201225959 、發明說明： 【發明所屬之技術領域】 本發明係提供一種人類自然殺手細胞之増殖方法，使自然殺 手細胞經本發明方法增殖過後’可達到至少高於增殖前五百倍的 細胞數目，並保持自然殺手細胞之毒殺活性，為人類自然殺手細 胞在用於製備腫瘤疫苗等醫療藥物組合時提供一種切實可行的 方法。201225959, invention description: [Technical field of the invention] The present invention provides a method for colonizing human natural killer cells, which enables natural killer cells to be at least five hundred times higher than the number of cells before proliferation by the method of the present invention. Maintaining the toxic activity of natural killer cells provides a practical method for human natural killer cells to be used in the preparation of medical drug combinations such as tumor vaccines.
【先前技術】 根據衛生署的資料，癌症一直高居台灣十大死因的第一位， 癌症影響層面之廣及所耗費社會成本之鉅可見一斑。目前為止人 麵熟知的抗癌絲至少有五大類’包括手術赠法、化學治療 及放射線治療等三種及其合併療法與民俗療法。 手術療法雖然效果很好，但有些腫瘤無法以外科手術切除，且手 術之後仍會有殘存之癌細胞容易造成復發或轉移，這正是癌症之 所以可怕以級人於死社要相，已娜或無法以外科手術 切除之癌細胞就必須以放射線療法或化學療法來治療，甚至無法 以現有的醫療方式達到治療效果。 可是放射線療法献學療法並沒有專—性’會將好的細胞以 及壞的細胞同時觀，導致嚴重_侧，網伽對病患本身 抗癌能力最為_的免疫系統破壞也最嚴重，這是目前醫學上一 直無法克服的蘭之—，因錢_治療無論是缺長壽命或降 低副作用方面_有大幅改善之空間。因此對於癌症治療，各施 3 201225959 都在積極尋求能抗癌，無副作用而又能加強免疫機能之療法。 自然殺手細胞(Natural killer cells，NK cells)是人體内 第一道與生倶來的免疫防線（Native immunity )成員之一， 也是對抗癌細胞或病毒最強、最有效的細胞。與其他抗癌免疫 細胞相比，例如毒殺性T細胞（Cytotoxic T cells )或樹突 細胞（Dendritic cells )，NK細胞之毒殺性較強、較具廣 譜性、不受組織相容性抗原限制（MHC-Restriction )且不需 要致敏作用（Priminig )之啟動就可直接發動對癌細胞之攻擊。 除此之外’ NK細胞還具有調節免疫能力及和神經系統交互作用 之能力。理論上NK細胞可以成為一個有效的癌症治療手段，然 而這種細胞平常在體内之數量本來就不多，只約佔血液中白血球 的2〜6%，再加上受到疾病、年齡、壓力、不良生活習慣及其它 環境因素等之影響，NK細胞之數量及活性常隨著年齡增長而逐 漸降低，直接或間接促成癌症之發生或惡化；而臨床上也常觀察 到癌症病人之NK細胞數量不足或活性下降之現象。習知技術無 法在體外短時間内將NK細胞大量的增殖，且同時保有NK細胞的 毒殺活性，再者，習知技術所採用的NK細胞，係取自於已經得 到癌症_患’錢祕下取得的NK細騎於癌細胞的毒殺能 力不夠強’即便·後再回輸於病人體内，所能達義癌細胞殺 傷效果亦不高’使得_ NK細胞絲療麵㈣法固然很有吸 引力，但是實際執行上卻礙於f知技術之不足而無法真正應用於 癌症治療。 201225959 【發明内容】 本毛月之目的’在於提供—種人類自然殺手細胞之體外增殖 方法’其係可以從冷絲存之周邊血液單核球細胞⑻ blood m〇n〇nuclear cells，pBMCs)中增殖c航廳+亞型自然殺 手細胞的方法’使自紐手細胞經本方法增_後，可達到至少 尚於增殖刖五百倍的細胞數目，並保持自然殺手細胞之毒殺活 性’為人類自然殺手細胞在製備_疫苗療藥物組合時 提供一種切實可行的方法。 本發明之又-目的，在料腫瘤Νκ細胞療法提供了一種切實 可行的醫療方案和技術，例如將使財發明之增雜術所培養而 得之NK細胞用於製備腫瘤疫苗等醫療組成物。 為更好的描述和理解本發明，我們將對本發明中所涉及的術 語做詳盡的解釋。 自然殺手細胞(Natural killer ceiis，cells) : NK細胞 疋-群不同於T、B淋巴細胞的大顆粒淋巴細胞，分佈於周邊各 淋巴器g及血液循環系統，無需抗原的預先刺激與活化即可發揮 細胞毋殺效應，並可分泌多種細胞因子及趨化因子。 NK細胞的啟動：NK細胞可表達一系列活化性受體及抑制性受 體兩者間的平衡是控制NK細胞是否被啟動的重要機制。同時， 干擾素及巨噬細胞來源的細胞介素均是細胞極強的活化因 子。本發明中是利用重組人類白血球間素(Rec〇mbi細七hu_[Prior Art] According to the Department of Health, cancer has consistently ranked first among the top ten causes of death in Taiwan. The extent of cancer impact and the cost of social costs are evident. There are at least five major categories of anti-cancer filaments that have been known so far, including surgery, chemotherapy, and radiation therapy, as well as combination therapy and folklore therapy. Although the surgical treatment is very effective, some tumors cannot be surgically removed, and there are still residual cancer cells that are likely to cause recurrence or metastasis after the operation. This is why the cancer is terrible. Cancer cells that cannot be surgically removed must be treated with radiotherapy or chemotherapy, or even in the existing medical treatment. However, radiotherapy offers no specificity. It will treat both good cells and bad cells at the same time, leading to serious _ side, and the immune system damage of the disease itself is the most serious. This is the most serious damage. At present, the medical science has been unable to overcome the blue - because of the money - treatment, whether it is lack of longevity or reduce side effects, there is room for significant improvement. Therefore, for cancer treatment, each application 3 201225959 is actively seeking treatments that can fight cancer without side effects and strengthen immune function. Natural killer cells (NK cells) are one of the first members of the body to be immune to the native immunity. They are also the strongest and most effective cells against cancer cells or viruses. Compared with other anti-cancer immune cells, such as Cytotoxic T cells or Dendritic cells, NK cells are more potent, more broad-spectrum, and are not restricted by histocompatibility antigens. (MHC-Restriction) does not require the initiation of sensitization (Priminig) to directly attack cancer cells. In addition, 'NK cells also have the ability to regulate immunity and interact with the nervous system. In theory, NK cells can be an effective treatment for cancer. However, the number of such cells in the body is usually not much, only about 2 to 6% of white blood cells in the blood, plus disease, age, stress, Due to adverse lifestyle habits and other environmental factors, the number and activity of NK cells often decrease with age, directly or indirectly contributing to the occurrence or deterioration of cancer. Clinically, it is often observed that the number of NK cells in cancer patients is insufficient. Or the phenomenon of decreased activity. The conventional technology cannot proliferate a large amount of NK cells in a short time in vitro, and at the same time retains the cytotoxic activity of NK cells. Furthermore, the NK cells used in the prior art are obtained from the cancer that has been obtained. The NK fine riding ability of the cancer cell is not strong enough. Even if it is returned to the patient, the cancer cell killing effect is not high enough to make the NK cell silk surface (4) method very attractive. Force, but the actual implementation is due to the lack of knowledge of the technology and can not be truly applied to cancer treatment. 201225959 [Summary of the Invention] The purpose of this month is to provide a method for in vitro proliferation of human natural killer cells, which can be obtained from peripheral blood mononuclear cells (8), blood m〇n〇nuclear cells (pBMCs). The method of proliferating c-airport + subtype natural killer cells' enables the self-nucleated cells to increase the number of cells at least 500 times of proliferation and maintain the poisoning activity of natural killer cells. Killer cells provide a practical method when preparing a vaccine combination. A further object of the present invention is to provide a practical medical solution and technique for the treatment of tumor Ν kappa cell therapy, for example, NK cells cultured by the augmentation of the invention for the preparation of a medical composition such as a tumor vaccine. For a better description and understanding of the present invention, we will explain the terms of the invention in detail. Natural killer ceiis (cells): Large-particle lymphocytes of NK cell 疋-group different from T and B lymphocytes, distributed in peripheral lymphatic g and blood circulation system, without pre-stimulation and activation of antigen It exerts a cell killing effect and can secrete a variety of cytokines and chemokines. Activation of NK cells: The balance between NK cells expressing a range of activating receptors and inhibitory receptors is an important mechanism for controlling whether NK cells are activated. At the same time, interferon and macrophage-derived interleukins are extremely strong cell activation factors. In the present invention, recombinant human leukocyte interleukin (Rec〇mbi fine seven hu_) is utilized.
Interleukin，rhIL) 2(rhIL-2)、12 (rhIL-12)、15 (rhIL-15)、 201225959 18 (rhIL-18)、與21 (rhIL-21)及其組合的應用來活化服細胞。 NK細胞的增殖：是指NK細胞在體外培養中通過一系列的細胞 分裂，而達到一定的增殖數目。本發明中所增殖的NK細胞主要是 CD3CD56+亞群的NK細胞。 細胞毒殺活性：是指由細胞引起的單純的細胞殺傷事件。NK 細胞的毋殺活性不單純偶限於腫瘤細胞，還包括對病毒感染細 胞、受損和功能異常細胞的殺傷作用。本發明中所涉及的M細胞 毋权活性主要是指其對腫瘤細胞的殺傷作用。Nj(細胞毒殺活性的 測定目前主要是應用對NK細胞敏感的骨髓白血病 (ery thro 1 eukem i a)細胞株K562和對NK細胞抗性的淋巴腫瘤細胞 株Raji為靶細胞，測定nk細胞對它們的殺傷率。本發明中應用 K562細胞株來測定NK細胞毒殺活性。 NK細胞療法：是一種利用高科技之細胞培養技術在短時間内 於體外（Ex vivo)大量繁殖病人本身可以對抗癌細胞的陬細胞 後，再將其注入病人體内，直接提高病人本身之抗癌能力，達到 抗癌、冶癌目標的一種醫療技術。NJ(細胞療法的前提是nk細胞的 啟動和大量增殖，並對腫瘤細胞保持高的細胞毒殺傷活性。 自體免疫細胞’尤其是NK細胞的免疫細胞治療法簡單的說， 是一種利用高科技之細胞培養技術在短時間内於體外（Εχ viv〇) 大量繁殖病人本身可以對抗癌細胞的胍細胞後，再將其注入病人 體内，直接提高病人本身之抗癌能力。實際的做法是病人經醫師 仔細評估且判定適合NK細胞療法後，從病人身上採集約3〇毫升之 201225959 周邊血液在符合GTP/GMP規範之無塵、無菌實驗室進行連續15天 的培養，在過程中以特殊之配方專門培養、增殖其中之陬細胞， 使其數量增加達原先之數百倍甚至上千倍後再注入體内，直接提 向NK細胞之數量進而達到治療癌症之目的。 利用NK細胞療法來治療癌症有下列各種學理上之優勢：一、 NK細胞是人體内抗癌活性最強的細胞，可直接殺死癌細胞抑制 腫瘤的生長及擴散；二、NK細胞會抑制腫瘤附近新血管的增 生，故可限制腫瘤取得所需要的養份進而限制腫瘤之生長；三、 NK細胞可直接改善並調節患者之免疫力及神經系統，間接提高 患者之生活品質；四、Νκ細胞會分泌—種叫做b_end〇_in的 腦内非可減少病患之疼痛令病患有愉悅感，間接提升生活品 貝，五、田Ij作用低或幾乎無副作用。目前一些研究已指出，周邊 企液自然、权手細胞所具有的毒殺範圍除了包括黑色素瘤、頭頸癌 之外’還有肺、即巢、子宮頸'膀胱、攝護腺、肝細胞、胰臟、 食道、乳歧許多合併的癌症。尤其對肺癌、淋巴癌、食道癌、 乳癌、肝鱗錢旺盛且血管較密減區之癌症較有效。 叙的狀況下，我們身體以部位計算，會有5到15顆的癌細 胞產生方pa寺’只要一顆NK細胞，就可以殺光這些癌細胞。但 〇 疋仁彳于了癌症，每—顆癌細胞，要250顆當時體内的NK細 匕才月匕杈死。因此’儲存未得癌症前的戰鬥力高的細胞， j萬一未來得辨’複製成多數，再注人身體以協助抗癌，是目 珂最有效'無副作用的輔助方法。 201225959 本發明提供職細胞增殖之方法，滿足NK細胞療法的條件， 為腫瘤NK細胞療法提供了 一種切實可行的醫療技術。 本發明人等欲解決腫瘤服細胞療法之課題經銳意研討結果， 發現從凍存周邊血液單核球細胞啟動、增殖NK細胞之方法。所增 殖NK細胞是具有高腫瘤細胞毒殺活性的(：1)3-(；：1)56+亞群。細胞培養 條件在貫麵巾詳細贿。根縣發明實細結果，周邊血液單 核球細胞在體外培養5天後，服細胞開始增殖，發明人等稱之為 啟動階段’在後續的1_天過程培養中，發明人等稱之為增殖 階段，NK細胞會繼續增殖至1〇〇到5〇〇倍，甚至到1〇〇〇倍。 根據本發明貫施例結果，啟動並增殖的胍細胞與非體外增殖 的NK細胞相比，細胞毒殺活性至少增加了 1〇〇%，較好者增加了 150%到200% ’甚至最好者增加了雌。本發明中非體外啟動並增 殖的Μ細胞是指·⑽抗體磁珠，陽性分選的新觸細胞。 啟動並增殖ΝΚ細胞的細胞毒殺活性之所以增高可能是由於結合 受體表達上調所介導的一種效應。 本發明的實施例中描述了前述服細胞啟動、增殖之具體方 法。需要_的是’在以冷絲存之周邊血液單核球細胞啟動、 增殖ΝΚ細胞的培養體系中，由於周邊錢單核球細胞是包含不同 細胞群的混合物，例如C航D56-亞群的ΝΚ細胞、附細胞 (CD3+CD56+)和Τ細胞⑽+) ’所以在啟動增殖後，除了大量择 殖的0)襲6+亞戰細胞外，還有其他細胞類型在啟動增殖_ 養體系中存在。 201225959 根據本發明實補結果，發明人等亦詳細設計了鼠細胞的臨 床加用方案。發明人等在列職細胞培養及其狀態的觀察過 程中，發現亂細胞的最錄態在培養的第9到14天。根據細胞狀 悲’發明人等將在第9-1〇天和13—14天換無血清培養基培養，48 小時後換液，再經24小時其狀態達到最佳，陬細胞的數目達到 10-80億之間。因此，此時為進行細胞回輸至人體的最佳時機。 根據本發明中NK細胞對腫瘤細胞的細胞毒殺活性，本發明包 括Μ細胞能與其他癌症治療手段聯合給藥或應用。例如與手術、 放療和細胞毒性藥物的聯合應用。同時，本發明相關的研究結果 提示：體外啟動增殖的Μ細胞與體内未啟動的胍細胞相比具有明 顯的抗腫瘤能力。因此本發明亦包括了陬細胞對腫瘤的殺傷作 用。而所說的腫瘤細胞是指自體腫瘤細胞。自體腫瘤細胞是指來 源和再植入的是同一個體的細胞，也就是說，供體和受體是同一 個人。上述的自體腫瘤細胞包含了血液腫瘤及實體腫瘤。 與以往ΝΚ細胞增殖發明之方法比較，本發明包含以下幾點明 顯的不同或優勢： 1. 本發明係抽取健康人類個體的周邊血液，取出其中之周邊血液 單核細胞（PBMCs)以進行ΝΚ細胞的增殖，較之習知技術可以確 保NK細胞之活力、品質、與毒殺活性皆處於健康而良好有效的 狀態。 2. 本發明在NK細胞增殖過程中，應用rhIL-2、rhIL-12、 rhIL-15、rhIL-18、rhIL-21及其組合刺激增殖NK細胞，更有 201225959 效的保證了 NK細胞具有高的細胞毒殺傷活性。 3·本發明之增殖方法可以使得陬細胞在增殖後達到至少高於增 殖前細胞數目五百倍的細胞數量。 4·本發明在ΝΚ細胞增殖過程中，採用自體血清進行培養，使胍 細胞腫瘤治療方案更加安全。 本發明中顧不畴細胞培養基混合培養ΝΚ細胞方案，有 效降低了 ΝΚ細胞增殖之成本，也為ΝΚ、細胞在用於製備腫瘤疫苗 等醫療藥物組合時提供一種切實可行的方法。 【實施方式】 在以下内容中，發明人將對如何從;東存周邊血液單核球細胞 中啟動增殖ΝΚ細胞做詳盡說明。同時，發明人會進一步說明所啟 動增殖ΝΚ細胞的細胞毒殺傷活性。這些數據為腫瘤的胍細胞療法 提供了堅實的依據。 實施例一：請參照圖1(A)、圖1 (Β)、與圖1 (〇。 研究樣本來源： 周邊血液來自5名健康獻血者，獻血者經臨床診斷無血液系 統惡性腫瘤和嚴重自身免疫性疾病。本研究獲得當地研究倫理委 員會同意’同時與所有獻血者簽署知情書。 周邊血液單核細胞（PBMCs)的提取、冷凍保存及復甦： 所有操作均嚴格遵守標準操作規程（s〇p)的程式。採集20—40 毫升受試者周邊血液（約含3-6 XI〇7個單個核細胞）。經 Ficoll-Hypaque密度梯度離心分離單個核細胞，分離的單個核細 201225959 胞採用90%的自體血清和1〇%二甲基亞砜混合液進行冷凍保存。 冷滚保存細胞時’較佳之實施態樣為先將其置於攝氏4度2小 時，再放置於攝氏零下20度過夜，最後放置於含有液態氮的冷 凍保存裝置中長期保存，或者是先將含有9〇%的自體血清和1〇% 二甲基亞職以及周邊血液單核細胞（pBMCs)的混合液放置於攝 氏4度10分鐘，再放置於攝氏零下20度30分鐘，接著再放置 於攝氏零下8〇度過夜’最後再放置於含有液態氮的冷凍保存裝 置中長期保存。另一種合適的降溫方法為將含有90%的自體血清 和10%二曱基亞砜以及周邊血液單核細胞（PBMCs)的混合液放入 使用粒式控制的降溫機_進行降溫。冷凌保存細胞時，降溫的速 度較佳應當維持在每分鐘下降攝氏1至3度，以免降溫速度過快 導致細胞破裂，或者降溫速度過慢使細胞喪失活性。增殖周邊血 液單核細胞（PBMCs)時，取出冷凍細胞小管，投入攝氏37至4〇 度水浴中復溫，小管内容物全部融化後，吸出細胞以5倍體積的 無血清RPMI 1640培養基稀釋，以uoorpm /min的速度離心5 分鐘，丟棄上清液，洗滌兩次，再用無血清RPMI 164〇培養基重 新懸浮。 體外啟動增殖冷凍保存之周邊血液ΡΒΜ(：0ΝΚ細胞的方法： 將所得之PBMCs離心，重新懸浮於含5%人自體血清幹細胞培 養基中，以0.5xl06/ml細胞密度鋪入培養瓶中，加入重組人類白 血球間素2號（Recombinant human Interleukin 2，rhIL-2)， 最終〉辰度為500-750U/ml和良抗人CD3 mAb，10-20ng/ml ’培養5 201225959 天。第6天計數細胞，Hanks液洗滌1-2次後，加入含3-5%人自體 血清的幹細胞培養基，該些幹細胞培養基可含有rhIL-2 (最終濃 度為500U/ml)、rhIL-12 (最終濃度為200U/ml)、rhIL-15 (最終 濃度為20-30ng/ml)、rhIL-18 (最終濃度為l〇〇U/ml)、rhIL-21 (最終濃度為l〇〇U/ml )、及其組合，隨後重新懸浮細胞，使細胞 密度維持在0. 5xl06/ml左右。較佳的實施態樣為，在〇、lo-ii、 14-15天計數細胞、用流式細胞儀檢測細胞比例並詳細記錄細胞 增殖數目（圖2) ’根據細胞的增殖情況酌情補加含3-5%人自體血 清的幹細胞培養基’該些幹細胞培養基可為rhIL-2 (最終濃度為 500U/ml)、rhIL-12 (最終濃度為 200U/ml)、rhIL-15 (最終濃度 為 20-30ng/ ml)、rhIL-18 (最終濃度為 loou/ml)、rhiL-21 (最 終礙度為lOOU/ml )、及其組合，使細胞密度維持在〇. 5xi〇6/mi 左右。 以本發明之方法增殖而得之自然殺手細胞，可以連同適當體 積之該人類自體血清與適當體積之生理食鹽水，運用於製備腫瘤 疫苗等醫療藥物組合之範疇，例如利用靜脈注射的方式回輸至該 人類個體體内。 為了驗證NK細胞啟動增殖的效率，發明人等在5名獻血者 的冷凍保存之周邊血液PBMCs中，進行了 NK細胞的增殖實驗。 在5名健賴血麵PBMcs細胞巾，啟動增殖祕流式細胞儀分 析，確認CD3—CD56+亞群NK細胞為總細胞的9· 7%±2· 1%，⑽細胞， 主要是T細胞為總數的6綱％。在_ 15 *的啟動增殖後，細 201225959 胞總數增殖了 135±11倍（圖3)。而NK細胞的比例由增殖前的 9· 7%±2.1%增長為62°/〇±3. 6% (圖4)，由於ΝΚ細胞比例的增高， 發明人等計算得出ΝΚ細胞的增殖倍數為888±165 (圖5)倍。以 上結果均證實發明人等可有⑽财存周邊錢pBMCs啟動增殖 NK細胞，而且所增殖的NK細胞主要是CD3-CD56+亞群細胞。 根據體外增殖NK細胞的實際製作，在細胞培養第9_丨6天即 可透過靜脈注射，施打於個案身上，其中較佳的實施態樣為細胞 培養第10-11天與第M-15天，預計個案體内可看到陬細胞的 大量持續增殖。 實施例二· NK細胞毒殺活性檢測方法： 收集對數生長期K562細胞、培養第5、15天啟動增殖NK細 胞及體外利用細胞磁珠陽性分選的Νκ細胞。以K562細胞為靶細 胞，調整細胞濃度為lx 105/ml。以ΝΚ細胞為效應細胞，根據不 同的政乾比分別調整細胞濃度為lx 105/m卜1.5x 105/m卜lx 10/ml。按不同效靶比（1:1、5: 1、1〇: 1)將效應細胞和靶細 胞此合’同時設相應的靶細胞孔、效應細胞孔和培養基空白對照 孔°每組均設3個平行孔。置37¾、5% C02、飽和濕度培養箱 中培養12小時後，每孔加入l〇ul的Cell Counting kit-8 (日 本同仁化工）’混勻，於37°C、5% C02、飽和濕度培養箱中繼續 培養4小時後用酶標儀測定每孔450nm波長處0D值。細胞毒殺 活性計算方法：求出平行孔的平均值，按以下方法計算各組NK 細胞毒殺活性，以殺傷率％表示。 13 201225959 細胞毒殺活性=[1-(靶細胞孔〇D值-效應細胞孔〇D值）/ 靶細胞孔0D值]xlOO% 本發明人等為檢測比較所啟動增殖NK細胞的細胞毒殺傷活 性，以K562細胞為靶細胞，對比了體外利用細胞磁珠陽性分選 的NK細胞和應用本發明增殖5及15天的NK細胞的細胞毒殺傷 活性。在靶效比為k10時，經15天啟動增殖的NK細胞毒殺傷 活性為65%±10%，經5天啟動增殖的NK細胞毒殺傷活性為51%± 6. 3% ’而細胞磁珠陽性分選的NK細胞毒殺傷活性僅為9%± 3. 2%。在靶效比為1:5時，三者的細胞毒殺傷活性分別為51缸 8. 2%、39%±5.1%、7°/d2. 4%。在靶效比為1:1時’三者的細胞毒 殺傷活性分別為31%±5. 6%、22%±3. 7%、4%±1. 4%(圖6)。以上結 果揭示本發明所啟動增殖的NK細胞，對腫瘤細胞具有高的殺傷 活性。 上述實施例所揭示者係藉以具體說明本發明，且文中雖透過 特定的術語進行說明，當不能以此限定本發明之專利範圍；熟弗 此項技術領域之人士當可在瞭解本發明之精神與原則後對其進 行變更與修改而達到等效之目的，而此等變更與修改，皆應涵蓋 於如後所述之申請專利範圍所界定範疇中。 14 201225959 【圖式簡單說明】 圖1(A)為本發明方法較佳實施例之流程圖。 圖1(B)為本發明方法中，冷;東保存周邊血液單核球細胞(酬⑶ 的較佳實施例之流程圖。 圖1(c)為本發明方法中，於體外增殖周邊血液單核球細胞 (PBMCs)中的自然殺手細胞的較佳實施例之流程圖。 圖2為根據本發明之方法增殖NK細胞，第η和15天nk細胞比 例流式細胞儀檢測圖。 圖3為根據本發明之方法使用5名健康人周邊血液單核球細胞 (PBMCs)培養增殖之結果。 圖4為根據本發明之方法使用5名健康人之周邊血液單核球細胞 (PBMCs)培養增殖過程中NK細胞比例變化之結果。 圖5為根據本發明之方法使用5名健康人之周邊血液單核球細 胞(PBMCs)中NK細胞培養增殖之結果。 圖6為根據本發明之方法增殖NK細胞，以K562細胞為靶細胞， 進行細胞毒殺傷活性檢測之結果。 【主要元件符號說明】 七、申請專利範圍： 1. 一種人類自然殺手細胞(Natural killer cells, MK cel Is)之體外 增殖方法，其係一種自冷凍之周邊血液單核球細胞(Peripheral blood mononuclear cells，PBMCs)中增殖 CD3 CD56+亞型自然殺手細Interleukin, rhIL) 2 (rhIL-2), 12 (rhIL-12), 15 (rhIL-15), 201225959 18 (rhIL-18), and 21 (rhIL-21) and combinations thereof were used to activate the cells. Proliferation of NK cells means that NK cells undergo a series of cell divisions in vitro to achieve a certain number of proliferation. The NK cells proliferated in the present invention are mainly NK cells of the CD3CD56+ subgroup. Cytotoxic activity: refers to a simple cell killing event caused by cells. The killing activity of NK cells is not limited to tumor cells, but also includes killing effects on virus-infected cells, damaged and dysfunctional cells. The M cell 毋-activity involved in the present invention mainly refers to its killing effect on tumor cells. Nj (the determination of cytotoxic activity is mainly based on the use of blast-sensitive myeloma leukemia (ery thro 1 eukem ia) cell line K562 and NK cell-resistant lymphoid tumor cell line Raji as target cells, and measuring nk cells against them Killing rate. In the present invention, K562 cell line is used to determine the killing activity of NK cells. NK cell therapy: is a high-tech cell culture technique that can multiply in vitro in vitro (Ex vivo) to fight cancer cells. After sputum cells, it is injected into the patient to directly improve the patient's own anti-cancer ability, and achieve a medical technology that targets cancer and cancer. NJ (the premise of cell therapy is the initiation and mass proliferation of nk cells, and Tumor cells maintain high cytotoxic activity. Autoimmune cell therapy of autologous immune cells, especially NK cells, is simply a high-tech cell culture technique that multiplies in vitro (Εχviv〇) in a short period of time. The patient itself can fight the cancer cells' sputum cells and then inject them into the patient to directly improve the patient's own anti-cancer ability. The procedure is that after the patient has carefully evaluated and determined that it is suitable for NK cell therapy, about 3 ml of 201225959 peripheral blood is collected from the patient for 15 consecutive days in a GTP/GMP-compliant dust-free, sterile laboratory. Specially formulated to specifically culture and proliferate the sputum cells, increase the number by hundreds or even thousands of times, and then inject them into the body, directly to the number of NK cells to achieve the purpose of treating cancer. Therapy to treat cancer has the following various academic advantages: First, NK cells are the most potent anti-cancer cells in the human body, which can directly kill cancer cells to inhibit the growth and spread of tumors; Second, NK cells can inhibit new blood vessels near the tumor. Hyperplasia, it can limit the tumor to obtain the nutrients needed to limit the growth of the tumor; Third, NK cells can directly improve and regulate the patient's immunity and nervous system, indirectly improve the quality of life of patients; Fourth, Ν κ cells will secrete - species The brain called b_end〇_in can reduce the pain of the patient and make the patient feel happy. Ij has low or almost no side effects. At present, some studies have pointed out that the range of poisoning of natural and right-handed cells is not only melanoma, head and neck cancer, but also lung, nest, cervix and bladder. Many combined cancers of the lining gland, liver cells, pancreas, esophagus, and milky gland are especially effective for cancers of lung cancer, lymphoma, esophageal cancer, breast cancer, liver scale, and blood vessels in dense areas. According to our body, there will be 5 to 15 cancer cells that produce Fang Pa Temple. As long as one NK cell can kill these cancer cells, but the sputum is smashed into cancer, every cancer cell, 250 NK fine sputum in the body at that time died suddenly. Therefore, 'storage cells that have high fighting power before cancer can be diagnosed, 'the future will be identified', copied into a majority, and then injected into the body to help fight cancer, is the purpose珂 The most effective 'no side effect' auxiliary method. 201225959 The present invention provides a method for cell proliferation, which satisfies the conditions of NK cell therapy and provides a practical medical technique for tumor NK cell therapy. The present inventors have attempted to solve the problem of tumor cell therapy, and have found a method for starting and proliferating NK cells from frozen peripheral blood mononuclear cells. The augmented NK cells are (:1)3-(;:1)56+ subpopulations with high tumor cell cytotoxic activity. Cell culture conditions are detailed in the face towel. According to the results of the invention in the county, the peripheral blood mononuclear cells were cultured for 5 days in vitro, and the cells began to proliferate. The inventors called it the start-up phase. In the subsequent 1 day process, the inventors called it During the proliferative phase, NK cells will continue to proliferate from 1〇〇 to 5〇〇, or even 1〇〇〇. According to the results of the present invention, the cytotoxic activity of the activated and proliferated sputum cells was increased by at least 1% compared to the non-extended NK cells, and the better was increased by 150% to 200%. Increased female. The sputum cells which are not activated and propagated in vitro in the present invention are the (10) antibody magnetic beads, and the positively-selected neo-tactile cells. The increased cytotoxic activity of the priming and proliferating sputum cells may be due to an effect mediated by up-regulation of binding receptor expression. The foregoing specific methods for initiating and proliferating cells are described in the Examples of the present invention. What is needed is that in a culture system in which blood mononuclear cells are activated and proliferated in the peripheral blood cells, the peripheral money mononuclear cells are a mixture containing different cell populations, such as the C-D56-subgroup. ΝΚ cells, attached cells (CD3+CD56+) and sputum cells (10)+) ' So after the initiation of proliferation, in addition to a large number of colonization 0) 6+ Asian war cells, there are other cell types in the initiation of proliferation _ system . 201225959 According to the results of the present invention, the inventors have also elaborated a clinical application scheme of mouse cells. The inventors found that the most recorded state of disordered cells was on the 9th to 14th day of culture during the observation of the cell culture and its state. According to the cell-like sorrow, the inventors will change the serum-free medium on the 9th day and the 13th day, and change the solution after 48 hours. The state will be optimal after 24 hours, and the number of sputum cells will reach 10- Between 8 billion. Therefore, this is the best time to return the cells to the human body. According to the cytotoxic activity of NK cells against tumor cells in the present invention, the present invention encompasses the administration or application of sputum cells in combination with other cancer treatments. For example, in combination with surgery, radiation therapy, and cytotoxic drugs. At the same time, the results of the present invention suggest that proliferating sputum cells in vitro have a significant anti-tumor ability compared to sputum cells that are not activated in vivo. Therefore, the present invention also encompasses the killing effect of sputum cells on tumors. The said tumor cell refers to an autologous tumor cell. Autologous tumor cells refer to cells that are sourced and reimplanted in the same individual, that is, the donor and recipient are the same person. The above autologous tumor cells include hematological tumors and solid tumors. Compared with the method of the invention of the sputum cell proliferation invention, the present invention comprises the following distinct differences or advantages: 1. The present invention extracts peripheral blood of a healthy human individual, and extracts peripheral blood mononuclear cells (PBMCs) therein for sputum cells. The proliferation of NK cells ensures that the vitality, quality, and toxicity of NK cells are in a healthy and effective state. 2. The present invention stimulates the proliferation of NK cells by using rhIL-2, rhIL-12, rhIL-15, rhIL-18, rhIL-21 and combinations thereof in the process of NK cell proliferation, and the 201225959 effect ensures that NK cells are high. Cytotoxic activity. 3. The proliferation method of the present invention allows the sputum cells to reach a cell number at least five times higher than the number of pre-proliferation cells after proliferation. 4. The present invention is cultured in the process of proliferation of sputum cells using autologous serum to make the treatment of sputum cell tumors safer. In the present invention, the cell culture medium is mixed with the cultured sputum cell program, which effectively reduces the cost of sputum cell proliferation, and also provides a practical method for sputum and cells in the preparation of a medical drug combination such as a tumor vaccine. [Embodiment] In the following, the inventors will explain in detail how to initiate proliferative sputum cells from the peripheral blood mononuclear cells of Dongcun. At the same time, the inventors will further explain the cytotoxic activity of the proliferating sputum cells. These data provide a solid basis for tumor cell therapy. Example 1: Please refer to Figure 1 (A), Figure 1 (Β), and Figure 1 (〇. Study sample source: peripheral blood comes from 5 healthy blood donors, blood donors are clinically diagnosed without hematological malignancies and Severe autoimmune disease. This study was approved by the local research ethics committee. 'At the same time, all donors signed an informed letter. Peripheral blood mononuclear cells (PBMCs) extraction, cryopreservation and resuscitation: All operations are in strict compliance with standard operating procedures ( S〇p) program. Collect 20-40 ml of peripheral blood (about 3-6 XI〇7 mononuclear cells). Separate mononuclear cells by Ficoll-Hypaque density gradient centrifugation, separate single nuclei 201225959 The cells were cryopreserved with 90% autologous serum and 1% dimethyl sulfoxide mixture. The best way to preserve cells in cold-rolling is to place them at 4 degrees Celsius for 2 hours before placing them in Celsius. Leave at minus 20 degrees overnight, and finally store in a cryopreservation device containing liquid nitrogen for a long time, or first contain 9%% autologous serum and 1% dimethyl sub-sector and peripheral blood mononuclear cells (pBMCs). Mixed Place the solution at 4 degrees Celsius for 10 minutes, then place it at minus 20 degrees Celsius for 30 minutes, then place it at minus 8 degrees Celsius overnight. Finally, place it in a cryopreservation device containing liquid nitrogen for long-term storage. Another suitable cooling The method is to reduce the temperature by using a mixture of 90% autologous serum and 10% dimercaptosulfoxide and peripheral blood mononuclear cells (PBMCs) using a particle-controlled cooling machine. The speed should preferably be maintained at 1 to 3 degrees Celsius per minute, so as not to cause the cell to rupture if the temperature is too fast, or the cell to lose activity when the temperature is too slow. When the peripheral blood mononuclear cells (PBMCs) are proliferated, the frozen cell tubules are taken out. After rewarming in a 37 to 4 degree water bath, the contents of the small tube were completely thawed, and the cells were aspirated and diluted in 5 volumes of serum-free RPMI 1640 medium, centrifuged at uoorpm /min for 5 minutes, and the supernatant was discarded and washed. Twice, and then resuspended in serum-free RPMI 164 〇 medium. In vitro initiation of proliferation and cryopreservation of peripheral blood sputum (: 0 ΝΚ cell method: the resulting P The BMCs were centrifuged, resuspended in 5% human autologous serum stem cell culture medium, and placed in a culture flask at a cell density of 0.5×10 6 /ml. Recombinant human Interleukin 2 (rhIL-2) was added. 〉Temperature is 500-750U/ml and good anti-human CD3 mAb, 10-20ng/ml 'culture 5 201225959 days. Count the cells on the 6th day, Hanks liquid wash 1-2 times, add 3-5% human self Serum stem cell culture medium containing rhIL-2 (final concentration 500 U/ml), rhIL-12 (final concentration 200 U/ml), rhIL-15 (final concentration 20-30 ng/ml), rhIL 5xl06/毫升左右。 -18 (final concentration of l〇〇U / ml), rhIL-21 (final concentration of l〇〇U / ml), and a combination thereof, and then resuspended cells, so that the cell density is maintained at about 0. 5xl06 / ml. In a preferred embodiment, the cells are counted in sputum, lo-ii, 14-15 days, the proportion of cells is detected by flow cytometry, and the number of cell proliferation is recorded in detail (Fig. 2) 'According to the proliferation of the cells, the content is added as appropriate. 3-5% human autologous serum stem cell medium's stem cell medium can be rhIL-2 (final concentration 500U/ml), rhIL-12 (final concentration 200U/ml), rhIL-15 (final concentration 20 -30 ng/ml), rhIL-18 (final concentration is loou/ml), rhiL-21 (final incompatibility is lOOU/ml), and combinations thereof, so that the cell density is maintained at about 5 xi 〇 6 / mi. The natural killer cells obtained by the method of the present invention can be used together with an appropriate volume of the human autologous serum and an appropriate volume of physiological saline to prepare a medical drug combination such as a tumor vaccine, for example, by intravenous injection. Lost into the human body. In order to verify the efficiency of NK cell initiation of proliferation, the inventors performed proliferation experiments of NK cells in peripheral blood PBMCs of five blood donors. In 5 healthy sputum blood surface PBMcs cell washes, the proliferating secret flow cytometry analysis was started, and it was confirmed that the CD3-CD56+ subgroup NK cells were 9.7%±2.1% of the total cells, and (10) cells, mainly T cells. 6% of the total. After the initiation of _ 15 * proliferation, the total number of cells in 201225959 increased by 135 ± 11 times (Fig. 3). The proportion of NK cells increased from 9.7%±2.1% before proliferation to 62°/〇±3.6% (Fig. 4). Due to the increase in the proportion of sputum cells, the inventors calculated the proliferation factor of sputum cells. It is 888±165 (Figure 5) times. The above results confirmed that the inventors and the like may have (10) the peripheral money pBMCs to initiate the proliferation of NK cells, and the proliferated NK cells are mainly CD3-CD56+ subpopulation cells. According to the actual production of NK cells in vitro, the cells can be administered intravenously on the 9th to 6th day of cell culture, and the preferred embodiment is cell culture day 10-11 and M-15. In the day, it is expected that a large number of sputum cells will continue to proliferate in the case. Example 2: Detection method of NK cell toxicity activity: K562 cells in logarithmic growth phase, NK cells activated on the 5th and 15th day of culture, and Νκ cells positively sorted by magnetic beads in vitro were collected. K562 cells were used as the target cells, and the cell concentration was adjusted to be lx 105/ml. Using sputum cells as effector cells, the cell concentration was adjusted to be lx 105/m b 1.5x 105/m b lx 10/ml according to different political-to-dry ratios. The effector cells and the target cells are combined according to different target ratios (1:1, 5:1, 1〇: 1). At the same time, corresponding target cell wells, effector cell wells and medium blank control wells are set. Parallel holes. After culturing for 12 hours in a 373⁄4, 5% C02, and saturated humidity incubator, add l〇ul of Cell Counting kit-8 (Japan Tongren Chemical) to each well and mix at 37 ° C, 5% CO 2 , and saturated humidity. After continuing to culture for 4 hours in the chamber, the 0D value at a wavelength of 450 nm per well was measured with a microplate reader. Calculation method of cytotoxic activity: The average value of parallel wells was determined, and the cytotoxic activity of each group was calculated by the following method, and the killing rate was expressed by %. 13 201225959 Cell cytotoxic activity = [1 - (target cell 〇 D value - effector cell 〇 D value) / target cell hole 0D value] x lOO% The present inventors examined and compared the cytotoxic activity of the activated proliferating NK cells. K562 cells were used as target cells, and NK cells positively sorted by cell magnetic beads in vitro and cytotoxic activity of NK cells proliferating for 5 and 15 days of the present invention were compared. When the target-effect ratio is k10, the NK cell cytotoxic activity that initiated proliferation in 15 days was 65% ± 10%, and the NK cell cytotoxic activity that initiated proliferation in 5 days was 51% ± 6.3% ' while the cell beads The positively sorted NK cell cytotoxic activity was only 9% ± 3.2%. At a target-to-effect ratio of 1:5, the cytotoxic activity of the three were 51 cylinders, 8. 2%, 39% ± 5.1%, and 7°/d2. 4%, respectively. The cytotoxic activity of the cytotoxicity was 31% ± 5.6%, 22% ± 3.7%, 4% ± 1.4% (Fig. 6), respectively. The above results revealed that the proliferating NK cells of the present invention have high killing activity against tumor cells. The above embodiments are intended to be illustrative of the present invention, and the specific scope of the present invention is not to be construed as limiting the scope of the invention; those skilled in the art can understand the spirit of the invention. Changes and modifications are made to the equivalent of the principles, and such changes and modifications are to be included in the scope of the patent application as described later. 14 201225959 [Simple Description of the Drawings] Figure 1 (A) is a flow chart of a preferred embodiment of the method of the present invention. Fig. 1(B) is a flow chart showing a preferred embodiment of cryopreservation of peripheral blood mononuclear cells in the method of the present invention. Fig. 1(c) is a method for proliferating peripheral blood in vitro in the method of the present invention. A flow chart of a preferred embodiment of natural killer cells in nucleated cells (PBMCs). Figure 2 is a flow cytometric test for the proliferation of NK cells, η and 15 days nk cells according to the method of the present invention. The results of the proliferation of 5 healthy human peripheral blood mononuclear cells (PBMCs) were cultured according to the method of the present invention. Fig. 4 is a diagram showing the proliferation process of peripheral blood mononuclear cells (PBMCs) of five healthy persons according to the method of the present invention. Results of changes in the proportion of NK cells in the cell. Figure 5 shows the results of NK cell culture proliferation in peripheral blood mononuclear cells (PBMCs) of five healthy individuals according to the method of the present invention. Figure 6 is a method for proliferating NK cells according to the method of the present invention. K562 cells are used as target cells for the detection of cytotoxic activity. [Key device symbol description] 7. Patent application scope: 1. An in vitro proliferation of human killer cells (MK cel Is) , Which is a self-refrigerating system of peripheral blood mononuclear cells balls (Peripheral blood mononuclear cells, PBMCs) proliferating CD3 CD56 + natural killer fine subtype