自然殺傷細胞為何極端重要?

［譯者按：本文基於今年年初生物醫學雜誌編輯關於CAR-NK細胞療法的述評編譯而成，希望對免疫治療有興趣的廣大朋友和（或）腫瘤患者有所幫助，不過需要你們耐心點看哈］

1 近10餘年來，免疫治療惡性腫瘤的快速進展，如抗-PD-1，抗PD-L1和抗CTLA-4等已經被證實可以治癒某些以往根本無法治癒晚期惡性腫瘤。

2 2017年美國食品藥品管理局（FDA）批准了第一個基於細胞的免疫療法 嵌合抗原受體（CAR）-T細胞療法，用於治療難治性B細胞急性淋巴細胞白血病；2018年，FDA又批准了第二個可用於治療B細胞非霍奇金淋巴瘤的CAR-T細胞免疫療法。

3 2018年，諾貝爾評獎委員會將當年的諾貝爾生理或醫學獎頒發給了兩位免疫學家 James P Allison and Tasuku Honjo。

4 毫無疑問，前面提到的FDA批准的兩種免疫療法都已經證明了它們在對抗癌症方面的重要性，但需要注意的是兩種方法都需要有足夠的功能正常的T細胞。當治療不含T細胞的所謂「冷腫瘤（cold tumours）」時，或當患者的T細胞幾乎完全因「一線治療（指化或放療）」而消失時，CAR-T治療可能就會因為Ｔ-細胞不足而面臨挑戰。設計捐贈的T細胞並擴增它們以獲得足夠的CAR-T細胞治療是非常耗時的，加之目前可用的CAR-T治療價格也是非常昂貴的。此外，CAR-T療法作為實體瘤（相對於「血液腫瘤「的腫瘤，如肝癌）的治療方法尚未完成成熟，也即仍然有一些工作有待完善。所有這些挑戰都凸顯了尋求其他免疫治療方案的必要性，最近的研究進展表明自然殺傷（NK）細胞治療是最有希望之星。

5 NK細胞是一種人體獲得性免疫必不可少的淋巴細胞之一，它們不需要抗體和主要組織相容性複合物（MHC），就能夠識別「非自身」細胞（或稱異體/外源細胞），從而啟動快速免疫反應，廣泛的細胞毒性和快速的殺滅能力使得NK細胞可望成為癌症免疫療法的理想選擇。

6 事實上，早在CAR-T問世之前，研究人員就試圖利用NK細胞來對抗癌症，這些嘗試可以追溯到20世紀80年代後期的臨床研究，但由於技術、後勤和財務等方面的限制，科學家們當時排除了人體血液中原本存在的NK細胞作為令人興奮和有希望的癌症治療手段。過去10多年時間以來，該領域已經見證了許多重要的發展。臨床前和臨床研究已充分證明了同種異體NK細胞對各種血液惡性腫瘤和實體瘤的安全性和有效性，其中有些研究目前還在進行臨床試驗中。

7 CAR-T細胞治療腫瘤的巨大成功引發了用CAR改良NK細胞的熱情，以提高其腫瘤殺傷能力。與CAR-T細胞療法相比，CAR-NK細胞療法具有幾個特別突出的優點。首先，與CAR-T細胞不同，CAR-NK細胞保留了通過其天然受體識別和靶向腫瘤細胞的固有能力，使得通過CAR靶抗原的下調不太可能使腫瘤細胞能夠逃逸CAR-NK細胞療法的攻擊；其次，CAR-NK細胞在數天至數周內不會發生克隆擴增或免疫排斥，因此它們不會出現相同的安全性問題，例如在許多CAR-T臨床試驗中觀察到的細胞因子釋放症候群（俗稱「細胞因子風暴「）在CAR-NK治療過程幾乎不存在；最後，NK細胞不需要嚴格的HLA匹配，並且缺乏引起移植物抗宿主病（GVHD）的可能性，GVHD是CAR-T細胞免疫療法所面臨的重大風險。CAR-NK細胞療法的這些特點，使得它能夠從實驗室走向臨床，最終成為同種異體的有效細胞免疫療法。

8 原代NK細胞難以分離，純化和轉導，通常產生擴增不良的異質細胞群。然而，NK細胞系NK-92可在體外容易且無限地擴增，並已在臨床中使用，這使其成為產生CAR-NK-92細胞的重要可再生資源。由於通常關於永生細胞系的關注，例如染色體異常和惡性轉化的風險，NK-92需要在輸注到患者之前進行照射，這可以抑制NK-92細胞的增殖，同時保持其完全細胞毒活性。誘導的多能幹細胞（iPSC）可以提供另一種可再生的和可能更好的NK細胞資源。最近在加利福尼亞大學聖地牙哥分校Dan Kaufman小組於2018年6月28日在Cell Stem Cell上發表的一項臨床前研究探索了這種可能性。研究人員在人類iPSCs中表達了一種優化的NK特異性CAR構建體，並將這些基因修飾的iPSC分化為功能性NK細胞。他們證明了這些NK-CAR-iPSC-NK細胞在卵巢癌異種移植小鼠模型中顯著抑制腫瘤生長。更重要的是，研究人員比較了iPSC-NK細胞與CAR-T細胞的體內抗腫瘤效力，並發現儘管兩種方法都達到了類似的腫瘤殺傷效力，但NK-CAR-iPSC-NK細胞處理的小鼠表現出顯著更長的存活率。與CAR-T治療的小鼠相比，沒有出現體重減輕，器官病理學改變或細胞因子水平增加，這表明CAR-NK治療可能比目前的CAR-T治療更安全。這種差異可能使治療患有多劑量CAR-NK細胞的患者變得可行，這可能導致比單劑量更好的臨床治療效果，由於用於CAR-T細胞療法的細胞的有限性和高成本。

9 儘管CAR-NK療法在癌症患者中的安全性和有效性需要在更多的臨床試驗中進一步檢測，但令人興奮的是，我們現在正在見證一種新的CAR-NK細胞療法，正在快速趕上CAR-T細胞療法。CAR-NK似乎不太可能取代CAR-T，但它可能是基於細胞的免疫療法的基本補充。雖然現在想像同時用CAR-NK和CAR-T細胞進行聯合治療還為時尚早，但最近發表於臨床研究雜誌（2018年9月10日）和細胞（2018年11月29日）的研究表明，NK細胞可能會發揮作用。在PD-1 / PD-L1阻斷免疫療法中的重要作用以及釋放NK細胞和T細胞同時增強抗腫瘤活性。

Natural killer cells for cancer immunotherapy: a new CAR is catching up （EBioMedicine. 2019 Jan; 39: 1–2.）

Immunotherapy has revolutionised cancer treatment. Since the approval of ipilimumab (an anti-cytotoxic T lymphocyte antigen [CTLA]-4 monoclonal antibody) in 2011 for the treatment of patients with malignant melanoma, immune checkpoint inhibitors such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 have demonstrated their power in the clinic to treat previously untreatable terminal tumours. These success stories finally led to James P Allison and Tasuku Honjo winning the Nobel Prize in Physiology or Medicine in 2018. The US Food and Drug Administration (FDA) approved the first cell-based immunotherapy, chimeric antigen receptor (CAR)-T cell therapy, for the treatment of refractory B-cell acute lymphoblastic leukaemia in 2017, and the second one for the treatment of B-cell non-Hodgkin lymphoma in 2018.

Undoubtedly both immunotherapy approaches have proved their own importance in fighting cancer, with a caveat: both require sufficient functional, primary T cells. This requirement can be challenging when treating so-called 「cold tumours」 that do not contain T cells to be unleashed, or when a patient’s T cells are almost entirely wiped out from a first-line therapy. It can be time-consuming to engineer donated T cells and expand them to obtain enough CAR-T cells, and currently available CAR-T treatments are expensive. Moreover, CAR-T therapy still has some way to go as a therapy for solid tumours. All these challenges highlight the need to seek other immunotherapy options, and recent developments have shown natural killer (NK) cell therapy as one of the most promising.

NK cells are a type of lymphoid cell essential for the innate immune system. They recognise 「non-self」 cells without the need for antibodies and major histocompatibility complex (MHC), executing a rapid immune reaction. The broad cytotoxicity and rapid killing make NK cells ideal for the use in cancer immunotherapy. Indeed, long before the era of CAR-T, researchers had attempted to harness NK cells to fight cancers. These attempts can be tracked back to clinical studies in the late 1980s, but technical, logistical and financial challenges excluded the application of blood NK cells as an exciting and promising cancer therapy at the time. Over the past decade, the field has witnessed numerous important developments. Pre-clinical and clinical studies have demonstrated the safety and efficacy of allogeneic NK cells against various hematological malignancies and solid tumours and several clinical trials are currently ongoing.

The huge success of CAR-T cells generated enthusiasm to genetically modify NK cells with CARs to sharpen their tumour-killing capacity. CAR-NK cells have several advantages over CAR-T cells. First, unlike CAR-T cells, CAR-NK cells retain an intrinsic capacity to recognise and target tumour cells through their native receptors, making the escaping of tumour cells through downregulation of the CAR target antigen less likely. Second, CAR-NK cells do not undergo clonal expansion or immune rejection within days to weeks, and thus they do not present the same safety concerns, such as cytokine release syndrome, observed in many CAR-T clinical trials. Lastly, NK cells do not require strict HLA matching and lack the potential to cause graft-versus-host disease, an important risk imposed by CAR-T cell immunotherapy, which make it possible for CAR-NK cells to be an off-the-shelf allogeneic therapeutic.

Primary NK cells are difficult to isolate, purify, and transduce, often producing a heterogeneous cell population that expands poorly. However, the NK cell line NK-92 can expand easily and indefinitely in vitro and has been used in the clinic, which makes it a great renewable resource to generate CAR-NK-92 cells. Due to the usual concerns regarding immortal cell lines, such as chromosomal abnormalities and the risk of malignant transformation, NK-92 requires irradiation before infusion into patients, which can suppress proliferation of NK-92 cells while maintaining their full cytotoxic activity. Induced pluripotent stem cells (iPSCs) can offer another renewable and potentially better resources of NK cells. A recent pre-clinical study published in Cell Stem Cell on June 28, 2018, from Dan Kaufman’s group at University of California, San Diego (USA) explored this possibility. The researchers expressed an optimised, NK-specific CAR construct in human iPSCs and differentiated these genetically modified iPSCs into functional NK cells. They were able to show that these NK-CAR-iPSC-NK cells significantly inhibited tumour growth in an ovarian cancer xenograft mouse model. More importantly, the authors compared in vivo antitumour efficacy between iPSC-NK cells with CAR-T cells and found that although both approaches achieved similar tumour-killing efficacy, NK-CAR-iPSC-NK cell-treated mice exhibited significantly longer survival and did not suffer from weight loss, organ pathology, or increased cytokine levels compared with CAR-T treated mice, indicating that CAR-NK therapy might be a safer option than current CAR-T therapy. This difference would probably make it feasible to treat patients with multiple doses of CAR-NK cells, which might lead to better clinical outcomes than with a single dose, which is used for CAR-T cell therapy owing to limited availability of cells and high cost.

While CAR-T cells have produced exciting clinical results, studies using CAR-NK cells have been largely pre-clinical until very recently. Right now, there are more than a dozen clinical trials registered on clinicaltrials.gov to test CAR-NK cell therapy in both haematological and solid tumours, including glioblastoma, prostate cancer, and ovarian cancer. China launched several clinical trials targeting multiple tumours in 2016, and results from one phase 1 clinical trial (published in the American Journal of Cancer Research on June 1, 2018) demonstrated the safety of CD33-CAR-NK-92 cells in patients with relapsed and refractory acute myeloid leukaemia. A European trial testing HER2-specific CAR-NK-92 cells in glioblastoma patients was launched last year, with results expected in the next 2 years. Although both the safety and efficacy of CAR-NK therapy in patients with cancer need to be further tested in more clinical trials, it is exciting that we are now witnessing a new CAR, with NK cells behind the wheel, that is rapidly catching up with CAR-T cells. It seems unlikely that CAR-NK would replace CAR-T, but it could be an addition to the armamentarium of cell-based immunotherapy. Although it is early to imagine a combinational treatment with CAR-NK and CAR-T cells, recent studies published in the Journal of Clinical Investigation (Sept 10, 2018) and Cell (Nov 29, 2018) have indicated that NK cells may play an important role in PD-1/PD-L1 blockade immunotherapy and that unleashing both NK cells and T cells simultaneously enhances anti-tumour activity.

Standing at the beginning of 2019, we are enthusiastic. We expect exciting news from CAR-NK therapy clinical trials. We look forward to embracing CAR-NK cells to join our continuing war against cancer in the coming years.

EBioMedicine