一、Challenges of Using Blood NK Cells as Cellular Effectors in Cancer Treatment（使用血液NK細(xì)胞作為癌癥治療的效應(yīng)細(xì)胞的挑戰(zhàn)）
Over the last few decades, there has been significant advancement of our knowledge concerning the biology of NK cells, which comprise about 10% of the circulating blood lymphocyte pool. A more comprehensive characterization, including gene and surface molecule expression, as well as proteomics, recently has become available [2 4]. It is now well accepted that NK cells are an important component of the rapid cellular immune response that can inhibit the cell-to-cell spread of malignant or infected cells. Not only can they execute immediate (“spontaneous”) killing upon contact with those cells (without the need to be “primed” like T-cells), but they also produce a host of cytokines that support the expansion and function of other immune cells in the blood circulation and the tumor microenvironment.
在過去的幾十年里，我們對NK細(xì)胞生物學(xué)的認(rèn)識取得了顯著進(jìn)展，NK細(xì)胞約占循環(huán)血液淋巴細(xì)胞池的10%。最近，包括基因和表面分子表達(dá)以及蛋白質(zhì)組學(xué)在內(nèi)的更全面的表征已經(jīng)可用。現(xiàn)在普遍認(rèn)為，NK細(xì)胞是快速細(xì)胞免疫反應(yīng)的重要組成部分，能夠抑制惡性或感染細(xì)胞的細(xì)胞間傳播。它們不僅可以在與這些細(xì)胞接觸時(shí)立即（“自發(fā)地”）進(jìn)行殺傷（無需像T細(xì)胞那樣“啟動(dòng)”），還可以產(chǎn)生一系列細(xì)胞因子，支持血液循環(huán)和腫瘤微環(huán)境中其他免疫細(xì)胞的擴(kuò)增和功能。
Through their FcgIII (CD16) receptor, NK cells are major effector cells of ADCC, which is the main effector arm for immunoglobulin G (IgG) 1 and IgG3 monoclonal antibodies (mAbs). Notably, NK cells that express the high-affinity (158V) variant of the CD16 receptor are more effective for mAb-mediated targeted cell killing, although only approximately 10% of the human population is homozygous for this variant [5].
通過其FcγIII（CD16）受體，NK細(xì)胞是ADCC的主要效應(yīng)細(xì)胞，這是免疫球蛋白G（IgG）1和IgG3單克隆抗體（mAbs）的主要效應(yīng)臂。值得注意的是，表達(dá)高親和力（158V）CD16受體變異體的NK細(xì)胞在mAb介導(dǎo)的靶向細(xì)胞殺傷中更為有效，盡管人類人群中只有大約10%的人是這種變異體的純合子。
The effector molecules that execute rapid NK cell mediated target cell killing are perforin and granzymes and to a lesser extent FasL and tumor necrosis factor related apoptosis inducing ligand (TRAIL) [6]. The former are released on contact with transformed cells and act within minutes, with perforin “punching holes” into the membrane of target cells so that granzymes can enter the cell and destroy it by degrading their genetic material. FasL and tumor necrosis factor related apoptosis inducing ligand seem to have a more delayed cytotoxic effect through activation of the death receptor pathways on target cells [6].
執(zhí)行快速NK細(xì)胞介導(dǎo)的靶細(xì)胞殺傷的效應(yīng)分子是穿孔素和顆粒酶，以及在較小程度上的FasL和腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體（TRAIL）。前者在與目標(biāo)細(xì)胞接觸時(shí)釋放，并在幾分鐘內(nèi)起作用，穿孔素在靶細(xì)胞膜上“打孔”，使顆粒酶能夠進(jìn)入細(xì)胞并通過降解其遺傳物質(zhì)來破壞細(xì)胞。FasL和腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體似乎通過激活靶細(xì)胞上的死亡受體途徑產(chǎn)生更延遲的細(xì)胞毒性效應(yīng)。
Enriching NK cells from blood (peripheral or cord) either for research or clinical applications usually is done by negative selection employing a number of monoclonal antibodies that can deplete and eliminate non-NK cells (reviewed in Kundo et al. [7]). NK cells also may be enriched from blood, based on their characteristic CD56 (neuronal cell adhesion molecule) surface expression. One of the concerns is, however, that the bound antibody may affect the NK cell function and/or can interfere with any further manipulation (engineering) of the cells. Another limitation of such enrichment is the unpredictable variability of yield, particularly for NK cells from patients with cancer whose NK cells often are also dysfunctional due to circulating suppressive serum factors or following chemotherapy. A further challenge is that the NK-cell donors must be connected to a leukapheresis device for several hours to collect their white blood cells, of which only about 10% are NK cells. To achieve sufficient NK cells for therapeutic infusion, NK cells need to be expanded on a cell line feeder layer, which may be genetically altered. In addition, cytokines need to be included to support expansion (reviewed in Saito et al. [8]).
從血液（外周或臍帶）中富集NK細(xì)胞用于研究或臨床應(yīng)用，通常通過負(fù)選擇進(jìn)行，使用多種單克隆抗體耗盡和消除非NK細(xì)胞。NK細(xì)胞也可以根據(jù)其特征性的CD56（神經(jīng)細(xì)胞黏附分子）表面表達(dá)從血液中富集。然而，其中一個(gè)問題是，結(jié)合的抗體可能會(huì)影響NK細(xì)胞的功能和/或干擾細(xì)胞的任何進(jìn)一步操作（工程改造）。這種富集的另一個(gè)限制是產(chǎn)量的不可預(yù)測性，特別是對于癌癥患者的NK細(xì)胞，因?yàn)檠h(huán)抑制血清因子或化療后，其NK細(xì)胞通常也會(huì)功能失調(diào)。進(jìn)一步的挑戰(zhàn)是，NK細(xì)胞供體必須連接到白細(xì)胞分離裝置數(shù)小時(shí)，以收集其白細(xì)胞，其中只有大約10%是NK細(xì)胞。為了獲得足夠數(shù)量的NK細(xì)胞用于治療輸注，需要在細(xì)胞系飼養(yǎng)層上擴(kuò)增NK細(xì)胞，該飼養(yǎng)層可能經(jīng)過基因改造。此外，還需要加入細(xì)胞因子以支持?jǐn)U增。
As an alternative to using a patient’s own (autologous) NK cells, allogeneic NK cells can be collected from the blood of a healthy related or unrelated person. Those allogeneic NK-cell collections need to be further treated to remove T lymphocytes, as those can cause the potentially life-threatening graft-versus-host reactions after infusion. This additional purification step adds to the cost of the cell preparation and also results in a further loss of NK cells. Regardless of the source, blood NK cells need to be expanded to have suffi cient numbers of cells available for therapeutic infusion [9]. Another challenge with blood-derived NK cells is the fact that those NK cells are more difficult to manipulate or engineer, requiring more complex, often virus-based gene transduction methods, which come with unpredictable and inconsistent efficacy [8].
作為使用患者自身（自體）NK細(xì)胞的替代方法，可以從健康相關(guān)或無關(guān)人員的血液中收集異體NK細(xì)胞。這些異體NK細(xì)胞收集后需要進(jìn)一步處理以去除T淋巴細(xì)胞，因?yàn)檫@些細(xì)胞在輸注后可能會(huì)導(dǎo)致潛在致命的移植物抗宿主反應(yīng)。這個(gè)額外的純化步驟增加了細(xì)胞制備的成本，并且也導(dǎo)致NK細(xì)胞的進(jìn)一步損失。無論來源如何，血液NK細(xì)胞都需要擴(kuò)增，以獲得足夠數(shù)量的細(xì)胞用于治療輸注。血液來源NK細(xì)胞的另一個(gè)挑戰(zhàn)是，這些NK細(xì)胞更難以操作或工程改造，需要更復(fù)雜、通?；诓《镜幕蜣D(zhuǎn)導(dǎo)方法，這些方法的效果難以預(yù)測且不一致。
Considering those drawbacks of blood-derived NK cells, including the costs of obtaining a consistent NK-cell population from blood for research and clinical applications, a continuously growing NK cell line is an appealing alternative. Although several human NK cell lines have been established [10], only the NK-92 cell line has shown consistently high cytotoxicity against a broad spectrum of cancer cells, and can easily be expanded ex vivo with a short doubling time of 24 36 h [11,12]. Importantly, NK-92 can be genetically engineered by plasmid electroporation to express a high-affinity Fc-receptor, CAR, or molecules that can modulate the tumor microenvironment [13,14].
鑒于血液來源NK細(xì)胞的這些缺點(diǎn)，包括從血液中獲得一致的NK細(xì)胞群用于研究和臨床應(yīng)用的成本，持續(xù)生長的NK細(xì)胞系是一個(gè)有吸引力的替代選擇。盡管已經(jīng)建立了幾種人類NK細(xì)胞系，但只有NK-92細(xì)胞系顯示出對廣泛癌癥細(xì)胞的一致高細(xì)胞毒性，并且可以在體外輕松擴(kuò)增，其倍增時(shí)間為24-36小時(shí)。重要的是，NK-92可以通過質(zhì)粒電穿孔進(jìn)行基因工程改造，以表達(dá)高親和力Fc受體、CAR或可以調(diào)節(jié)腫瘤微環(huán)境的分子。
二、Early Clinical Studies with NK-92 （NK-92的早期臨床研究）
In 1997, the license and rights for NK-92 were transferred from the University of British Columbia to Rush Medical Center in Chicago, where in 2001 a phase I trial with NK-92 in patients with advanced cancer was conducted [31]. Due to the responses seen with LAK cell infusions in patients with renal cell cancer and melanoma [32], those two diseases were selected as initial indications for NK-92 infusions.
1997年，NK-92的許可和權(quán)利從不列顛哥倫比亞大學(xué)轉(zhuǎn)移到芝加哥的拉什醫(yī)學(xué)中心，2001年在那里進(jìn)行了NK-92在晚期癌癥患者中的I期試驗(yàn)。由于在腎細(xì)胞癌和黑色素瘤患者中使用LAK細(xì)胞輸注看到了反應(yīng)，這兩種疾病被選為NK-92輸注的最初適應(yīng)癥。
In that study, NK-92 cells were infused on days 1, 3 and 5 with the rationale that it usually takes a few days to trigger a T-cell mediated allogeneic immune response that could potentially lead to rejection of the infused NK-92 cells. In this phase I dose-escalation study, 12 patients were enrolled and received a starting dose of 1 x109 NK-92 cells/m2 . The greatest dose administered was 5 x 109 NK-92 cells/m2 cells. None of the patients experienced grade 3 or 4 side effects, and only a few patients developed a mild fever or a rash. Although phase I studies are not designed to assess efficacy, it is noteworthy that the majority of the patients with renal cell cancer experienced a prolonged disease-free survival and overall survival compared with historical controls. The single patient with melanoma in that study had a partial response with significant tumor reduction after the third infusion. In contrast to CAR/T-cell therapy that generally includes some form of immunosuppressive chemotherapy before infusion, NK-92 cells in those and all other studies (including with CAR modified NK-92) were given without any preparative chemotherapy.
在該研究中，NK-92細(xì)胞在第1、3和5天進(jìn)行輸注，理由是通常需要幾天時(shí)間才能觸發(fā)T細(xì)胞介導(dǎo)的異體免疫反應(yīng)，這可能會(huì)導(dǎo)致輸注的NK-92細(xì)胞被排斥。在這項(xiàng)I期劑量遞增研究中，共納入了12名患者，起始劑量為每平方米1×10^9個(gè)NK-92細(xì)胞。給予的最大劑量為每平方米5×10^9個(gè)NK-92細(xì)胞。沒有患者出現(xiàn)3級或4級副作用，只有少數(shù)患者出現(xiàn)輕度發(fā)熱或皮疹。盡管I期研究并非旨在評估療效，但值得注意的是，大多數(shù)腎細(xì)胞癌患者的無病生存期和總生存期比歷史對照組有所延長。該研究中唯一的黑色素瘤患者在第三次輸注后出現(xiàn)了部分反應(yīng)，腫瘤顯著縮小。與通常在輸注前進(jìn)行某種形式免疫抑制化療的CAR/T細(xì)胞療法不同，NK-92細(xì)胞在這些以及所有其他研究（包括CAR修飾的NK-92）中都是在沒有任何準(zhǔn)備性化療的情況下給予的。
Contemporaneous with the aforementioned study, Dr. TorstenTonn, who had previously performed post-doctoral work in the author’s laboratory, initiated a phase I trial in Frankfurt, Germany, in pediatric and adult patients with advanced, mostly solid cancers [26]. The schedule consisted of two infusions, given on day 1 and 3. The dose of 1 x 1010 cells/m2 was considered dose-limiting, but not because of side effects, but rather because of logistical challenges of expanding larger numbers of NK-92 cells at that time.
與此同時(shí)，托爾斯滕·托恩博士（Torsten Tonn）在德國法蘭克福啟動(dòng)了一項(xiàng)I期試驗(yàn)，研究對象為患有晚期癌癥（主要是實(shí)體瘤）的兒童和成人患者。該試驗(yàn)的安排是在第1天和第3天進(jìn)行兩次輸注。每平方米1×10^10個(gè)細(xì)胞的劑量被認(rèn)為是劑量限制性的，但這并非由于副作用，而是因?yàn)楫?dāng)時(shí)擴(kuò)大NK-92細(xì)胞數(shù)量存在物流挑戰(zhàn)。
Another phase I trial was conducted under the guidance of Dr. Armand Keating, at Ontario Cancer Center in Toronto [15]. Among the 12 patients with advanced hematologic malignancies enrolled in the study, two patients (with myeloma and Hodgkin disease) experienced long-lasting clinical remissions. Patients received multiple infusions over time (planned six monthly cycles). Only one-half of the patients developed HLA antibodies, and none had a positive mixed lymphocyte culture when NK-92 cells were used as stimulators.
在多倫多安大略癌癥中心，阿曼德·基廷博士（Armand Keating）指導(dǎo)進(jìn)行了另一項(xiàng)I期試驗(yàn)。在該研究中，共有12名患有晚期血液系統(tǒng)惡性腫瘤的患者接受治療，其中兩名患者（分別患有骨髓瘤和霍奇金?。┙?jīng)歷了長期的臨床緩解。患者接受了多次輸注（計(jì)劃進(jìn)行六次每月）。只有一半的患者產(chǎn)生了HLA抗體，沒有任何患者的混合淋巴細(xì)胞培養(yǎng)物呈陽性反應(yīng)。
In another phase I trial conducted at the University of Pittsburgh, seven patients with treatment-resistant acute myeloid leukemia were enrolled and received NK-92 [33]. Although none of thepatients experienced serious adverse effects, no significant clinical responses were noted, prompting the group to investigate possible reasons for the lack of response. In a subsequent publication, they reported that acute myeloid leukemia cell derived exosomes collected pre-therapy from all seven patients had the ability to inhibit the cytotoxic anti-leukemia effects of NK-92 in co-incubation assays [34].
在匹茲堡大學(xué)進(jìn)行的另一項(xiàng)I期試驗(yàn)中，共有7名治療抵抗性的急性髓系白血病患者接受NK-92治療。盡管沒有患者出現(xiàn)嚴(yán)重的不良反應(yīng)，但也沒有觀察到顯著的臨床反應(yīng)，這促使該團(tuán)隊(duì)調(diào)查缺乏反應(yīng)的可能原因。在隨后的出版物中，他們報(bào)告稱，所有7名患者在治療前收集的細(xì)胞來源的外泌體在共培養(yǎng)試驗(yàn)中能夠抑制NK-92對白血病的細(xì)胞毒性效應(yīng)。
Pooled response data from all 4 phase I studies reveal that approximately 36% of the treated patients showed some tumor reduction, ranging from tumor shrinkage to partial remission. Most importantly, repeated infusions with NK-92 cells, even at high cell numbers, did not induce any side effects greater than grade 2. In all trials the NK-92 cells were expanded in flasks, bags or G-Rex bioreactors using X-Vivo 10 medium with 5% human serum. For currently ongoing trials with engineered NK-92, the same medium is used but production is scaled up by use of large bioreactors.
所有4項(xiàng)I期研究的匯總反應(yīng)數(shù)據(jù)顯示，大約36%的接受治療的患者顯示出某種程度的腫瘤縮小，范圍從腫瘤縮小到部分緩解。最重要的是，即使在高細(xì)胞數(shù)量下，重復(fù)輸注NK-92細(xì)胞也沒有引起超過2級的副作用。在所有試驗(yàn)中，NK-92細(xì)胞都是在含有5%人血清的X-Vivo 10培養(yǎng)基中，使用培養(yǎng)瓶、袋子或G-Rex生物反應(yīng)器進(jìn)行擴(kuò)增。對于目前正在開展的基于改造后的NK-92細(xì)胞的臨床試驗(yàn)，使用了相同的培養(yǎng)基，但通過使用大型生物反應(yīng)器擴(kuò)大了生產(chǎn)規(guī)模。
三、The Next Generation of Engineered NK-92: haNK, taNK, t-haNK, qt-haNK （下一代工程化NK-92：haNK、taNK、t-haNK、qt-haNK）
(1) High-affinity fc-receptor expressing NK-92 (haNK) （高親和力Fc受體表達(dá)NK-92（haNK）
Largely as the result of predictable rapid proliferation (doubling time of 24 36 h) and ease of expansion, several genetically engineered variants of NK-92 cells have been generated, including a line expressing a high-affinity Fc-receptor, haNK. NK cells are the main effector cells for mAbs of IgG1 or IgG3 type such as trastuzumab, rituximab or avelumab that engage the CD16 Fc-receptor on NK cells for ADCC. The Fc-receptor on NK cells can have low, intermediate or high affinity for IgG. However, only approximately 10% of the general population expresses the high-affinity Fc-receptor for mAbs on their NK cells, with the majority of people expressing a low- or intermediate-affinity Fc-receptor [5]. This also implies that the majority of the population lacks the most relevant effector mechanism for mAb mediated cytotoxicity. Moreover, the Fc-receptor on blood NK cells is sensitive to the enzymatic cleavage by the ADAM17 enzyme, which can result in reduced efficacy of mAbs [35].
由于NK-92可預(yù)測的快速增殖（倍增時(shí)間為24-36小時(shí)）和易于擴(kuò)增，已經(jīng)產(chǎn)生了幾種基因工程改造的NK-92細(xì)胞變體，包括表達(dá)高親和力Fc受體的haNK。NK細(xì)胞是IgG1或IgG3型單克隆抗體（如曲妥珠單抗、利妥昔單抗或阿維魯單抗）的主要效應(yīng)細(xì)胞，這些抗體通過與NK細(xì)胞上的CD16 Fc受體結(jié)合來介導(dǎo)ADCC。NK細(xì)胞上的Fc受體對IgG的親和力可以是低、中或高。然而，在一般人群中，只有大約10%的人在其NK細(xì)胞上表達(dá)高親和力Fc受體，大多數(shù)人表達(dá)低或中等親和力的Fc受體。這也意味著大多數(shù)人缺乏mAb介導(dǎo)細(xì)胞毒性效應(yīng)的最相關(guān)效應(yīng)機(jī)制。此外，血液NK細(xì)胞上的Fc受體對ADAM17酶的酶切敏感，這可能導(dǎo)致mAb的療效降低。
With these considerations in mind, haNK cells were generated from NK-92 that express a high-affinity Fc-receptor (CD16A, 158V) genetically linked to endoplasmatic reticulum IL-2 [35]. The endoplasmatic reticulum linked IL-2 guarantees that only low amounts of IL-2 are secreted by haNK cells, but the intra-cellular concentration is sufficient enough to maintain cell viability, expansion and cytotoxicity. Importantly, the Fc-receptor on haNK cells is resistant to ADAM17-mediated degradation [35]. Moreover, studies from the National Cancer Institute have shown that haNK cells do not lose cytotoxicity under low oxygen (hypoxic) conditions, which is in contrast to blood derived NK cells [18]. Hypoxia is one of the significant immunosuppressive factors in the tumor microenvironment.
鑒于這些考慮，從NK-92中產(chǎn)生了haNK細(xì)胞，它們表達(dá)高親和力Fc受體（CD16A，158V），并且該受體與內(nèi)質(zhì)網(wǎng)IL-2基因連接。內(nèi)質(zhì)網(wǎng)連接的IL-2確保haNK細(xì)胞只分泌少量IL-2，但細(xì)胞內(nèi)濃度足以維持細(xì)胞的活性、擴(kuò)增和細(xì)胞毒性。重要的是，haNK細(xì)胞上的Fc受體對ADAM17介導(dǎo)的降解具有抵抗力。此外，美國國家癌癥研究所的研究表明，haNK細(xì)胞在低氧（缺氧）條件下不會(huì)失去細(xì)胞毒性，這與血液來源的NK細(xì)胞形成對比。缺氧是腫瘤微環(huán)境中一個(gè)重要的免疫抑制因素。
In a phase II clinical trial (NCT03853317) at the University of Washington, haNK cells were given in combination with Avelumab (anti-programmed death-ligand 1 [PD-L1] mAb) to patients with refractory Merkel cell cancer [36]. The haNK infusions were well tolerated, and despite the very advanced disease state of these patients, objective responses were seen in two of seven patients, including reversal of programmed cell death protein 1 (PD-1) refractoriness in one patient.
在美國華盛頓大學(xué)進(jìn)行的一項(xiàng)II期臨床試驗(yàn)（NCT03853317）中，haNK細(xì)胞與阿維魯單抗（抗程序性死亡配體1 [PD-L1]單克隆抗體）聯(lián)合用于治療耐藥的默克爾細(xì)胞癌患者。haNK輸注耐受性良好，盡管這些患者的疾病狀態(tài)非常晚期，但在7名患者中有2名患者觀察到客觀反應(yīng)，其中1名患者逆轉(zhuǎn)了對PD-1的耐藥性。
NK-92 cells have been further engineered to express a recombinant receptor containing the extracellular portion of the high-affinity Fc-receptor CD64 with the transmembrane and intracellular region of CD16A (referred to as CD64/16A). According to preliminary in vitro studies, ADCC is further improved over NK cells that express the CD16A variant [37].
NK-92細(xì)胞還被進(jìn)一步工程化改造，以表達(dá)一個(gè)重組受體，該受體包含高親和力Fc受體CD64的胞外部分以及CD16A的跨膜和細(xì)胞內(nèi)區(qū)域（稱為CD64/16A）。根據(jù)初步體外研究，與表達(dá)CD16A變體的NK細(xì)胞相比，ADCC得到了進(jìn)一步改善。
The main mechanism of action for most clinically effective mAbs is through ADCC with direct cytotoxicity and complement-mediated cytotoxicity playing a less important role. For that reason, the high-affinity FcR expressing NK-92 cells continue to be used by numerous biotech companies and research laboratories for development and testing of mAbs (available from www.BrinkBiologics.com).
大多數(shù)臨床上有效的單克隆抗體的主要作用機(jī)制是通過ADCC的直接細(xì)胞毒性，補(bǔ)體介導(dǎo)的細(xì)胞毒性作用較小。因此，高親和力FcR表達(dá)的NK-92細(xì)胞繼續(xù)被眾多生物技術(shù)公司和研究實(shí)驗(yàn)室用于開發(fā)和測試單克隆抗體（可通過www.BrinkBiologics.com獲得）。
(2) Targeted NK-92 (taNK) cells expressing CARs （表達(dá)CAR的靶向NK-92（taNK）細(xì)胞）
The features of NK-92 cells have stipulated the generation of CARexpressing variants. Table 2 lists the variants of NK-92 cells, including NK-92ci, NK-92mi, haNK, taNK, t-haNK and qt-haNK. Much of the research with CAR-engineered NK-92 cells has been summarized in review papers [38 40]. Investigators have used first and second generation CAR constructs generally delivered by lentiviral or retroviral constructs. Although several studies confirmed the efficacy of CAR-modified NK-92, ImmunityBio, Inc. (which now holds worldwide rights to NK-92 variants) developed CAR-modified NK-92 cells by use of plasmid-based CAR gene constructs and transfected by simple electroporation to generate the NK-92 variants currently used in clinical studies [38,41].
NK-92細(xì)胞的特性促使產(chǎn)生了表達(dá)CAR的變體。表2列出了NK-92細(xì)胞的變體，包括NK-92ci、NK-92mi、haNK、taNK、t-haNK和qt-haNK。關(guān)于CAR工程化NK-92細(xì)胞的許多研究已在綜述論文中進(jìn)行了總結(jié)。研究人員使用第一代和第二代CAR構(gòu)建體，這些構(gòu)建體通常通過慢病毒或逆轉(zhuǎn)錄病毒載體傳遞。盡管幾項(xiàng)研究證實(shí)了CAR修飾的NK-92的療效，但I(xiàn)mmunityBio公司（現(xiàn)持有NK-92變體的全球權(quán)利）使用基于質(zhì)粒的CAR基因構(gòu)建體，并通過簡單的電穿孔轉(zhuǎn)染來產(chǎn)生目前用于臨床研究的NK-92變體。
Over the last few decades, there has been significant advancement of our knowledge concerning the biology of NK cells, which comprise about 10% of the circulating blood lymphocyte pool. A more comprehensive characterization, including gene and surface molecule expression, as well as proteomics, recently has become available [2 4]. It is now well accepted that NK cells are an important component of the rapid cellular immune response that can inhibit the cell-to-cell spread of malignant or infected cells. Not only can they execute immediate (“spontaneous”) killing upon contact with those cells (without the need to be “primed” like T-cells), but they also produce a host of cytokines that support the expansion and function of other immune cells in the blood circulation and the tumor microenvironment.
在過去的幾十年里，我們對NK細(xì)胞生物學(xué)的認(rèn)識取得了顯著進(jìn)展，NK細(xì)胞約占循環(huán)血液淋巴細(xì)胞池的10%。最近，包括基因和表面分子表達(dá)以及蛋白質(zhì)組學(xué)在內(nèi)的更全面的表征已經(jīng)可用。現(xiàn)在普遍認(rèn)為，NK細(xì)胞是快速細(xì)胞免疫反應(yīng)的重要組成部分，能夠抑制惡性或感染細(xì)胞的細(xì)胞間傳播。它們不僅可以在與這些細(xì)胞接觸時(shí)立即（“自發(fā)地”）進(jìn)行殺傷（無需像T細(xì)胞那樣“啟動(dòng)”），還可以產(chǎn)生一系列細(xì)胞因子，支持血液循環(huán)和腫瘤微環(huán)境中其他免疫細(xì)胞的擴(kuò)增和功能。
Through their FcgIII (CD16) receptor, NK cells are major effector cells of ADCC, which is the main effector arm for immunoglobulin G (IgG) 1 and IgG3 monoclonal antibodies (mAbs). Notably, NK cells that express the high-affinity (158V) variant of the CD16 receptor are more effective for mAb-mediated targeted cell killing, although only approximately 10% of the human population is homozygous for this variant [5].
通過其FcγIII（CD16）受體，NK細(xì)胞是ADCC的主要效應(yīng)細(xì)胞，這是免疫球蛋白G（IgG）1和IgG3單克隆抗體（mAbs）的主要效應(yīng)臂。值得注意的是，表達(dá)高親和力（158V）CD16受體變異體的NK細(xì)胞在mAb介導(dǎo)的靶向細(xì)胞殺傷中更為有效，盡管人類人群中只有大約10%的人是這種變異體的純合子。
The effector molecules that execute rapid NK cell mediated target cell killing are perforin and granzymes and to a lesser extent FasL and tumor necrosis factor related apoptosis inducing ligand (TRAIL) [6]. The former are released on contact with transformed cells and act within minutes, with perforin “punching holes” into the membrane of target cells so that granzymes can enter the cell and destroy it by degrading their genetic material. FasL and tumor necrosis factor related apoptosis inducing ligand seem to have a more delayed cytotoxic effect through activation of the death receptor pathways on target cells [6].
執(zhí)行快速NK細(xì)胞介導(dǎo)的靶細(xì)胞殺傷的效應(yīng)分子是穿孔素和顆粒酶，以及在較小程度上的FasL和腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體（TRAIL）。前者在與目標(biāo)細(xì)胞接觸時(shí)釋放，并在幾分鐘內(nèi)起作用，穿孔素在靶細(xì)胞膜上“打孔”，使顆粒酶能夠進(jìn)入細(xì)胞并通過降解其遺傳物質(zhì)來破壞細(xì)胞。FasL和腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體似乎通過激活靶細(xì)胞上的死亡受體途徑產(chǎn)生更延遲的細(xì)胞毒性效應(yīng)。
Enriching NK cells from blood (peripheral or cord) either for research or clinical applications usually is done by negative selection employing a number of monoclonal antibodies that can deplete and eliminate non-NK cells (reviewed in Kundo et al. [7]). NK cells also may be enriched from blood, based on their characteristic CD56 (neuronal cell adhesion molecule) surface expression. One of the concerns is, however, that the bound antibody may affect the NK cell function and/or can interfere with any further manipulation (engineering) of the cells. Another limitation of such enrichment is the unpredictable variability of yield, particularly for NK cells from patients with cancer whose NK cells often are also dysfunctional due to circulating suppressive serum factors or following chemotherapy. A further challenge is that the NK-cell donors must be connected to a leukapheresis device for several hours to collect their white blood cells, of which only about 10% are NK cells. To achieve sufficient NK cells for therapeutic infusion, NK cells need to be expanded on a cell line feeder layer, which may be genetically altered. In addition, cytokines need to be included to support expansion (reviewed in Saito et al. [8]).
從血液（外周或臍帶）中富集NK細(xì)胞用于研究或臨床應(yīng)用，通常通過負(fù)選擇進(jìn)行，使用多種單克隆抗體耗盡和消除非NK細(xì)胞。NK細(xì)胞也可以根據(jù)其特征性的CD56（神經(jīng)細(xì)胞黏附分子）表面表達(dá)從血液中富集。然而，其中一個(gè)問題是，結(jié)合的抗體可能會(huì)影響NK細(xì)胞的功能和/或干擾細(xì)胞的任何進(jìn)一步操作（工程改造）。這種富集的另一個(gè)限制是產(chǎn)量的不可預(yù)測性，特別是對于癌癥患者的NK細(xì)胞，因?yàn)檠h(huán)抑制血清因子或化療后，其NK細(xì)胞通常也會(huì)功能失調(diào)。進(jìn)一步的挑戰(zhàn)是，NK細(xì)胞供體必須連接到白細(xì)胞分離裝置數(shù)小時(shí)，以收集其白細(xì)胞，其中只有大約10%是NK細(xì)胞。為了獲得足夠數(shù)量的NK細(xì)胞用于治療輸注，需要在細(xì)胞系飼養(yǎng)層上擴(kuò)增NK細(xì)胞，該飼養(yǎng)層可能經(jīng)過基因改造。此外，還需要加入細(xì)胞因子以支持?jǐn)U增。
As an alternative to using a patient’s own (autologous) NK cells, allogeneic NK cells can be collected from the blood of a healthy related or unrelated person. Those allogeneic NK-cell collections need to be further treated to remove T lymphocytes, as those can cause the potentially life-threatening graft-versus-host reactions after infusion. This additional purification step adds to the cost of the cell preparation and also results in a further loss of NK cells. Regardless of the source, blood NK cells need to be expanded to have suffi cient numbers of cells available for therapeutic infusion [9]. Another challenge with blood-derived NK cells is the fact that those NK cells are more difficult to manipulate or engineer, requiring more complex, often virus-based gene transduction methods, which come with unpredictable and inconsistent efficacy [8].
作為使用患者自身（自體）NK細(xì)胞的替代方法，可以從健康相關(guān)或無關(guān)人員的血液中收集異體NK細(xì)胞。這些異體NK細(xì)胞收集后需要進(jìn)一步處理以去除T淋巴細(xì)胞，因?yàn)檫@些細(xì)胞在輸注后可能會(huì)導(dǎo)致潛在致命的移植物抗宿主反應(yīng)。這個(gè)額外的純化步驟增加了細(xì)胞制備的成本，并且也導(dǎo)致NK細(xì)胞的進(jìn)一步損失。無論來源如何，血液NK細(xì)胞都需要擴(kuò)增，以獲得足夠數(shù)量的細(xì)胞用于治療輸注。血液來源NK細(xì)胞的另一個(gè)挑戰(zhàn)是，這些NK細(xì)胞更難以操作或工程改造，需要更復(fù)雜、通?；诓《镜幕蜣D(zhuǎn)導(dǎo)方法，這些方法的效果難以預(yù)測且不一致。
Considering those drawbacks of blood-derived NK cells, including the costs of obtaining a consistent NK-cell population from blood for research and clinical applications, a continuously growing NK cell line is an appealing alternative. Although several human NK cell lines have been established [10], only the NK-92 cell line has shown consistently high cytotoxicity against a broad spectrum of cancer cells, and can easily be expanded ex vivo with a short doubling time of 24 36 h [11,12]. Importantly, NK-92 can be genetically engineered by plasmid electroporation to express a high-affinity Fc-receptor, CAR, or molecules that can modulate the tumor microenvironment [13,14].
鑒于血液來源NK細(xì)胞的這些缺點(diǎn)，包括從血液中獲得一致的NK細(xì)胞群用于研究和臨床應(yīng)用的成本，持續(xù)生長的NK細(xì)胞系是一個(gè)有吸引力的替代選擇。盡管已經(jīng)建立了幾種人類NK細(xì)胞系，但只有NK-92細(xì)胞系顯示出對廣泛癌癥細(xì)胞的一致高細(xì)胞毒性，并且可以在體外輕松擴(kuò)增，其倍增時(shí)間為24-36小時(shí)。重要的是，NK-92可以通過質(zhì)粒電穿孔進(jìn)行基因工程改造，以表達(dá)高親和力Fc受體、CAR或可以調(diào)節(jié)腫瘤微環(huán)境的分子。
二、Early Clinical Studies with NK-92 （NK-92的早期臨床研究）
In 1997, the license and rights for NK-92 were transferred from the University of British Columbia to Rush Medical Center in Chicago, where in 2001 a phase I trial with NK-92 in patients with advanced cancer was conducted [31]. Due to the responses seen with LAK cell infusions in patients with renal cell cancer and melanoma [32], those two diseases were selected as initial indications for NK-92 infusions.
1997年，NK-92的許可和權(quán)利從不列顛哥倫比亞大學(xué)轉(zhuǎn)移到芝加哥的拉什醫(yī)學(xué)中心，2001年在那里進(jìn)行了NK-92在晚期癌癥患者中的I期試驗(yàn)。由于在腎細(xì)胞癌和黑色素瘤患者中使用LAK細(xì)胞輸注看到了反應(yīng)，這兩種疾病被選為NK-92輸注的最初適應(yīng)癥。
In that study, NK-92 cells were infused on days 1, 3 and 5 with the rationale that it usually takes a few days to trigger a T-cell mediated allogeneic immune response that could potentially lead to rejection of the infused NK-92 cells. In this phase I dose-escalation study, 12 patients were enrolled and received a starting dose of 1 x109 NK-92 cells/m2 . The greatest dose administered was 5 x 109 NK-92 cells/m2 cells. None of the patients experienced grade 3 or 4 side effects, and only a few patients developed a mild fever or a rash. Although phase I studies are not designed to assess efficacy, it is noteworthy that the majority of the patients with renal cell cancer experienced a prolonged disease-free survival and overall survival compared with historical controls. The single patient with melanoma in that study had a partial response with significant tumor reduction after the third infusion. In contrast to CAR/T-cell therapy that generally includes some form of immunosuppressive chemotherapy before infusion, NK-92 cells in those and all other studies (including with CAR modified NK-92) were given without any preparative chemotherapy.
在該研究中，NK-92細(xì)胞在第1、3和5天進(jìn)行輸注，理由是通常需要幾天時(shí)間才能觸發(fā)T細(xì)胞介導(dǎo)的異體免疫反應(yīng)，這可能會(huì)導(dǎo)致輸注的NK-92細(xì)胞被排斥。在這項(xiàng)I期劑量遞增研究中，共納入了12名患者，起始劑量為每平方米1×10^9個(gè)NK-92細(xì)胞。給予的最大劑量為每平方米5×10^9個(gè)NK-92細(xì)胞。沒有患者出現(xiàn)3級或4級副作用，只有少數(shù)患者出現(xiàn)輕度發(fā)熱或皮疹。盡管I期研究并非旨在評估療效，但值得注意的是，大多數(shù)腎細(xì)胞癌患者的無病生存期和總生存期比歷史對照組有所延長。該研究中唯一的黑色素瘤患者在第三次輸注后出現(xiàn)了部分反應(yīng)，腫瘤顯著縮小。與通常在輸注前進(jìn)行某種形式免疫抑制化療的CAR/T細(xì)胞療法不同，NK-92細(xì)胞在這些以及所有其他研究（包括CAR修飾的NK-92）中都是在沒有任何準(zhǔn)備性化療的情況下給予的。
Contemporaneous with the aforementioned study, Dr. TorstenTonn, who had previously performed post-doctoral work in the author’s laboratory, initiated a phase I trial in Frankfurt, Germany, in pediatric and adult patients with advanced, mostly solid cancers [26]. The schedule consisted of two infusions, given on day 1 and 3. The dose of 1 x 1010 cells/m2 was considered dose-limiting, but not because of side effects, but rather because of logistical challenges of expanding larger numbers of NK-92 cells at that time.
與此同時(shí)，托爾斯滕·托恩博士（Torsten Tonn）在德國法蘭克福啟動(dòng)了一項(xiàng)I期試驗(yàn)，研究對象為患有晚期癌癥（主要是實(shí)體瘤）的兒童和成人患者。該試驗(yàn)的安排是在第1天和第3天進(jìn)行兩次輸注。每平方米1×10^10個(gè)細(xì)胞的劑量被認(rèn)為是劑量限制性的，但這并非由于副作用，而是因?yàn)楫?dāng)時(shí)擴(kuò)大NK-92細(xì)胞數(shù)量存在物流挑戰(zhàn)。
Another phase I trial was conducted under the guidance of Dr. Armand Keating, at Ontario Cancer Center in Toronto [15]. Among the 12 patients with advanced hematologic malignancies enrolled in the study, two patients (with myeloma and Hodgkin disease) experienced long-lasting clinical remissions. Patients received multiple infusions over time (planned six monthly cycles). Only one-half of the patients developed HLA antibodies, and none had a positive mixed lymphocyte culture when NK-92 cells were used as stimulators.
在多倫多安大略癌癥中心，阿曼德·基廷博士（Armand Keating）指導(dǎo)進(jìn)行了另一項(xiàng)I期試驗(yàn)。在該研究中，共有12名患有晚期血液系統(tǒng)惡性腫瘤的患者接受治療，其中兩名患者（分別患有骨髓瘤和霍奇金?。┙?jīng)歷了長期的臨床緩解。患者接受了多次輸注（計(jì)劃進(jìn)行六次每月）。只有一半的患者產(chǎn)生了HLA抗體，沒有任何患者的混合淋巴細(xì)胞培養(yǎng)物呈陽性反應(yīng)。
In another phase I trial conducted at the University of Pittsburgh, seven patients with treatment-resistant acute myeloid leukemia were enrolled and received NK-92 [33]. Although none of thepatients experienced serious adverse effects, no significant clinical responses were noted, prompting the group to investigate possible reasons for the lack of response. In a subsequent publication, they reported that acute myeloid leukemia cell derived exosomes collected pre-therapy from all seven patients had the ability to inhibit the cytotoxic anti-leukemia effects of NK-92 in co-incubation assays [34].
在匹茲堡大學(xué)進(jìn)行的另一項(xiàng)I期試驗(yàn)中，共有7名治療抵抗性的急性髓系白血病患者接受NK-92治療。盡管沒有患者出現(xiàn)嚴(yán)重的不良反應(yīng)，但也沒有觀察到顯著的臨床反應(yīng)，這促使該團(tuán)隊(duì)調(diào)查缺乏反應(yīng)的可能原因。在隨后的出版物中，他們報(bào)告稱，所有7名患者在治療前收集的細(xì)胞來源的外泌體在共培養(yǎng)試驗(yàn)中能夠抑制NK-92對白血病的細(xì)胞毒性效應(yīng)。
Pooled response data from all 4 phase I studies reveal that approximately 36% of the treated patients showed some tumor reduction, ranging from tumor shrinkage to partial remission. Most importantly, repeated infusions with NK-92 cells, even at high cell numbers, did not induce any side effects greater than grade 2. In all trials the NK-92 cells were expanded in flasks, bags or G-Rex bioreactors using X-Vivo 10 medium with 5% human serum. For currently ongoing trials with engineered NK-92, the same medium is used but production is scaled up by use of large bioreactors.
所有4項(xiàng)I期研究的匯總反應(yīng)數(shù)據(jù)顯示，大約36%的接受治療的患者顯示出某種程度的腫瘤縮小，范圍從腫瘤縮小到部分緩解。最重要的是，即使在高細(xì)胞數(shù)量下，重復(fù)輸注NK-92細(xì)胞也沒有引起超過2級的副作用。在所有試驗(yàn)中，NK-92細(xì)胞都是在含有5%人血清的X-Vivo 10培養(yǎng)基中，使用培養(yǎng)瓶、袋子或G-Rex生物反應(yīng)器進(jìn)行擴(kuò)增。對于目前正在開展的基于改造后的NK-92細(xì)胞的臨床試驗(yàn)，使用了相同的培養(yǎng)基，但通過使用大型生物反應(yīng)器擴(kuò)大了生產(chǎn)規(guī)模。
三、The Next Generation of Engineered NK-92: haNK, taNK, t-haNK, qt-haNK （下一代工程化NK-92：haNK、taNK、t-haNK、qt-haNK）
(1) High-affinity fc-receptor expressing NK-92 (haNK) （高親和力Fc受體表達(dá)NK-92（haNK）
Largely as the result of predictable rapid proliferation (doubling time of 24 36 h) and ease of expansion, several genetically engineered variants of NK-92 cells have been generated, including a line expressing a high-affinity Fc-receptor, haNK. NK cells are the main effector cells for mAbs of IgG1 or IgG3 type such as trastuzumab, rituximab or avelumab that engage the CD16 Fc-receptor on NK cells for ADCC. The Fc-receptor on NK cells can have low, intermediate or high affinity for IgG. However, only approximately 10% of the general population expresses the high-affinity Fc-receptor for mAbs on their NK cells, with the majority of people expressing a low- or intermediate-affinity Fc-receptor [5]. This also implies that the majority of the population lacks the most relevant effector mechanism for mAb mediated cytotoxicity. Moreover, the Fc-receptor on blood NK cells is sensitive to the enzymatic cleavage by the ADAM17 enzyme, which can result in reduced efficacy of mAbs [35].
由于NK-92可預(yù)測的快速增殖（倍增時(shí)間為24-36小時(shí)）和易于擴(kuò)增，已經(jīng)產(chǎn)生了幾種基因工程改造的NK-92細(xì)胞變體，包括表達(dá)高親和力Fc受體的haNK。NK細(xì)胞是IgG1或IgG3型單克隆抗體（如曲妥珠單抗、利妥昔單抗或阿維魯單抗）的主要效應(yīng)細(xì)胞，這些抗體通過與NK細(xì)胞上的CD16 Fc受體結(jié)合來介導(dǎo)ADCC。NK細(xì)胞上的Fc受體對IgG的親和力可以是低、中或高。然而，在一般人群中，只有大約10%的人在其NK細(xì)胞上表達(dá)高親和力Fc受體，大多數(shù)人表達(dá)低或中等親和力的Fc受體。這也意味著大多數(shù)人缺乏mAb介導(dǎo)細(xì)胞毒性效應(yīng)的最相關(guān)效應(yīng)機(jī)制。此外，血液NK細(xì)胞上的Fc受體對ADAM17酶的酶切敏感，這可能導(dǎo)致mAb的療效降低。
With these considerations in mind, haNK cells were generated from NK-92 that express a high-affinity Fc-receptor (CD16A, 158V) genetically linked to endoplasmatic reticulum IL-2 [35]. The endoplasmatic reticulum linked IL-2 guarantees that only low amounts of IL-2 are secreted by haNK cells, but the intra-cellular concentration is sufficient enough to maintain cell viability, expansion and cytotoxicity. Importantly, the Fc-receptor on haNK cells is resistant to ADAM17-mediated degradation [35]. Moreover, studies from the National Cancer Institute have shown that haNK cells do not lose cytotoxicity under low oxygen (hypoxic) conditions, which is in contrast to blood derived NK cells [18]. Hypoxia is one of the significant immunosuppressive factors in the tumor microenvironment.
鑒于這些考慮，從NK-92中產(chǎn)生了haNK細(xì)胞，它們表達(dá)高親和力Fc受體（CD16A，158V），并且該受體與內(nèi)質(zhì)網(wǎng)IL-2基因連接。內(nèi)質(zhì)網(wǎng)連接的IL-2確保haNK細(xì)胞只分泌少量IL-2，但細(xì)胞內(nèi)濃度足以維持細(xì)胞的活性、擴(kuò)增和細(xì)胞毒性。重要的是，haNK細(xì)胞上的Fc受體對ADAM17介導(dǎo)的降解具有抵抗力。此外，美國國家癌癥研究所的研究表明，haNK細(xì)胞在低氧（缺氧）條件下不會(huì)失去細(xì)胞毒性，這與血液來源的NK細(xì)胞形成對比。缺氧是腫瘤微環(huán)境中一個(gè)重要的免疫抑制因素。
In a phase II clinical trial (NCT03853317) at the University of Washington, haNK cells were given in combination with Avelumab (anti-programmed death-ligand 1 [PD-L1] mAb) to patients with refractory Merkel cell cancer [36]. The haNK infusions were well tolerated, and despite the very advanced disease state of these patients, objective responses were seen in two of seven patients, including reversal of programmed cell death protein 1 (PD-1) refractoriness in one patient.
在美國華盛頓大學(xué)進(jìn)行的一項(xiàng)II期臨床試驗(yàn)（NCT03853317）中，haNK細(xì)胞與阿維魯單抗（抗程序性死亡配體1 [PD-L1]單克隆抗體）聯(lián)合用于治療耐藥的默克爾細(xì)胞癌患者。haNK輸注耐受性良好，盡管這些患者的疾病狀態(tài)非常晚期，但在7名患者中有2名患者觀察到客觀反應(yīng)，其中1名患者逆轉(zhuǎn)了對PD-1的耐藥性。
NK-92 cells have been further engineered to express a recombinant receptor containing the extracellular portion of the high-affinity Fc-receptor CD64 with the transmembrane and intracellular region of CD16A (referred to as CD64/16A). According to preliminary in vitro studies, ADCC is further improved over NK cells that express the CD16A variant [37].
NK-92細(xì)胞還被進(jìn)一步工程化改造，以表達(dá)一個(gè)重組受體，該受體包含高親和力Fc受體CD64的胞外部分以及CD16A的跨膜和細(xì)胞內(nèi)區(qū)域（稱為CD64/16A）。根據(jù)初步體外研究，與表達(dá)CD16A變體的NK細(xì)胞相比，ADCC得到了進(jìn)一步改善。
The main mechanism of action for most clinically effective mAbs is through ADCC with direct cytotoxicity and complement-mediated cytotoxicity playing a less important role. For that reason, the high-affinity FcR expressing NK-92 cells continue to be used by numerous biotech companies and research laboratories for development and testing of mAbs (available from www.BrinkBiologics.com).
大多數(shù)臨床上有效的單克隆抗體的主要作用機(jī)制是通過ADCC的直接細(xì)胞毒性，補(bǔ)體介導(dǎo)的細(xì)胞毒性作用較小。因此，高親和力FcR表達(dá)的NK-92細(xì)胞繼續(xù)被眾多生物技術(shù)公司和研究實(shí)驗(yàn)室用于開發(fā)和測試單克隆抗體（可通過www.BrinkBiologics.com獲得）。
(2) Targeted NK-92 (taNK) cells expressing CARs （表達(dá)CAR的靶向NK-92（taNK）細(xì)胞）
The features of NK-92 cells have stipulated the generation of CARexpressing variants. Table 2 lists the variants of NK-92 cells, including NK-92ci, NK-92mi, haNK, taNK, t-haNK and qt-haNK. Much of the research with CAR-engineered NK-92 cells has been summarized in review papers [38 40]. Investigators have used first and second generation CAR constructs generally delivered by lentiviral or retroviral constructs. Although several studies confirmed the efficacy of CAR-modified NK-92, ImmunityBio, Inc. (which now holds worldwide rights to NK-92 variants) developed CAR-modified NK-92 cells by use of plasmid-based CAR gene constructs and transfected by simple electroporation to generate the NK-92 variants currently used in clinical studies [38,41].
NK-92細(xì)胞的特性促使產(chǎn)生了表達(dá)CAR的變體。表2列出了NK-92細(xì)胞的變體，包括NK-92ci、NK-92mi、haNK、taNK、t-haNK和qt-haNK。關(guān)于CAR工程化NK-92細(xì)胞的許多研究已在綜述論文中進(jìn)行了總結(jié)。研究人員使用第一代和第二代CAR構(gòu)建體，這些構(gòu)建體通常通過慢病毒或逆轉(zhuǎn)錄病毒載體傳遞。盡管幾項(xiàng)研究證實(shí)了CAR修飾的NK-92的療效，但I(xiàn)mmunityBio公司（現(xiàn)持有NK-92變體的全球權(quán)利）使用基于質(zhì)粒的CAR基因構(gòu)建體，并通過簡單的電穿孔轉(zhuǎn)染來產(chǎn)生目前用于臨床研究的NK-92變體。
When CAR/T-cell therapies were being developed and the first clinical trial results were made available, it became clear that the high costs of producing them as well as the CRS and ICANS were sufficient motivation to explore NK cells and particularly NK-92 cells as an alternative source of CAR engineered cytotoxic immune cells.
當(dāng)CAR/T細(xì)胞療法正在開發(fā)并且首次臨床試驗(yàn)結(jié)果公布時(shí)，生產(chǎn)CAR/T的高成本以及CRS和ICANS因素促使人們探索將NK細(xì)胞，特別是NK-92細(xì)胞，作為CAR工程化細(xì)胞毒性免疫細(xì)胞的替代來源。
In collaboration with the German group in Frankfurt, lentivirus based transduction was used to generate a clinical grade NK-92 variant that expresses a CAR for HER2 (ErbB2) [42]. The group went on to start a clinical trial (NCT03383978) in patients with HER2-positive glioblastoma. Patients are receiving intracranial HER2 CAR expressing NK-92 cells injected into the resection margin during relapse surgery. The single-dose escalation part of the phase I study in nine patients has been completed, and no significant side effects were observed [43]. In the current extension of the study, a more permanent micro catheter is placed into the post-surgical cavity, and patients receive repeated injections of HER2 CAR-engineered NK-92 cells. Since approximately 40% of glioblastoma tumors are positive for HER2 expression, it will be important to see whether this treatment can prolong remissions and maintain quality of life for patients with this devastating brain cancer.
在與德國法蘭克福小組合作中，使用慢病毒介導(dǎo)的轉(zhuǎn)導(dǎo)產(chǎn)生了表達(dá)針對HER2（ErbB2）的CAR的臨床級NK-92變體。該小組隨后啟動(dòng)了一項(xiàng)臨床試驗(yàn)（NCT03383978），針對HER2陽性膠質(zhì)母細(xì)胞瘤患者?；颊咴诮邮軓?fù)發(fā)手術(shù)期間，將HER2 CAR表達(dá)的NK-92細(xì)胞注入切除邊緣。該I期研究的單劑量遞增部分已在9名患者中完成，未觀察到顯著的副作用。在當(dāng)前研究的擴(kuò)展中，將更永久的微導(dǎo)管放置在術(shù)后腔內(nèi)，患者接受重復(fù)注射HER2 CAR工程化的NK-92細(xì)胞。由于大約40%的膠質(zhì)母細(xì)胞瘤腫瘤表達(dá)HER2，因此將重要的是看看這種治療是否能夠延長緩解期并維持這些患有這種毀滅性腦癌患者的生活質(zhì)量。
To further dissect why HER2 taNK cells kill their tumor target so effectively, the group at the University in Dresden, Germany, used special confocal microscopy for live imaging of NK-92 cells and its HER2 CAR variant. The purpose of the study was to determine the different steps of lytic granule movement in both cell lines [44]. They found that although unmodified NK-92 cells are able to form conjugates with Her-2 expressing cancer cells, their lytic granules do not polarize toward the synapse with cancer targets. In contrast, CAR expressing NK-92 cells or haNK cells, expressing a high-affinity Fc receptor in combination with a HER2 specific antibody, were able to
effectively polarize their granules toward the synapse and release them for effective killing of the cancer target.
為了進(jìn)一步剖析為什么HER2 taNK細(xì)胞能如此有效地殺死其腫瘤靶標(biāo)，德國德累斯頓大學(xué)的小組使用特殊的共聚焦顯微鏡對NK-92細(xì)胞及其HER2 CAR變體進(jìn)行活體成像。該研究的目的是確定兩種細(xì)胞系中溶酶體顆粒運(yùn)動(dòng)的不同步驟。他們發(fā)現(xiàn)，盡管未修飾的NK-92細(xì)胞能夠與表達(dá)Her-2的癌細(xì)胞形成共軛物，但它們的溶酶體顆粒不會(huì)極化朝向與癌細(xì)胞靶標(biāo)的突觸。相比之下，表達(dá)CAR的NK-92細(xì)胞或表達(dá)高親和力Fc受體與HER2特異性抗體結(jié)合的haNK細(xì)胞能夠有效地將它們的顆粒極化朝向突觸并釋放它們，以有效地殺死癌細(xì)胞靶標(biāo)。
In addition to systemic infusion of NK-92 cells/variants, intra tumoral injection has been studied. Our group has shown in a murine model that intra-tumor injection of CD19 CAR-modified NK-92 cells can induce regression of subcutaneous lymphoma [45]. Remarkably, upon re-challenge of the mice with the same lymphoma cell line, no tumor re-growth occurred. This strongly suggest that intra-tumor injection of NK-92 cells can induce a systemic memory-like immune response. In a murine glioblastoma model, intra-tumor injection of ErbB2 CAR-NK-92 cells the Frankfurt group confirmed that specific IgG antibodies against the tumor had developed in treated mice that together with T cells provided long-term protection against rechallenge with the same tumor [42].
除了全身輸注NK-92細(xì)胞/變體外，還研究了瘤內(nèi)注射。我們的小組在小鼠模型中表明，瘤內(nèi)注射CD19 CAR修飾的NK-92細(xì)胞可以誘導(dǎo)皮下淋巴瘤的消退。值得注意的是，在用相同淋巴瘤細(xì)胞系重新挑戰(zhàn)小鼠時(shí)，沒有腫瘤重新生長。這強(qiáng)有力地表明，NK-92細(xì)胞的瘤內(nèi)注射可以誘導(dǎo)系統(tǒng)性記憶樣免疫反應(yīng)。在小鼠膠質(zhì)母細(xì)胞瘤模型中，法蘭克福小組證實(shí)，在接受ErbB2 CAR-NK-92細(xì)胞治療的小鼠中，針對腫瘤產(chǎn)生了特異性IgG抗體，這些抗體與T細(xì)胞一起為小鼠提供了長期的保護(hù)，使其免受相同腫瘤的復(fù)發(fā)。
(3) t-haNK (targeted high-affinity NK-92) cells expressing a high-affinity FcR and a CAR （表達(dá)高親和力FcR和CAR的t-haNK（靶向高親和力NK-92）細(xì)胞）
The t-haNK modification of NK-92 uses a polycistronic plasmid comprising FcR (158V) and erIL-2 as well as a first-generation CAR. In contrast to CAR/T cells, these t-haNK cells not only provide CAR-specific target cell recognition and killing but also have additional target cell killing mechanisms in place including spontaneous NK-mediated cytotoxicity as well as ADCC through their high-affinity FcR [38,41].
t-haNK對NK-92的修飾使用包含F(xiàn)cR（158V）和erIL-2的多順反子質(zhì)粒。與CAR/T細(xì)胞不同，這些t-haNK細(xì)胞不僅提供CAR特異性靶細(xì)胞識別和殺傷，還具有其他靶細(xì)胞殺傷機(jī)制，包括自發(fā)的NK介導(dǎo)的細(xì)胞毒性以及通過其高親和力FcR進(jìn)行的ADCC效應(yīng)。
A number of clinical-grade t-haNK clones have been generated expressing the CARs for PD-L1, CD19, Her-2 and epidermal growth factor receptor (EGFR) [4,38]. PD-L1 t-haNK cells have been given to a number of patients with solid tumors in a phase I/II study. In some patients with triple-negative breast cancer and pancreatic cancer, some remarkable responses have been observed. Importantly, no significant side effects were noted, in particular no CRS/ICANS (ImmunityBio, internal data from ongoing studies).
已經(jīng)產(chǎn)生了一系列表達(dá)針對PD-L1、CD19、Her-2和表皮生長因子受體（EGFR）的CAR的臨床級t-haNK細(xì)胞。PD-L1 t-haNK細(xì)胞已在一項(xiàng)I/II期研究中給予多名實(shí)體瘤患者。在一些患有三陰性乳腺癌和胰腺癌的患者中，觀察到了一些顯著的反應(yīng)。重要的是，沒有觀察到顯著的副作用，特別是沒有CRS/ICANS（ImmunityBio正在進(jìn)行的內(nèi)部研究數(shù)據(jù)）。
The next generation of t-haNK clones introduces a fourth gene coding for a molecule of choice. In the CD19 t-haNK cell line, the gene for the CCR7 homing receptor for lymphatic tissue was incorporated into such a quadrocistronic construct [46] (qt-haNK). Encouraging preclinical data including a reduction of human lymphoma growth were noted in NOD SCID mice compared with a CD19 t-haNK without CCR7. The quadrocistronic concept also allows to incorporate genes coding for proteins that can positively affect the tumor microenvironment such as a TGF-beta trap or IL-12 (ImmunityBio, internal data).
下一代t-haNK克隆引入了編碼選定分子的第四個(gè)基因。在CD19 t-haNK細(xì)胞系中，將編碼CCR7淋巴組織歸巢受體的基因納入了這種四順反子構(gòu)建體中（qt-haNK）。在NOD SCID小鼠中，與沒有CCR7的CD19 t-haNK相比，觀察到了令人鼓舞的臨床前數(shù)據(jù)，如人類淋巴瘤生長的抑制。四順反子還可以接入編碼可以積極影響腫瘤微環(huán)境的蛋白的基因，例如TGF-beta陷阱或IL-12（ImmunityBio內(nèi)部數(shù)據(jù)）。
銷售咨詢
技術(shù)支持
