之方法，尤指用於產生抗皰疹病毒之免疫細胞的方法。 The present invention relates to a method of producing immune cells, and more particularly to a method for producing immune cells against herpesvirus.
1964年Achong與Barr等人首次在伯奇氏淋巴瘤病人細胞中分離並發現艾普斯坦-巴爾二氏病毒(Epstein-Barr Virus,EBV)，中文名稱為「人類皰疹病毒第四型」(Human herpesvirus-4,HHV-4)，屬於皰疹病毒科線型雙股DNA病毒。 In 1964, Achong and Barr et al. first isolated Epstein-Barr Virus (EBV) in the cells of patients with Birch's lymphoma, and the Chinese name was "Human Herpesvirus Type 4" ( Human herpesvirus-4, HHV-4), belongs to the herpesvirus family of linear double-stranded DNA viruses.
EBV像大多數的皰疹病毒一樣有兩種生活史：細胞裂解時期(Lytic phase)和潛伏期(Latent phase)。其中，細胞裂解時期多發生在上皮細胞，因為病毒會在細胞內大量複製，最後導致細胞破裂，釋出大量的病毒。而潛伏期多發生在B淋巴球，EBV藉由鑲嵌在B淋巴球的基因體中，以逃避免疫細胞的攻擊，同時也有機會造成淋巴球不正常增生而引起癌化與其他慢性感染症狀，例如「伯奇氏淋巴瘤」、「胃腺癌」、「鼻咽癌」、「何傑金氏病」、「T淋巴瘤」、「淋巴球增生性疾病」、「愛滋病相關淋巴癌」與「移植後淋巴球增生疾病」。 EBV has two life histories like most herpes viruses: the Lytic phase and the Latent phase. Among them, the cell lysis period occurs mostly in epithelial cells, because the virus will replicate in the cells in large quantities, eventually causing the cells to rupture and release a large amount of virus. The incubation period occurs mostly in the B lymphocytes. EBV is embedded in the genome of the B lymphocytes to escape the attack of immune cells, and also has the opportunity to cause abnormal proliferation of lymphocytes and cause cancer and other chronic infection symptoms, such as " Birch's lymphoma, gastric adenocarcinoma, nasopharyngeal carcinoma, Hodgkin's disease, T lymphoma, lymphocytic proliferative disease, AIDS-associated lymphoma and after transplantation Lymphocytosis disease."
從分子生物學及EBV結構研究發現，近100個病毒基因會在細胞複製的過程中被表現出來，然而只有10個基因會在被潛伏感染的B細胞(latently infected B cells)表現出來，其中包含有兩種未轉譯RNA(例如EBV-encoded RNA；EBER)、6種核蛋白(例如核蛋白-1(EBV nuclear antigen-1，EBNA1)及核蛋白-3c(EBV nuclear antigen-3c，EBNA3c)與兩種膜蛋白(包含潛伏性膜蛋白-1(latent membrane protein-1；LMP1)及潛伏性膜蛋白-2(latent membrane protein-2；LMP2))。在不同潛伏期的病毒會表現不同的蛋白質，這些病毒蛋白亦會在上述惡性腫瘤細胞中大量表 現。 From molecular biology and EBV structure studies, nearly 100 viral genes are expressed during cell replication, but only 10 genes are expressed in latently infected B cells, including There are two untranslated RNAs (eg EBV-encoded RNA; EBER), six nuclear proteins (eg EBV nuclear antigen-1, EBNA1 and EBV nuclear antigen-3c, EBNA3c) Two membrane proteins (latent membrane protein-1 (LMP1) and latent membrane protein-2 (LMP2)). Viruses with different incubation periods exhibit different proteins. These viral proteins will also be abundant in the above malignant tumor cells. Now.
目前現行治療EBV相關感染疾病的治療上，除了外科手術與放射線治療外，有傳統的抗病毒藥物(例如acyclovir)與抗腫瘤用藥物，及新興的抗體藥物(例如Gp350 vaccine；Rituximab)。然而前述藥物都無法突破血腦障壁(blood-brain barrier；BBB)，亦即無法經由體循環(systemic circulation)而到達腦部感染部位，此外，即使這類抗病毒藥物有效抑制EBV增生，但是病患往往無法負荷藥物所產生的副作用因而降低或停止用藥，以致於無法有效治療。 Currently, in addition to surgery and radiation therapy, there are conventional antiviral drugs (such as acyclovir) and antitumor drugs, and emerging antibody drugs (such as Gp350 vaccine; Rituximab) in the treatment of EBV-associated diseases. However, none of the above drugs can break through the blood-brain barrier (BBB), that is, it cannot reach the brain infection site via systemic circulation. In addition, even if such antiviral drugs effectively inhibit EBV proliferation, patients It is often impossible to load the side effects of the drug and thus reduce or stop the medication, so that it cannot be effectively treated.
目前對於移植受者之皰疹病毒的感染主要是使用抗病毒試劑進行先行性治療或預防治療。然而，這些藥理學方式具有其限制，諸如藥物毒性、抗藥性的發生、不佳的口服生物可利用性(oral bioavailability)以及低效力等問題。死亡率在即使已利用抗病毒試劑治療之情況下仍然偏高，特別是於療程中未盡早開始治療。此外，當病患長期使用這些試劑，對於抗病毒試劑的抗藥性會逐漸產生。 At present, the infection of herpesviruses in transplant recipients is mainly by using antiviral agents for prophylactic or preventive treatment. However, these pharmacological approaches have limitations such as drug toxicity, emergence of drug resistance, poor oral bioavailability, and low efficacy. Mortality is still high even if it has been treated with antiviral agents, especially if treatment is not initiated as early as possible during the course of treatment. In addition, when patients use these agents for a long time, resistance to antiviral agents will gradually develop.
為克服前述傳統藥物所具有的缺點，具有專一性高、副作用低、不易產生抗藥性，以及可個體化等優點的免疫療法便應運而生。現有針對皰疹病毒感染疾病之免疫療法是使用以活病毒為基礎的皰疹病毒抗原來源(herpes virus antigen sources based on live virus)、經皰疹病毒感染的細胞(herpes virus-infected cells)、皰疹病毒基因表現載體(hepresherpes virus gene expression vector)或者合成的皰疹病毒蛋白或胜肽，藉以活化對皰疹病毒有專一性免疫反應的抗原呈現細胞(antigen presenting cells)，希望藉由已活化的抗原呈現細胞可以在活體外(ex vivo)或活體內(in vivo)刺激個體產生對EBV有專一性的毒殺型T細胞(CTL)及細胞免疫反應。實驗證實，在曾經感染EBV且有血清反應的人體(非急性感染)主要係藉由第一型主要組織相容複體(MHC class I)的毒殺型T細胞控制EBV，故EBV專一毒殺型T細胞也經常可以從曾經感染過EBV個體的 周邊血液中發現。此外，潛伏性膜蛋白1及潛伏性膜蛋白2抗原專一性的毒殺型T細胞在體外試驗中也已經被證實可以有效的毒殺藉由第一型主要組織相容複體表現這兩種抗原細胞株。但是使用病毒或經病毒感染的細胞作為抗原來源，會有病毒污染以及導致感染的潛在風險；而使用全長皰疹病毒基因或蛋白質作為抗原來源則又有誘發免疫抑制作用(immune suppression)或免疫耐受性(immune tolerance)的問題。 In order to overcome the shortcomings of the aforementioned conventional drugs, immunotherapy with high specificity, low side effects, resistance to drug resistance, and individualization has emerged. The existing immunotherapy for herpes virus-infected diseases is the use of herpes virus antigen sources based on live virus, herpes virus-infected cells, and vesicles. A herpesheres virus gene expression vector or a synthetic herpesvirus protein or peptide to activate antigen presenting cells with a specific immune response to herpesvirus, hopefully by activated The antigen presenting cells can stimulate the individual to produce cytotoxic T cells (CTLs) and cellular immune responses that are specific for EBV either ex vivo or in vivo. Experiments have confirmed that humans (non-acute infections) who have been infected with EBV and have a serum response mainly control EBV by the type 1 major histocompatibility complex (MHC class I), so EBV-specific toxic T Cells can also often be from individuals who have been infected with EBV Found in the surrounding blood. In addition, latent membrane protein 1 and latent membrane protein 2 antigen-specific toxic T cells have also been shown to be effective in in vitro tests by the first major histocompatibility complex. Strain. However, the use of viruses or virus-infected cells as a source of antigen has the potential for virus contamination and infection, and the use of full-length herpesvirus genes or proteins as antigen sources induces immune suppression or immune tolerance. The problem of immunity tolerance.
為克服上述問題，本發明係提供一種生產免疫細胞的方法，包含將免疫細胞以及免疫原組成物予以混合培養，其中免疫原組成物包含有至少兩種衍生自人類皰疹病毒第四型之胜肽。 In order to overcome the above problems, the present invention provides a method for producing immune cells comprising mixing an immune cell and an immunogenic composition, wherein the immunogenic composition comprises at least two types derived from the human herpesvirus type IV. Peptide.
因此，本發明之主要目的在於提供一種生產免疫細胞的方法，可用於誘發針對皰疹病毒之免疫反應，藉以用來預防以及治療皰疹病毒相關疾病。 Accordingly, it is a primary object of the present invention to provide a method of producing immune cells which can be used to induce an immune response against herpesviruses for the prevention and treatment of herpes virus-associated diseases.
本發明另一目的在於提供一種生產免疫細胞的方法，由於使用兩種、三種或四種EBV胜肽庫組成的混合庫作為免疫原組成物，相較於使用僅包含有單一種EBV蛋白質衍生之胜肽的免疫原組成物，本方法可以更為有效的活化抗-EBV免疫反應。 Another object of the present invention is to provide a method for producing immune cells, which uses a mixed library consisting of two, three or four EBV peptide libraries as an immunogen composition, which is derived from a single EBV protein. The immunogenic composition of the peptide, the method can more effectively activate the anti-EBV immune response.
本發明另一目的在於提供一種生產免疫細胞的方法，由於沒有使用活病毒、經EBV感染細胞，所使用之抗原亦非全長EBV蛋白質，因此不會產生非所欲的感染問題，且亦不具有誘發免疫抑制作用及免疫耐受等潛在風險。 Another object of the present invention is to provide a method for producing immune cells. Since no live virus or EBV-infected cells are used, the antigen used is not a full-length EBV protein, so that an unintended infection problem does not occur, and there is no Potential risks such as induction of immunosuppression and immune tolerance.
本發明中亦提供一種誘發產生免疫作用細胞(immune effector cells)的方法，其包含下列步驟：將樹狀細胞以及以及免疫原組成物予以混合成培養物；以及令前述培養物和免疫細胞共同培養於適當的培養基中，以獲取活化之免疫作用細胞，其中免疫原組成物包含有至少兩種衍生自人類皰疹病毒第四型之胜肽。 The present invention also provides a method for inducing immune effector cells, comprising the steps of: mixing dendritic cells and immunogenic compositions into a culture; and co-cultivating the aforementioned culture and immune cells In an appropriate medium, an activated immune cell is obtained, wherein the immunogenic composition comprises at least two peptides derived from human herpesvirus type IV.
因此，本發明之另一目的在於提供一種誘發產生免疫作用細胞的方法，可用於預防以及治療皰疹病毒相關疾病。 Accordingly, it is another object of the present invention to provide a method for inducing the production of immune cells for use in the prevention and treatment of herpes virus-associated diseases.
本發明另一目的在於提供一種誘發產生免疫作用細胞的方法，由於使用兩種、三種或四種EBV胜肽庫組成的混合庫作為免疫原組成物，相較於使用僅包含有單一EBV蛋白質衍生之胜肽的免疫原組成物，本方法可以更為有效的活化抗-EBV免疫反應。 Another object of the present invention is to provide a method for inducing the production of immune cells by using a mixed library consisting of two, three or four EBV peptide libraries as an immunogen composition, which comprises only a single EBV protein derivative compared to the use. The immunogenic composition of the peptide, the method can more effectively activate the anti-EBV immune response.
本發明另一目的在於提供一種誘發產生免疫作用細胞的方法，由於沒有使用活病毒、經EBV感染細胞，所使用之抗原亦非全長EBV蛋白質，因此不會產生非所欲的感染問題，且亦不具有誘發免疫抑制作用及免疫耐受等潛在風險。 Another object of the present invention is to provide a method for inducing the production of immune cells. Since no live virus or EBV-infected cells are used, the antigen used is not a full-length EBV protein, so that an unintended infection problem does not occur, and It does not have the potential risk of inducing immunosuppressive effects and immune tolerance.
本發明另一目的在於提供一種誘發產生免疫作用細胞的方法，可選擇使用自體淋巴細胞，以降低後續進行細胞治療時所可能產生的免疫排斥問題。 Another object of the present invention is to provide a method for inducing the production of immune cells, optionally using autologous lymphocytes to reduce the immune rejection problem that may occur in subsequent cell therapy.
由於本發明係揭露一種生產免疫細胞的方法以及誘發產生免疫作用細胞的方法，其中所利用之免疫學原理、細胞培養、細胞染色及偵測等相關技術，已為相關技術領域具有通常知識者所能明瞭，故以下文中之說明，不再作完整描述。同時，以下文中所對照之圖式，係表達與本發明特徵有關之示意，並未亦不需要依據實際情形完整繪製，合先敘明。 Since the present invention discloses a method for producing immune cells and a method for inducing cells for producing immune cells, related technologies such as immunological principles, cell culture, cell staining, and detection have been used by those having ordinary knowledge in the related art. It can be understood, so the description below will not be fully described. At the same time, the drawings in the following texts are indicative of the features related to the features of the present invention, and are not required to be completely drawn according to the actual situation.
本發明第一實施例係提供一種生產免疫細胞的方法，包含有下列步驟：將免疫細胞以及免疫原組成物予以混合培養，而所使用的免疫原組成物包含有至少兩種衍生自人類皰疹病毒第四型的胜肽。免疫細胞可為抗原呈現細胞(antigen presenting cell；APC)或淋巴細胞(lymphocyte)。 A first embodiment of the present invention provides a method for producing an immune cell comprising the steps of: mixing an immune cell and an immunogenic composition, and using the immunogenic composition comprising at least two derived from human herpes The peptide of the fourth type of virus. The immune cell can be an antigen presenting cell (APC) or a lymphocyte.
前述之混合培養可採用活體外(ex vivo)方式進行，並在適當的培養基中進行培養，培養基則可進一步包含有GM-CSF、IL-4、IL-15等細胞激素，或是前述細胞激素的任意組合。 The above mixed culture can be carried out in an ex vivo manner and cultured in an appropriate medium, and the medium may further contain cytokines such as GM-CSF, IL-4, IL-15, or the aforementioned cytokines. Any combination.
本發明所提供之免疫原組成物，可包含兩種、三種，甚或是四種以上之胜肽。請參考第1A圖，為本發明生產免疫細胞的方法流程示意圖。如圖所示，免疫原組成物1包含有胜肽11、胜肽12、胜肽13，及胜肽14。在適當的培養環境下，將免疫原組成物1加入抗原呈現細胞30的培養環境中，促使抗原呈現細胞30活化成為已活化抗原呈現細胞(activated APC)31。 The immunogenic composition provided by the present invention may comprise two, three or even more than four peptides. Please refer to FIG. 1A , which is a schematic flow chart of a method for producing immune cells according to the present invention. As shown, the immunogenic composition 1 contains a peptide 11, a peptide 12, a peptide 13, and a peptide 14. The immunogenic composition 1 is added to the culture environment of the antigen presenting cells 30 in an appropriate culture environment to promote activation of the antigen presenting cells 30 into activated activated APCs 31.
胜肽11、胜肽12、胜肽13、及胜肽14均衍生自人類皰疹病毒第四型(human herpesvirus-4；HHV-4，又稱「艾普斯坦-巴爾二氏病毒」Epstein-Barr virus；EBV)。這些胜肽的種類較佳是由EBV潛伏性膜蛋白-2、EBV核蛋白-1、EBV核蛋白-3c以及EBV-BZLF-1的多肽中，兩種或兩種以上的多肽所衍生的胜肽片段組合而成。其中EBV潛伏性膜蛋白-2具有與SEQ ID NO 1實質上相同之序列，EBV核蛋白-1具有與SEQ ID NO 2實質上相同之序列，EBV核蛋白-3c具有與SEQ ID NO 3實質上相同之序列，而EBV-BZLF-1具有與SEQ ID NO 4實質上相同之序列。 Peptide 11, peptide 12, peptide 13, and peptide 14 are all derived from human herpesvirus-4 (HHV-4, also known as "Epstein-Barr virus" Epstein- Barr virus; EBV). The types of these peptides are preferably derived from EBV latent membrane protein-2, EBV nuclear protein-1, EBV nuclear protein-3c, and EBV-BZLF-1 polypeptides, which are derived from two or more polypeptides. Peptide fragments are combined. Wherein EBV latent membrane protein-2 has a sequence substantially identical to SEQ ID NO: 1, EBV nucleoprotein-1 has a sequence substantially identical to SEQ ID NO 2, and EBV nucleoprotein-3c has substantially the same as SEQ ID NO 3 The same sequence, while EBV-BZLF-1 has a sequence substantially identical to SEQ ID NO 4.
其中本實施例中所使用的EBV核蛋白-1多肽，並非為全長的EBV核蛋白-1，而為截頭型(N-terminal truncated)，截斷片段為全長成熟的EBV核蛋白-1其N端第1個胺基酸至第392個胺基酸，故其序列相當於全長成熟EBV核蛋白-1的第393個胺基酸至第641個胺基酸，具有與SEQ ID NO 2實質相同之序列。 The EBV nucleoprotein-1 polypeptide used in the present example is not a full-length EBV nucleoprotein-1 but a truncated type (N-terminal truncated), and the truncated fragment is a full-length mature EBV nuclear protein-1. The first amino acid to the 392th amino acid, so the sequence corresponds to the 393th amino acid to the 641th amino acid of the full-length mature EBV nuclear protein-1, and has the same as SEQ ID NO 2 The sequence.
此處表示之本發明所提供之免疫原組成物1，其所包含之胜肽種類個數可為兩種、三種，或四種，但實際上根據本發明之精神，其所包含之胜肽種類個數除上述所列之情形之外，實可至五種甚或以上。 The immunogenic composition 1 provided by the present invention represented herein may have two, three, or four types of peptides, but actually contains the peptides according to the spirit of the present invention. In addition to the cases listed above, the number of types can be five or more.
本實施例所提供的免疫原組成物1可用於活體外誘發針對皰疹病毒(herpes virus)之免疫反應上，亦可用於製造治療皰疹病毒感染疾病之醫藥品。 The immunogenic composition 1 provided in the present embodiment can be used for inducing an immune response against herpes virus in vitro, and can also be used for the manufacture of a medicament for treating a herpes virus-infected disease.
前述「所衍生的胜肽片段」係指具有所述蛋白質序列的全部或一部分序列之胜肽或胜肽片段。而前述「實質上相同」係指只要胺基酸序列之具有一定程度的相似度，但不影響其編碼的蛋白質之活性。上述一定程度的相似度較佳的是70%以上；更佳的是80%以上；以及又更佳的是90%以上。 The aforementioned "derived peptide fragment" means a peptide or peptide fragment having all or a part of the sequence of the protein sequence. The above "substantially the same" means that as long as the amino acid sequence has a certain degree of similarity, it does not affect the activity of the protein encoded thereby. The above degree of similarity is preferably 70% or more; more preferably 80% or more; and still more preferably 90% or more.
本實施例使用的免疫原組成物，以包含有從EBV潛伏性膜蛋白-2、EBV核蛋白-1、EBV核蛋白-3c以及EBV-BZLF-1多肽中，選擇兩種或兩種以上的多肽所衍生的胜肽庫的組合，其誘發免疫反應的效果較使用單一種多肽衍生胜肽庫的效果為佳；其中又以包含有EBV潛伏性膜蛋白-2、EBV核蛋白-1、EBV核蛋白-3c以及EBV-BZLF-1四種多肽所衍生的胜肽庫的組合為最佳。而所謂的「胜肽庫(peptide pool)」係指由複數個胜肽片段組成的混合庫，同一胜肽庫中的胜肽片段序列是依同一基因產物的序列所設計，並且延擴(spanning)此基因產物的整體序列，而各相鄰片段間具有部分胺基酸重疊(overlapping)，各胜肽片段的長度較佳為(但不必然)完全相同。 The immunogenic composition used in the present embodiment comprises two or more selected from the group consisting of EBV latent membrane protein-2, EBV nuclear protein-1, EBV nuclear protein-3c, and EBV-BZLF-1 polypeptide. The combination of the peptide library derived from the polypeptide has an effect of inducing an immune response better than using a single peptide-derived peptide library; wherein the EBV latent membrane protein-2, EBV nuclear protein-1, EBV are included. The combination of the peptide libraries derived from nucleoprotein-3c and the four polypeptides of EBV-BZLF-1 is optimal. The so-called "peptide pool" refers to a mixed library consisting of a plurality of peptide fragments. The sequence of the peptide fragments in the same peptide library is designed according to the sequence of the same gene product, and is extended (spanning) The overall sequence of this gene product, with partial amino acid overlap between adjacent fragments, and the length of each peptide fragment is preferably (but not necessarily) identical.
本發明所使用的胜肽庫中各胜肽片段以介於12至20個胺基酸之間為佳，其中又以為15個胺基酸為最佳。此外，在相鄰的胜肽片段間，具有互相重疊的序列，而互相重疊長度以介於10至15個連續胺基酸殘基為佳，其中又以具有11個連續胺基酸殘基重疊為最佳。 Preferably, each peptide fragment in the peptide library used in the present invention is between 12 and 20 amino acids, with 15 amino acids being preferred. In addition, between adjacent peptide fragments, there are overlapping sequences, and overlapping lengths are preferably between 10 and 15 contiguous amino acid residues, wherein there are 11 contiguous amino acid residues overlapping. For the best.
例如，請參考第1B圖，為本發明中所使用的「EBV潛伏性膜蛋白-2十五胜肽庫(pentadecapeptides)」組成示意圖。舉例而言，本實施例所提供之免疫原組成物1中之胜肽11係為EBV潛伏性膜蛋白-2十五胜肽庫110。如圖所示，EBV潛伏性膜蛋白-2十五胜肽庫110包含有複數個長度為15個胺基酸的胜肽片段，其中胜肽片段111其序列由EBV潛伏性膜蛋白-2完整胜肽2的第1個胺基酸至第15個胺基酸，胜肽片段112由於必須滿足長度為15個胺基酸且與胜肽片段111之間有11個連續胺 基酸殘基重疊的條件，故其序列是由EBV潛伏性膜蛋白-2完整胜肽2的第4個胺基酸至第19個胺基酸；而為滿足上述條件，胜肽片段113其序列則是由EBV潛伏性膜蛋白-2第8個胺基酸至第23個胺基酸，後續以此類推，直至EBV潛伏性膜蛋白-2的最後一個胺基酸。但是最後若是不足4個連續的胺基酸時，則藉由增加重疊部分之胺基酸序列，以滿足各胜肽為15員(15-mer)的條件。因此，此胜肽庫中的所有胜肽片段，其序列總長即延擴EBV潛伏性膜蛋白-2基因產物整體序列。 For example, please refer to FIG. 1B, which is a schematic diagram showing the composition of "EBV latent membrane protein-2 pentadecapeptides" used in the present invention. For example, the peptide 11 in the immunogenic composition 1 provided in the present embodiment is the EBV latent membrane protein-2 fifteen peptide library 110. As shown, the EBV latent membrane protein-2 fifteen peptide library 110 comprises a plurality of peptide fragments of 15 amino acids in length, wherein the peptide fragment 111 is sequenced from EBV latent membrane protein-2. The first amino acid of the peptide 2 to the 15th amino acid, the peptide fragment 112 must have a length of 15 amino acids and 11 consecutive amines with the peptide fragment 111 The condition in which the base acid residues overlap, so the sequence is from the 4th amino acid of the EBV latent membrane protein-2 intact peptide 2 to the 19th amino acid; and in order to satisfy the above conditions, the peptide fragment 113 The sequence is from the 8th amino acid of the EBV latent membrane protein-2 to the 23rd amino acid, and so on, up to the last amino acid of the EBV latent membrane protein-2. However, in the end, if it is less than 4 consecutive amino acids, the conditions of each peptide are 15 members (15-mer) by increasing the amino acid sequence of the overlapping portion. Therefore, the total length of all peptide fragments in this peptide library is extended by the overall sequence of the EBV latent membrane protein-2 gene product.
本實施例中所使用的免疫細胞可以是樹狀細胞或淋巴細胞，較佳係由下列細胞所衍生而得：週邊血液單核細胞(peripheral blood mononuclear cell)、骨髓細胞(bone marrow cell)、造血前趨細胞(hematopoietic progenitor cell)或幹細胞(stem cell)。 The immune cells used in the present embodiment may be dendritic cells or lymphocytes, preferably derived from the following cells: peripheral blood mononuclear cells, bone marrow cells, hematopoiesis Hematopoietic progenitor cell or stem cell.
本實施例所提供的免疫原組成物由於包含有其中兩種、三種或四種EBV胜肽庫組成的混合庫(mixed pool)，相較於單一的EBV蛋白質衍生之胜肽可以更為有效的活化抗-EBV免疫反應，在下列的實驗中，實驗結果證實使用本實施例所提供的免疫原組成物，最終得到的活化CD8+以及CD4+T細胞對於EBV具有專一性，並具有高作用功能(effector functions)。 The immunogenic composition provided in this embodiment can be more effective than a single EBV protein-derived peptide because it contains a mixed pool composed of two, three or four EBV peptide libraries. Activation of anti-EBV immune response, in the following experiments, the experimental results confirmed that using the immunogenic composition provided in this example, the resulting activated CD8 + and CD4 + T cells are specific to EBV and have high function. (effector functions).
為了要製成治療用途的醫藥品，本實施例中的免疫原組成物可進一步包含生理上可接受之載劑或賦型劑、免疫促進劑(immunostimulant)，其中免疫促進劑可進一步包含有佐劑(adjuvant)。前述佐劑可為完全(complete)或不完全(incomplete)佐劑，並可由避免抗原快速代謝之物質及/或免疫反應刺激物(stimulator of immune responses)所組成，例如由氫氧化鋁及/或礦物油組成的避免抗原快速代謝之物質。而前述免疫反應刺激物則可為介面活性劑(surface active agent)的共聚合物(copolymer)、由百日咳桿菌所衍生之蛋白質(Bordetalla pertussis derived proteins)、由結核分枝桿菌(Mycobacterium tuberculosis)所衍生之蛋白質、 -galatosylceramide(-GalCer)及其衍生物以及由CpG所衍生的聚核苷酸(polynucleotides)，或是由上述物質任意混合而組成。 In order to prepare a pharmaceutical for therapeutic use, the immunogenic composition of the present embodiment may further comprise a physiologically acceptable carrier or excipient, an immunostimulant, wherein the immunostimulating agent may further comprise Agent (adjuvant). The aforementioned adjuvant may be a complete or incomplete adjuvant and may consist of substances and/or stimulator of immune responses that avoid rapid metabolism of the antigen, such as aluminum hydroxide and/or A substance consisting of mineral oil that avoids rapid metabolism of antigen. The aforementioned immune response stimulator may be a surfactant of a surface active agent, a protein derived from Bordetella pertussis derived proteins, and derived from Mycobacterium tuberculosis. Protein, -galatosylceramide (-GalCer) and its derivatives, and polynucleotides derived from CpG, or composed of any of the above substances.
本發明第二較佳實施例係提供一種誘發產生免疫作用細胞之方法，包含下列步驟： A second preferred embodiment of the present invention provides a method of inducing the production of immune cells, comprising the steps of:
A.將抗原呈現細胞以及免疫原組成物予以混合成培養物，其中免疫原組成物之組成特徵如第一較佳實施例中所述。 A. The antigen presenting cells and the immunogenic composition are mixed into a culture, wherein the immunogenic composition is characterized as described in the first preferred embodiment.
B.將前述培養物和淋巴細胞共同培養於適當的培養基中，以獲取活化之免疫作用細胞。其中淋巴細胞以T淋巴細胞為佳，而適當的培養基則可添加IL-2、IL-7、IL-15等細胞激素，或是添加前述細胞激素的組合。 B. The aforementioned culture and lymphocytes are co-cultured in an appropriate medium to obtain activated immune cells. Among them, lymphocytes are preferably T lymphocytes, and a suitable medium may be added with cytokines such as IL-2, IL-7, IL-15, or a combination of the aforementioned cytokines.
請繼續參考第1A圖，在適當的培養環境下，將免疫原組成物1加入抗原呈現細胞30的培養環境中，促使抗原呈現細胞30活化成為已活化抗原呈現細胞31。再將上述已活化抗原呈現細胞31與單純淋巴細胞(naïve lymphocytes)40共同培養，使單純免疫細胞40增殖(cell expansion)以及活化(cell activation)成為免疫作用細胞(effector cell)41。由於此種方式取得之免疫作用細胞41具有在活體內辨識及清除免疫原組成物1之功能，故可利用適當方式將免疫作用細胞41注射至患者5體內，促使患者5產生後天免疫反應(adaptive immunity)，用以治療皰疹病毒感染疾病。 Referring to Figure 1A, the immunogenic composition 1 is added to the culture environment of the antigen-presenting cells 30 in an appropriate culture environment to promote activation of the antigen-presenting cells 30 into activated antigen-presenting cells 31. Further, the activated antigen-presenting cells 31 are co-cultured with naïve lymphocytes 40 to cause cell expansion and cell activation of the immune cells 40 to become effector cells 41. Since the immune action cells 41 obtained in this manner have the function of identifying and scavenging the immunogenic composition 1 in vivo, the immune cells 41 can be injected into the patient 5 in an appropriate manner to promote the acquired immune response in the patient 5 (adaptive). Immunity) for the treatment of herpes virus infections.
此外，本實施例中所使用之淋巴細胞可選用自體淋巴細胞(autologous lymphocytes)，以降低後續進行活體細胞治療時，所可能產生的免疫排斥風險。 In addition, the lymphocytes used in the present embodiment may be selected from autologous lymphocytes to reduce the risk of immunological rejection that may occur when subsequent living cell therapy is performed.
實驗結果顯示，經由本實施例所得到免疫作用細胞(不論是CD4或CD8族群)，具有針對EBV的專一性。此外，實驗結果進一步顯示，相 較於受到僅含單一種胜肽庫，包含有兩種、三種的免疫原組成物刺激而得的樹突細胞的活化效果較佳，而受到包含有四種胜肽庫(EBV潛伏性膜蛋白-2、EBV核蛋白-1、EBV核蛋白-3c及EBV-BZLF-1)免疫原組成物刺激後所得的樹突細胞，其誘發出的免疫細胞其作用效果最強。 The experimental results show that the immune cells obtained by the present example (whether the CD4 or CD8 population) have specificity for EBV. In addition, the experimental results further show that the phase Dendritic cells stimulated by a combination of two or three immunogenic compositions with a single peptide library are preferred, and are supported by four peptide libraries (EBV latent membrane proteins). -2, EBV nuclear protein-1, EBV nuclear protein-3c and EBV-BZLF-1) dendritic cells obtained after stimulation with the immunogenic composition, the immune cells induced by the immune cells have the strongest effect.
本發明將進一步藉由下面的實驗來作說明，但並非用以限定本發明之申請專利範圍權利。 The invention is further illustrated by the following experiments, but is not intended to limit the scope of the invention.
使用於下面各實驗中的15員胜肽庫(15-mer peptide pools)是購自於JPT Peptide Technologies GmbH(Berlin,Germany)，其包括延擴(spanning)以下列者整體之具有11個胺基酸部分重疊的序列之十五胜肽庫(pentadecapeptides)：EBV潛伏性膜蛋白-2(SEQ ID NO：1)(497個胺基酸，其所衍生之胜肽庫含122種胜肽)、EBV核蛋白-1(SEQ ID NO：2)(249個胺基酸，其所衍生之胜肽庫含60種胜肽)、EBV核蛋白-3C(SEQ ID NO：3)(641個胺基酸，所衍生之胜肽庫含265種胜肽)以及EBV-BZLF-1(SEQ ID NO：4)(245個胺基酸，所衍生之胜肽庫含59種胜肽)。其他抗原及胜肽可以藉由不同方法合成。其中由WT-1(Wilm’s tumor 1)(Swiss prot：P19544)所衍生之胜肽庫(WT33；JPT Peptide Technologies GmbH,Berlin,Germany)以及由C型肝炎病毒之核心病毒(core protein of Hepatitis C virus)(NCBI ACCESSION：ABV46234(1-191//product="core protein")所衍生之胜肽庫，並以Peptide Scan 15/11之方式，自JPT Peptide Technologies公司(JPT Peptide Technologies GmbH,Berlin,Germany訂購，其等係被用來作為非專一性胜肽庫。 The 15-mer peptide pools used in the following experiments were purchased from JPT Peptide Technologies GmbH (Berlin, Germany), which included spanning with 11 amine groups as a whole. Pentadecapeptides of the acid-overlapping sequence: EBV latent membrane protein-2 (SEQ ID NO: 1) (497 amino acids, the peptide library derived from the peptide contains 122 peptides), EBV nucleoprotein-1 (SEQ ID NO: 2) (249 amino acids containing 60 peptides), EBV nuclear protein-3C (SEQ ID NO: 3) (641 amino groups) The acid, the derived peptide library contains 265 peptides) and EBV-BZLF-1 (SEQ ID NO: 4) (245 amino acids, the derived peptide library contains 59 peptides). Other antigens and peptides can be synthesized by different methods. Among them, the peptide library derived from WT-1 (Wilm's tumor 1) (Swiss prot: P19544) (WT33; JPT Peptide Technologies GmbH, Berlin, Germany) and the core protein of Hepatitis C virus (NCBI ACCESSION: ABV46234 (1-191//product="core protein") derived peptide library, and Peptide Scan 15/11, from JPT Peptide Technologies GmbH (JPT Peptide Technologies GmbH, Berlin, Germany Ordering, etc. is used as a non-specific peptide library.
下列各實驗中所使用的血漿析離的血液(apheresis blood)以及全血係取自於健康的捐贈者。下列實驗所使用的週邊血液單核細胞係用Ficoll-Hypaque(GE Healthcare Bio-Sciences AB,NJ,USA)藉由梯度密度離心予以製備(Han S.et al.,2008,Molecular Therapy,16：269-279)。製備而得的週邊血液單核細胞的存活率藉由錐蟲藍染色(trypan blue staining)予以測定，只有細胞存活率大於80%之組別的細胞會繼續使用於下列實驗。 The plasma apheresis blood and whole blood used in the following experiments were taken from healthy donors. Peripheral blood mononuclear cell lines used in the following experiments were prepared by Gradient Density Centrifugation using Ficoll-Hypaque (GE Healthcare Bio-Sciences AB, NJ, USA) (Han S. et al., 2008, Molecular Therapy, 16:269) -279). The survival rate of the prepared peripheral blood mononuclear cells was determined by trypan blue staining, and only cells in the group with a cell survival rate greater than 80% continued to be used in the following experiments.
將週邊血液單核細胞以1x107細胞/孔(cells/well)之細胞密度置於6孔盤中，以將週邊血液單核細胞培養於AIM-V培養基(Gibco-BRL,CA,USA)中歷時2小時進行貼附。接而，溫和的移除未貼附之細胞，並且進行冷凍作為用於後續共培養實驗中之T淋巴細胞的來源。將貼附的細胞予以培養於添加有50 ng/mL之GM-CSF(Biosource,CA,USA)以及25 ng/mL之IL-4(Biosource,CA,USA)的AIM-V培養基中。為了產生樹狀細胞，細胞以GM-CSF以及IL-4培養歷時24小時，再以IFN-gamma(20 ng/mL)(Gentaur)、TNF-alpha(50 ng/mL)(R&D systems,MN,USA)、IL-1 beta(10 ng/mL)(R&D systems,MN,USA)、IL-6(10 ng/ml)(R&D systems)以及PGE2(1 μM)(Sigma-Aldrich,MO,USA)培育24小時，作為成熟化反應(maturation)。樹狀細胞之表現型(phenotype)藉由流式細胞分析法分析。 Peripheral blood mononuclear cells were placed in a 6-well plate at a cell density of 1×10 7 cells/well to culture peripheral blood mononuclear cells in AIM-V medium (Gibco-BRL, CA, USA). Attached for 2 hours. In turn, unattached cells were gently removed and frozen as a source of T lymphocytes for use in subsequent co-culture experiments. The attached cells were cultured in AIM-V medium supplemented with 50 ng/mL of GM-CSF (Biosource, CA, USA) and 25 ng/mL of IL-4 (Biosource, CA, USA). To generate dendritic cells, cells were cultured with GM-CSF and IL-4 for 24 hours, followed by IFN-gamma (20 ng/mL) (Gentaur), TNF-alpha (50 ng/mL) (R&D systems, MN, USA), IL-1 beta (10 ng/mL) (R&D systems, MN, USA), IL-6 (10 ng/ml) (R&D systems), and PGE2 (1 μM) (Sigma-Aldrich, MO, USA) Incubate for 24 hours as a maturation. The phenotype of dendritic cells was analyzed by flow cytometry.
經樹狀細胞活化的免疫細胞的分離及培養方法是依據Han等人所提出之方法(Han S.et al.,2008,Molecular Therapy,16：269-279)。簡言 之，經由前段方式所製得的成熟樹狀細胞，被裝載(loaded)以胜肽(5 μg/mL)或其他抗原歷時3小時，並予以輻射照射(2,500 rads)。樹狀細胞與同源性非貼附性週邊血液單核細胞(PBMC)以1：20的比例予以共培養於具有2-5%人類AB血清之AIM-V培養基中。於第三天，予以添加IL-2(Gentaur,Aachen,Germany)、IL-7(Gentaur)以及IL-15(Gentaur)。接著，每隔一天予以添加新鮮的具有該等細胞激素(cytokines)之培養基。共培養物中之T細胞的功能分析是透過細胞內細胞激素染色(intracellular cytokine staining)來進行。 The isolation and culture method of immune cells activated by dendritic cells is based on the method proposed by Han et al. (Han S. et al., 2008, Molecular Therapy, 16:269-279). Brief The mature dendritic cells prepared by the anterior method were loaded with peptides (5 μg/mL) or other antigens for 3 hours and irradiated with radiation (2,500 rads). Dendritic cells and homologous non-adherent peripheral blood mononuclear cells (PBMC) were co-cultured in AIM-V medium with 2-5% human AB serum at a ratio of 1:20. On the third day, IL-2 (Gentaur, Aachen, Germany), IL-7 (Gentaur) and IL-15 (Gentaur) were added. Next, fresh medium with these cytokines is added every other day. Functional analysis of T cells in co-cultures was performed by intracellular cytokine staining.
細胞染色之分析是如Han等人之著作所述者予以進行(Han et al.，同上述)。簡言之，3x105個經擴增的EBV專一性免疫作用細胞(expanded EBV immune effector,IE)使用經抗原裝載的同源性樹狀細胞(anitgen-loaded autologous DCs)或單核細胞於有或沒有抗-CD107a-FITC的AIM-V培養基中予以進行刺激。於刺激反應1小時後，予以添加孟寧素(Monensin)(Sigma)。於5小時後，細胞針對CD4及CD8染色、固定(fixed)、穿孔(permeablized)並且利用針對IFN-gamma的抗體(全部來自於BD Bioscience)並使用FIX/PERM以及PERM/Wash solution(BD)予以染色。 Analysis of cell staining was performed as described in the work of Han et al. (Han et al., supra). Briefly, 3x10 5 expanded EBV immune effectors (IEs) use anitgen-loaded autologous DCs or monocytes at or Stimulation was performed in AIM-V medium without anti-CD107a-FITC. After 1 hour of the stimulation reaction, Monensin (Sigma) was added. After 5 hours, cells were stained, fixed, permeablized against CD4 and CD8 and antibodies against IFN-gamma (all from BD Bioscience) and FIX/PERM and PERM/Wash solution (BD) were used. dyeing.
新鮮分離的PBMC以1 x 105 cell/well的濃度盛盤於塗布有10 μg/ml anti-IFN-gamma(1-D1K；MabTech)之96-孔盤並且使用5%去活性人類AB血清(inactivated human AB serum)(Valley Biomedical,USA)予以阻隔 (block)。細胞利用不同的胜肽庫之組合以二重複的方式進行刺激反應。於37℃於5% CO2下培育18至20小時後，將細胞予以沖離並將1 μg/ml經生物素標記的抗-IFN-gamma(biotin labeled anti-IFN-gamma)(7-B6-1；MabTech)予以加入。藉由加入鏈黴抗生物素標記的鹼性磷酸酯酶(Streptavidin conjugated alkaline phosphatase,Streptavidin conjugated ALP)(MabTech)以產生呈色反應[斑點(spots)]，繼而加入將受質BCIP/NBT-plus(Bio-Rad)。斑點的數目藉由ELISPOT讀取儀(AutoImmune Diagnostika)計算出，以每1x106的PBMC中含有斑點形成細胞(spot-forming cell,SFC)表示。 Freshly isolated PBMCs were plated at a concentration of 1 x 10 5 cells/well in 96-well plates coated with 10 μg/ml anti-IFN-gamma (1-D1K; MabTech) and 5% deactivated human AB serum was used ( Inactivated human AB serum) (Valley Biomedical, USA) was blocked. The cells are stimulated in a two-fold manner using a combination of different peptide libraries. After incubation for 18 to 20 hours at 37 ° C in 5% CO 2 , the cells were washed away and 1 μg/ml biotin-labeled anti-IFN-gamma (biotin labeled anti-IFN-gamma) (7-B6) -1; MabTech) to join. By adding Streptavidin conjugated alkaline phosphatase (Streptavidin conjugated ALP) (MabTech) to produce a color reaction [spots], followed by the addition of the BCIP/NBT-plus (Bio-Rad). The number of spots was calculated by an ELISPOT reader (AutoImmune Diagnostika) and expressed as a spot-forming cell (SFC) per 1×10 6 PBMC.
選擇5位健康的捐贈者，取得其周邊血液單核細胞，以EBV潛伏性膜蛋白-2胜肽庫(Lmp2)、EBV核蛋白-3c胜肽庫(EBNA3c)及EBV潛伏性膜蛋白-2、EBV核蛋白-1、EBV核蛋白-3c及EBV-BZLF-1四種組合胜肽庫(4mix)分別刺激，進行培養20小時後，以ELISpot分析方法進行IFN-gamma分析。實驗結果如第2圖所示，其中實驗結果顯示不論哪一位捐贈者，使用四種組合(4mix)胜肽庫刺激後所產生的IFN-gamma效果最佳。 Five healthy donors were selected to obtain peripheral blood mononuclear cells, EBV latent membrane protein-2 peptide library (Lmp2), EBV nuclear protein-3c peptide library (EBNA3c) and EBV latent membrane protein-2 EBV nucleoprotein-1, EBV nuclear protein-3c and EBV-BZLF-1 were combined and stimulated for 20 hours. The IFN-gamma analysis was performed by ELISpot analysis. The experimental results are shown in Fig. 2, and the experimental results show that regardless of which donor, the IFN-gamma produced by the four combinations (4mix) peptide library is the best.
取兩位健康捐贈者的周邊血液單核細胞，分別用下列包含不同胜肽肽庫組合的免疫原組成物進行刺激，培養20小時後，以ELISpot分析方法，進行IFN-gamma分析。各組合分別為：1：EBV潛伏性膜蛋白-2，2：EBV核蛋白-3c，3：EBV核蛋白-1，4：EBV-BZLF-1，5：EBV潛伏性膜蛋白-2+EBV核蛋白-3c，6：EBV潛伏性膜蛋白-2+EBV核蛋白-1，7：EBV核蛋白-3c+EBV核蛋白-1，8：EBV潛伏性膜蛋白-2+EBV 核蛋白-1+EBV核蛋白-3c，9：EBV潛伏性膜蛋白-2+EBV核蛋白-1+EBV核蛋白-3c+EBV-BZLF-1。其結果如第3A圖及第3B圖所示，分別為捐贈者1及2的實驗結果。實驗結果進一步顯示使用四種胜肽庫組合(組合9)，刺激周邊血液單核細胞產生IFN-gamma的效果強於使用單一種胜肽庫(組合1-4)、任兩種胜肽庫組合(組合5-7)或三種胜肽庫組合(組合8)。 Peripheral blood mononuclear cells from two healthy donors were stimulated with the following immunogen compositions containing different peptide peptide pool combinations. After 20 hours of culture, IFN-gamma analysis was performed by ELISpot analysis. The combinations are: 1: EBV latent membrane protein-2, 2: EBV nuclear protein-3c, 3: EBV nuclear protein-1, 4: EBV-BZLF-1, 5: EBV latent membrane protein-2+EBV Nuclear protein-3c,6: EBV latent membrane protein-2+EBV nuclear protein-1,7:EBV nuclear protein-3c+EBV nuclear protein-1,8:EBV latent membrane protein-2+EBV Nucleoprotein-1 + EBV nucleoprotein-3c, 9: EBV latent membrane protein - 2+EBV nucleoprotein-1 + EBV nucleoprotein-3c + EBV-BZLF-1. The results are shown in Figures 3A and 3B, and are the experimental results of donors 1 and 2, respectively. The experimental results further show that the use of four peptide library combinations (combination 9), the stimulation of peripheral blood mononuclear cells to produce IFN-gamma is stronger than the use of a single peptide library (combination 1-4), any combination of two peptide libraries (Combination 5-7) or a combination of three peptide libraries (combination 8).
依照前述的實驗方法，以單一種的胜肽庫：EBV潛伏性膜蛋白-2(Lmp2)、EBV核蛋白-1(ENBA1)、EBV核蛋白-3c(EBNA3c)或EBV-BZLF-1(BZLF1)，及包含EBV潛伏性膜蛋白-2+EBV核蛋白-1+EBV核蛋白-3c+EBV-BZLF-1四種組合胜肽庫(4 mix)，分別處理樹突細胞進而刺激T細胞共同培養15天後，將依上述五種方式刺激活化所得的五種免疫作用細胞，各自針對帶有專一性抗原的樹突細胞再次進行刺激5小時，同時以非專一性抗原(WT-1)處理之同源的樹突細胞作為對照組。前述受到再刺激的免疫細胞，觀察其細胞內IFN-gamma及CD107a的產生量，再將各組中專一性抗原所得到IFN-gamma及CD107a表現的值扣除非專一性抗原的背景值，分CD4及CD8族群繪製成柱狀圖，藉以對EBV專一性作用細胞的功能進行評估。實驗結果如第4圖所示，單一種胜肽庫刺激免疫作用細胞在功能檢測上具有些微的效果，但以四種胜肽庫混合刺激方式所得的免疫作用細胞，則表現出最高的IFN-gamma細胞激素及CD107a，免疫效果為最好，這樣的現象在CD4及CD8族群中都可以看到。 According to the above experimental method, a single peptide library: EBV latent membrane protein-2 (Lmp2), EBV nuclear protein-1 (ENBA1), EBV nuclear protein-3c (EBNA3c) or EBV-BZLF-1 (BZLF1) ), and contains four combinations of EBV latent membrane protein-2+EBV nucleoprotein-1+EBV nucleoprotein-3c+EBV-BZLF-1 (4 mix), respectively processing dendritic cells to stimulate T cells After 15 days of culture, the five immune cells obtained by activation were stimulated in the above five ways, and each of the dendritic cells with the specific antigen was stimulated again for 5 hours while being treated with a non-specific antigen (WT-1). The homologous dendritic cells were used as a control group. The above-mentioned re-stimulated immune cells were observed for the production of IFN-gamma and CD107a in the cells, and the values of IFN-gamma and CD107a obtained by the specific antigens in each group were subtracted from the background value of the non-specific antigen, and the CD4 was divided into CD4. And the CD8 population was drawn into a histogram to evaluate the function of EBV-specific cells. As shown in Fig. 4, a single peptide library stimulated immune cells to have a slight effect on function detection, but the immune cells obtained by the mixed stimulation of the four peptide libraries showed the highest IFN- The gamma cytokine and CD107a have the best immune effect, and this phenomenon can be seen in both the CD4 and CD8 populations.
首先將樹突細胞以EBV潛伏性膜蛋白-2+EBV核蛋白-1+EBV核蛋 白-3c+EBV-BZLF-1四種組合胜肽庫處理，並與未活化之T細胞進行共同培養15天，以得出活化的免疫作用細胞。將依前述方式所活化的免疫作用細胞，分別使用帶有單一種胜肽庫：EBV潛伏性膜蛋白-2、EBV核蛋白-1、EBV核蛋白-3c或EBV-BZLF-1，以及EBV潛伏性膜蛋白-2+EBV核蛋白-1+EBV核蛋白-3c+EBV-BZLF-1四種組合胜肽庫(4 pepmix)的樹突細胞再次刺激。正向的對照組為以佛波醇酯(phorbol-12-myristate-13-acetate)及離子黴素(Inonmycin)(PMA+Inomysin)刺激樹突細胞來活化前述得出的免疫作用細胞，負向對照組則使用帶有非專一性抗原(WT-1)的樹突細胞。而以未處理的免疫作用細胞(IE only)作為基準。經由前述再刺激處理所得的免疫細胞，觀察其細胞內IFN-gamma及CD107a的產生量，藉以評估對抗原專一性作用細胞的功能。實驗結果為第5A圖、第5B圖及第5C圖所示，其中第5A圖及第5B圖分別為經流式細胞分析儀所得之CD4及CD8族群分析圖。第5C圖則表示同時表現有IFN-gamma及CD107a的細胞所佔細胞全體中的百分比，結果顯示，不管是在CD4或是CD8族群中，使用四種胜肽庫混合處理的樹突細胞所活化得出的免疫作用細胞針對帶有任何單一種EBV胜肽庫的再刺激都有反應，其中又以受到帶有4種四種EBV胜肽庫混合的樹突細胞的再刺激效果最佳。 First, dendritic cells are EBV latent membrane protein-2+EBV nucleoprotein-1+EBV nuclear egg White-3c+EBV-BZLF-1 was treated with four combinations of peptide libraries and co-cultured with unactivated T cells for 15 days to obtain activated immune cells. The immune cells activated in the foregoing manner were used with a single peptide library: EBV latent membrane protein-2, EBV nuclear protein-1, EBV nuclear protein-3c or EBV-BZLF-1, and EBV latent The dendritic cells of the four combination peptide libraries (4 pepmix) of the membrane protein-2+EBV nucleoprotein-1+EBV nucleoprotein-3c+EBV-BZLF-1 were stimulated again. The positive control group stimulated dendritic cells with phorbol-12-myristate-13-acetate and Inonmycin (PMA+Inomysin) to activate the aforementioned immune cells, negative Dendritic cells with a non-specific antigen (WT-1) were used in the control group. Untreated immune cells (IE only) were used as a benchmark. The amount of IFN-gamma and CD107a produced in the cells was observed by the immunocytes obtained by the above-described re-stimulation treatment, thereby evaluating the function of the cells which specifically act on the antigen. The experimental results are shown in Fig. 5A, Fig. 5B and Fig. 5C, wherein Fig. 5A and Fig. 5B are graphs of CD4 and CD8 populations obtained by flow cytometry, respectively. Figure 5C shows the percentage of cells that have both IFN-gamma and CD107a, and the results show that they are activated by dendritic cells mixed with four peptide libraries, whether in the CD4 or CD8 population. The resulting immune cells responded to re-stimulation with any single EBV peptide library, with the best re-stimulation by dendritic cells with a mixture of four four EBV peptide libraries.
以上所述僅為本發明較佳實施例而已，並非用以限定本發明申請專利權利；同時以上的描述對於熟之本技術領域之專門人士應可明瞭與實施，因此其他未脫離本發明所揭示之精神下所完成的等效改變或修飾，均應包含於下述之申請專利範圍。 The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention. The above description is to be understood by those skilled in the art, and thus the other embodiments are not disclosed. Equivalent changes or modifications made in the spirit of the invention are to be included in the scope of the claims below.
1‧‧‧免疫原組成物 1‧‧‧ immunogenic composition
11、12、13、14‧‧‧胜肽 11, 12, 13, 14 ‧ ‧ peptides
110‧‧‧EBV潛伏性膜蛋白-2十五胜肽庫 110‧‧‧EBV latent membrane protein-2 fifteen peptide library
111、112、113‧‧‧胜肽片段 111, 112, 113‧‧‧ peptide fragments
2‧‧‧EBV潛伏性膜蛋白-2完整胜肽 2‧‧‧EBV latent membrane protein-2 intact peptide
30‧‧‧抗原呈現細胞 30‧‧‧ antigen presenting cells
31‧‧‧已活化抗原呈現細胞 31‧‧‧ Activated antigen presenting cells
40‧‧‧單純淋巴細胞 40‧‧‧Simple lymphocytes
41‧‧‧免疫作用細胞 41‧‧‧Immune cells
5‧‧‧患者 5‧‧‧ patients
第1A圖為本發明生產免疫細胞的方法流程示意圖。 Figure 1A is a schematic flow chart of the method for producing immune cells of the present invention.
第1B圖為本發明所使用的EBV潛伏性膜蛋白-2十五胜肽庫組成 示意圖。 Figure 1B is a composition of the EBV latent membrane protein-2 fifteen peptide library used in the present invention. schematic diagram.
第2圖為本發明實驗1中IFN-gamma分析圖。 Fig. 2 is a graph showing the analysis of IFN-gamma in Experiment 1 of the present invention.
第3A圖為本發明實驗2中捐贈者1之IFN-gamma分析圖。 Figure 3A is a graph showing the IFN-gamma analysis of Donor 1 in Experiment 2 of the present invention.
第3B圖為本發明實驗2中捐贈者2之IFN-gamma分析圖。 Figure 3B is a graph showing the IFN-gamma analysis of Donor 2 in Experiment 2 of the present invention.
第4圖為本發明實驗3中細胞內表現IFN-gamma及CD107a比例分析圖。 Fig. 4 is a graph showing the ratio of intracellular expression of IFN-gamma and CD107a in Experiment 3 of the present invention.
第5A圖為本發明實驗4CD4族群中表現IFN-gamma及CD107a流式細胞分析圖。 Figure 5A is a flow cytometric analysis of IFN-gamma and CD107a in the experimental 4CD4 population of the present invention.
第5B圖為本發明實驗4CD8族群中表現IFN-gamma及CD107a流式細胞分析圖。 Figure 5B is a flow cytometric analysis of IFN-gamma and CD107a in the experimental 4CD8 population of the present invention.
第5C圖為本發明中實驗4中細胞內表現IFN-gamma及CD107a比例分析圖。 Fig. 5C is a graph showing the ratio of intracellular expression of IFN-gamma and CD107a in Experiment 4 of the present invention.
<110> 鑫品生醫科技股份有限公司 <110> Xinpin Biomedical Technology Co., Ltd.
<120> 生產免疫細胞之方法及誘發產生免疫作用細胞的方法 <120> Method for producing immune cells and method for inducing immune cells
<160> 4 <160> 4
<210> 1 <210> 1
<211> 497 <211> 497
<212> PRT <212> PRT
<213> 人類皰疹病毒第四型B95-8(Human herpesvirus 4 strain B95-8) <213> Human herpesvirus 4 strain B95-8
<300> <300>
<308> swiss-prot P13285 <308> swiss-prot P13285
<309> 1990-01-01 <309> 1990-01-01
<400> 1 <400> 1
<210> 2 <210> 2
<211> 249 <211> 249
<212> PRT <212> PRT
<213> 人類皰疹病毒第四型AG876(Human herpesvirus 4 strain AG876) <213> Human herpesvirus 4 strain AG876
<220> <220>
<223> 人類皰疹病毒第四型AG876(Human herpesvirus 4 strain AG876)核蛋白-1(EBNA-1)經N端裁切之蛋白片段，相當於全長成熟EBV核蛋白-1的第393個胺基酸至第641個胺基酸 <223> Human herpesvirus type 4 AG876 (Human herpesvirus 4 strain AG876) nuclear protein-1 (EBNA-1) N-terminally cut protein fragment corresponding to the 393th amine of full-length mature EBV nuclear protein-1 Base acid to the 641th amino acid
<400> 2 <400> 2
<210> 3 <210> 3
<211> 1069 <211> 1069
<212> PRT <212> PRT
<213> 人類皰疹病毒第四型AG876(Human herpesvirus 4 strain AG876) <213> Human herpesvirus 4 strain AG876
<300> <300>
<308> NCBI ABB89245 <308> NCBI ABB89245
<309> 2006-06-14 <309> 2006-06-14
<400> 3 <400> 3
<210> 4 <210> 4
<211> 245 <211> 245
<212> PRT <212> PRT
<213> 人類皰疹病毒第四型B95-8(Human herpesvirus 4 strain B95-8) <213> Human herpesvirus 4 strain B95-8
<300> <300>
<308> swiss-prot P03206 <308> swiss-prot P03206
<309> 1986-07-21 <309> 1986-07-21
<400> 4 <400> 4
1‧‧‧免疫原組成物 1‧‧‧ immunogenic composition
11、12、13、14‧‧‧胜肽 11, 12, 13, 14 ‧ ‧ peptides
30‧‧‧抗原呈現細胞 30‧‧‧ antigen presenting cells
31‧‧‧已活化抗原呈現細胞 31‧‧‧ Activated antigen presenting cells
40‧‧‧單純淋巴細胞 40‧‧‧Simple lymphocytes
41‧‧‧免疫作用細胞 41‧‧‧Immune cells
5‧‧‧患者 5‧‧‧ patients
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Non-Patent Citations (3)
| Title |
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| Blake N., et al., "Differential Immunogenicity of Epstein-Barr Virus Latent-Cycle Proteins for Human CD41 T-Helper 1 Responses.", J. Virol., Sep. 2001, Vol.75, No.18, page 8649-8659 * |
| Khanna R., et al., "The immunology of Epstein-Barr virus infection.",Phil. Trans. R. Soc. Lond. B Bio. Sci. Apr. 29 2001, Vol.365, page 475-488 * |
| Rickinson A. B., et al., "Dual Stimulation of Epstein-Barr Virus (EBV)-Specific CD4+- and CD8+-T-Cell Responses by a Chimeric Antigen Construct: Potential Therapeutic Vaccine for EBV-Positive Nasopharyngeal Carcinoma.", J. Virol., Jan. 2004, Vol.78, No. 2, page 768–778 * |
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