自2015年1月20日美國總統奧巴馬高調宣布啟動“精准醫療計划（Precision Medicine Initiative）”以來，全球范圍內掀起一股精准醫療熱。在國內，精准醫療也風生水起，受到業內學者、葯企代表、患者等相關人群的廣泛關注。精准醫療本質上是一種更為精確的個性化醫療，非常適用於惡性腫瘤的臨床治療。而相對於其他精准醫療策略，精准細胞免疫治療（precision cell immunotherapy，PCIT）具有開發周期相對較短、投入相對較低的優勢，適合我國的國情，具有巨大的應用前景，有望成為我國惡性腫瘤精准醫療的一大突破口。
精准醫療（precision medicine）是通過基因組、蛋白質組等組學技術和醫學前沿技術，對疾病進行精細分類及精確診斷，從而對疾病和特定患者進行個性化精准療的新型醫學概念與醫療模式。2011年，在“人類基因組計划”完成近10年后，這一概念由美國著名基因組學家Olson博士在其參與起草的美國國家智庫報告《走向精准醫療》中首次提出。精准醫療模式集合了諸多現代醫學科技發展的知識與技術體系，體現了醫學科學發展趨勢，也代表了臨床實踐發展的方向，將帶來一場新的醫療革命並將深刻響未來醫療模式。正是基於此考慮，2015年1月20日，美國總統奧巴馬在白宮高調宣布啟動“精准醫療計划”，擬通過分析100萬名志願者的基因信息，研究遺傳性變異在疾病發生發展中的作用，了解疾病治療的分子基礎，為葯物研發與患者“精准治療”明確方向，以推動個性化醫療的發展，並希望以此“引領一個醫學新時代”。
在美國提出的精准醫療計划中，惡性腫瘤的精准醫療是“重中之重”。美國國立衛生研究院下設的國家癌症研究所，將接受重點資助開展解碼腫瘤基因及開發精准治療研究。那么，為什么要從腫瘤着手開展精准醫療計划呢？誠然，這與當前日趨嚴峻的腫瘤防治形勢相關，另一個重要的原因是提升 腫瘤療效的迫切需求。眾所周知，腫瘤本質上是一種由一系列基因變異的積累導致的復雜遺傳疾病，這意味着腫瘤的基因組是動態變化的，且存在着高度異質性。不同的疾病進展階段以及不同的腫瘤細胞可能攜帶不同的變異信息，從而對以大規模人群為基礎開發和測試葯物的治療模式構成了顛覆性挑戰。據一項覆蓋9個國家和地區的1217例患者的泛亞洲科研顯示：如果沒有基因檢測鑒定相關的靶標卻接受了靶向治療，死亡風險將增加185%。 而新一代測序技術能夠無假設、高分辨率地分析基因組，獲知這些不同的變異信息，能為制定更具針對性和有效性的防治措施提供准確依據，指導醫生對患者采取個性化用葯。基於上述原因，精准醫療模式已然成為癌症治療刻不容緩的任務，是惡性腫瘤治療的大勢所趨。
在具體操作中，腫瘤精准醫療通常可划分為以下三部曲：基因檢測，大數據分析和用葯指導。第一步，基因檢測是患者變異信息的獲知過程，如通過高通量測序方法獲得腫瘤單核苷酸有義突變、拷貝數變異、基因移位和融合基因等海量基因變異信息，該環節中相關檢測技術的精確性及所檢測對象（如腫瘤組織標本）所反映信息的全面性是關鍵。第二步，大數據分析是相關變異信息的解碼與提煉過程，即從海量的組學數據中抽絲剝繭、去粗存精，提取有 價值信息，發揮前后兩個環節之間承上啟下的作用，該環節相應分析模型與分析方法的精確性是關鍵。第三步，用葯指導是以大數據分析結果作為參考，制定因人因病而異的治療方案的過程；而治療的結果也可以反饋到第一個環節，通過新的環路保證治療能隨病情的變化而做出相應的調整。候選葯物可涵蓋所有類型惡性腫瘤臨床用葯，甚至用於其他疾病治療的葯物。該環節中，可供選擇的治療葯物的豐富度直接關系到實施精准醫療治療的成敗。
精准醫療直指惡性腫瘤臨床治療的軟肋，其好處不言而喻，已在臨床治療中越來越顯現其價值。 然而，每個患者多個癌細胞在癌變過程中與之相關的基因突變位點有成千上萬處，而其中起決定性作用的基因突變往往不足十處，如何從每個患者成千上萬處體細胞突變中找到每個腫瘤細胞真正的阿喀琉斯之踵，即引發癌變的關鍵基因，並非是一件容易的事；由於腫瘤的異質性，同一腫瘤患者不同癌細胞的基因突變並不一定相同，不同關鍵基因突變的隨機組合，導致癌症治療難度大為增加；更為嚴重的事是癌症細胞周期檢查點已破壞，各種新突變及融合基因仍在不斷累積，這些新突變及融合基因可能會破壞這些靶向葯物的靶點及其下游信號，從而使靶向治療葯物失效。因此，看似已抑制了關鍵基因，但癌症細胞又建立新的關鍵基因並產生旁路。按精准醫學模式，希望將癌症變為一種慢性病，但從發現靶點—使用靶向葯物—靶點突變或建立新旁路—癌症復發—尋找新靶點—使用新靶向葯物，這種反復的貓捉老鼠的游戲，傳統葯物開發手段難以開發滿足所有變異信息的治療葯物，同時腫瘤基因突變的速度可導致費盡心思尋找到的葯物在幾個月時間內失效，患者只能輾轉於不同葯物的變換，對患者家庭乃至整個醫療保險體系造成巨大經濟負擔。腫瘤精准醫療的這一系統性缺陷應值得引起充分的重視。
人類的免疫系統具有高度的特異性，能正確區分 正常和惡性細胞，能以高度的敏感性和特異性識別 “非自我的”分子或細胞，功能正常的T淋巴細胞能 通過其細胞表面TCR受體（T cell receptor）正確識別 腫瘤細胞中“非自我”改變，清除腫瘤細胞。因而，從 這個意義上說，通過激活、修復、改構、甚至重建患者 抗腫瘤免疫細胞反應的治療方法，尤其腫瘤細胞免疫 治療，天然具有精准治療的特征。通過激活患者體內 殘存腫瘤特異性T細胞的治療方式，已被證實具有良 好的臨床療效。更值得慶幸的是，不同T細胞所 攜帶的TCR受體千差萬別，具有高度的多樣性，為實 施針對不同腫瘤變異信息的精准醫學治療提供了足 夠的廣度。而且，免疫細胞來源於患者自體，作為一 種“活的葯物”，具有自主性與自我適應能力，能有效 縮短開發時間。因而，精准細胞免疫治療有望成為 腫瘤精准醫療的一個重要突破口。
本文定義的精准細胞免疫治療是通過高通量基 因測序及大數據分析，獲得針對癌細胞特異性新抗 原（neo-antigens）和具有高效應的精准T細胞（precision T cell for neoantigen，簡稱為PNA-T），富集 PNA-T細胞對腫瘤患者進行精准免疫治療。涉及的 步驟（圖1）包括：（1）基因檢測：高通量基因檢測手 段獲取患者的癌細胞特有的基因變異信息（包括突 變、融合基因等），從中篩選出能高效激活免疫反應 的腫瘤特異性新抗原，這種新抗原可以來自細胞核、 細胞質、細胞膜任何部位；（2）免疫靶點的篩選：根 據患者的主要組織相容性復合體（major histocom patibility complex，MHC）分型，尋找能引發強烈免疫 的新表位（neo-epitopes）；（3）尋找並富集針對新抗 原表位的PNA-T細胞：主要通過負載新表位的樹突 狀細胞（dendritic cells，DC）刺激，標記后的MHC-新 表位耦聯體流式/磁珠分選富集PNA-T細胞，克隆 PNA-T細胞的TCR基因，通過轉基因修飾手段快速 獲得轉基因PNA-T細胞；（4）過繼細胞回輸治療：大 量擴增PNA-T細胞，實施過繼回輸治療，並跟蹤 PNA-T細胞的變化規律與腫瘤關系。因而，腫瘤精 准細胞免疫治療是更為個性化的免疫細胞治療技 術，屬於第三代免疫細胞治療技術（三代免疫細胞 治療技術的比較見表）。
雖然腫瘤精准細胞免疫治療是一個新概念，但 該領域內研究者已進行了一些探索研究。 2013年，rosenberg領導的團隊率先采用外顯子測 序技術，鑒別在患者中表達的突變蛋白，並用一種 MHC分子-抗原表位親和力算法進行模擬預測評 估，進而合成候選的抗原表位，開展免疫反應驗證。 通過此方法，研究人員能快速鑒別出了在患者腫瘤 細胞上表達，能被腫瘤浸潤淋巴細胞（tumor-infiltrating lymphocytes，TIL）識 別 的 突 變 抗 原 。2014 年，Rosenberg團隊將該方法成功應用到臨床，他們 通過高深度外顯子測序技術、免疫反應功能驗證，篩 選出一位轉移性膽管癌患者的高頻突變基因，並鑒 定到其對應的TIL克隆，通過大量擴增該TIL克隆 並實施回輸治療，使患者的病情得到有效控制。 2014年底，另一個研究團隊聯合應用外顯子測序技 術、轉錄組測序技術、高通量蛋白質譜分析技術，及 MHC分子-抗原表位親和力模擬預測技術，尋找到 能被T細胞識別從而高效激活免疫反應的多肽疫 苗，該個性化腫瘤疫苗兼具預防性疫苗與治療性疫 苗的效能。筆者研究團隊作為全國第一家獲得 細胞治療應用批文的單位，對精准醫療在免疫細胞 治療方向中應用的重要性具有深刻的體會，前瞻性 地開展了精准細胞免疫治療的技術開發，搭建了高 通量測序平台，采用了患者循環腫瘤細胞的富集與 單細胞分離技術、免疫新靶點的生物信息學篩選技 術等，為實施精准細胞免疫治療打下堅實的基礎。
精准細胞免疫治療的大致流程
CTC:循環腫瘤細胞(circulating tumor cell)；ctDNA:循環腫瘤DNA(circulating tumor DNA);MHC:主要組織相容性復合體(major histocompatibility complex)
三代免疫細胞治療技術的比較
如前所述，免疫治療的理想靶點具有區別於其 他靶向治療策略的特征：小分子靶向葯物注重的是 能有效干預對腫瘤細胞生長、侵襲、轉移等細胞行為 至關重要的基因及調控通路，而細胞免疫治療的關 注點是其能否有效地被免疫系統識別，引起有效的 免疫反應。所以，同樣從基因檢測出發，精准細胞免 疫治療的側重點具有其特殊性，目前仍有幾大技術 難題亟待解決。
方便快速地獲取腫瘤患者的基因組變異信息
實際上，這是腫瘤基因檢測所面臨的共性問題。 腫瘤基因檢測最直接的對象是患者原代組織標本， 但對於那些未進行過手術的腫瘤患者，腫瘤標本不 易獲取，而活檢穿刺的技術雖已較為成熟，但患者接 受度相對較低，尤其對那些已發生多處轉移的患者。 即便之前留有標本，但往往是幾個月前甚至是幾年 前保存的局部標本，鑒於腫瘤基因組的動態性與異 質性，它們反映的信息已經是過時的或者代表的信 息不全。循環腫瘤細胞(circulating tumor cell，CTC)和循環腫瘤DNA(circulating tumor DNA,ctDNA)由 腫瘤發生的各個部位釋放入血，能良好地反映患者 整體的腫瘤負荷、惡性程度、轉移能力以及實時的基 因突變信息。因而，選擇CTC和ctDNA作為基 因檢測的樣品來源，可以保證腫瘤治療在取樣信息 上的全面精准，且與組織活檢相比具有檢查微創小、 無放射性污染、經濟等優點，並允許對治療反應進行 實時監測。
然而，如何獲取高純度的CTC細胞並進行基因 測序，以及如何在外周血巨大噪音背景的情況下准 確檢測ctDNA是一項具有挑戰的工作。筆者研究 團隊通過分離介質、抗體捕獲、熒光掃描顯微技術、 激光顯微捕獲等整合技術平台可以高效獲得單個CTC細胞用於基因檢測；同時開發了通過油滴PCR實現在一個油滴內單個CTC基因檢測技術，以及利 用納米孔徑的芯片進行ctDNA的腫瘤突變基因檢 測技術。發展類似於CAPP-Seq的超靈敏測序方 法，可以實現100%地檢出2~4期NSCLC患者50%的ctDNA，可以特異性（96%）檢出等位基因突變，並將錯 誤率降低至約0.02%水平。這類技術平台有望利 用CTC和ctDNA進行外周血腫瘤基因的精准檢測， 為精准細胞免疫治療提供可靠的檢測依據。
快速准確地篩選免疫細胞治療的適合靶點
在腫瘤表觀遺傳修飾變異尚無法被有效利用的現實條件下，細胞免疫治療更多的着眼點是腫瘤基 因組遺傳變異，而只有那些能形成新的氨基酸序列 且在腫瘤細胞中有效表達的變異信息才能被免疫有 效識別。因而，通過外顯子組測序探知腫瘤的基因 組變異信息，通過RNA轉錄組測序確定發生變異的DNA的轉錄情況，是尋找適合免疫治療新抗原的常 規方法。獲得一系列候選新抗原信息后，如何從中 篩選出能被抗原提呈細胞有效提呈的抗原，即新表 位是至關重要的一步。通常的觀點認為，能與MHC分子高效結合的抗原序列，能更好地形成MHC分 子-抗原復合物，從而具備更高的概率被提呈到細胞 表面，成為新抗原表位。因而，預測MHC分子與抗 原的親和力是該環節的關鍵要素。隨着MHC分子 的空間結構越來越清晰化、准確化，多種MHC分子- 抗原復合物的數學模型已被建立，通過計算機模擬 運算能預估出每種抗原與MHC分子的親和力數 值。但鑒於MHC分子亞型的多樣性，新抗原前后 氨基酸序列以不同組合、不同長度形成表位的多樣 性，數據龐大，目前該預測方法仍不夠成熟，假陽性 或假陰性仍普遍存在，需要后續耗時耗力的驗證工 作。因而，MHC分子與抗原的親和力的准確預估， 仍有待於通過數據的不斷積累、預測模型的不斷優 化來實現。
高效尋找PNA-T細胞的TCR組學技術
腫瘤精准細胞免疫治療最終的效應細胞是PNA-T細胞。然而，雖然正常人的TCR多樣性巨 大，但腫瘤患者經過長期的免疫編輯，或經過其他非 特異性治療策略處理后，其TCR組多樣性較正常人 明顯降低,TCR多樣性的降低預示着患者體內預留 的識別特定表位的TCR豐富度降低，即使經過上述 兩個步驟成功尋找到合適的新抗原表位，但可能無 法在患者體內找到與之對應的PNA-T細胞（除非通 過轉基因TCR-T技術實現），精准細胞免疫治療仍 將以失敗告終。因而，除了對腫瘤變異信息進行高 通量檢測分析外，還需從免疫T細胞角度進行考 量，通過高通量測序的方法評估腫瘤患者體內是否 存留能對腫瘤抗原起反應的PNA-T細胞。目前，利 用TCR組學高通量測序技術可以較為准確地獲得 患者的TCR多樣性數據，可以分析腫瘤患者和正常 人之間TCR多樣性的差異。但由於TCR與表位的 作用並不是一一對應關系的，即同一個TCR可以結 合不同的表位，而同一個表位也可能被不同的TCR所識別，顯然它們之間親和力會有所差異。而且TCR組庫測序得到的是大量單獨的α鏈和β鏈信 息，這些α鏈和β鏈理論上可以通過不同的組合構成千差萬別的TCR。因而，以目前的技術水平仍難 以解析出哪個TCR-T細胞具有識別特定抗原表位 的功能。可以預想，如果通過技術進步以及抗原表 位-TCR配對大數據的積累，能最終實現獲得針對特 定抗原表位，能快速地鑒別出哪些T細胞攜帶的TCR基因能對其有效識別並發揮治療作用，那么將 大大縮短腫瘤免疫治療的開發進程，為患者的治療 贏得寶貴的時間，並可以通過測序或數字PCR等手 段檢測體內這一群或者單個PNA-T細胞的克隆增 殖情況，實時監測治療的獲益情況。筆者所在團隊 正致力於發展基於CTC和ctDNA為樣本來源的腫 瘤抗原測序技術以及TCR組庫的測序技術，包括通 過油滴技術實現高通量的單個T細胞的微乳滴PCR（emulsion-PCR，emPCR）測序來獲得α鏈和β鏈的匹配信息，以此打開篩選特定抗原反應性TCR受體的方便之門。
新表位特異性PNA-T細胞的富集與擴增
實施精准細胞免疫治療的最后階段是PNA-T細胞克隆的富集與擴增，有三個策略可供選擇：（1） 將新表位負載到患者自體的DC細胞中，然后應用 成熟的DC刺激相應的PNA-T亞群特異性增殖。該 策略的優點是相對成熟，但由於涉及DC的抗原負 載、DC內的抗原加工、DC對T細胞的提呈等一系 列過程，各個環節的技術障礙均會影響相應T細胞 的富集效率；且DC細胞擴增不易，導致該流程耗費 時間較長。（2）PNA-T細胞的直接分選。通過在體 外合成HLA-抗原肽四聚體（HLA-peptide tetramer）能夠有效地被特異性T細胞識別，配合流式細胞分 選術，可以從淋巴細胞中分選出抗原特異性的T細 胞克隆，再通過成熟的T細胞培養方案可大量擴增 相應的T細胞克隆。但分選流式儀器價格昂貴，細 胞通量有限，長時間分選會影響細胞活力，對后續細 胞培養造成不良影響。為此，筆者實驗室建立結合HLA-抗原肽四聚體與免疫磁珠法的T細胞分選技 術，有效提高細胞分選通量，且磁珠可通過后續切除 消除其對細胞的影響，整個流程符合臨床應用規范。 下一步將設法將該技術集成到單一儀器設備中，提 高操作的便捷性與穩定性。（3）通過轉基因修飾手 段，將克隆到的PNA-T細胞的TCR基因導入初始T細胞，使其快速具有識別並殺傷攜帶相應新表位的 腫瘤細胞的能力。
基於PNA-T細胞變化規律的療效的實時監控 技術
療效評估是過繼細胞免疫治療的一大技術難 點。與其他治療方式不同，經行過繼細胞治療后，腫瘤負載可能不會立即縮小，甚至可能暫時增大，不利 於臨床醫生對病情的准確掌控。因而，應用PNA-T細胞對腫瘤患者實施過繼細胞治療，在治療過程需 要跟蹤血液中PNA-T細胞及其來源記憶性T細胞 的數量變化，對比觀察其與腫瘤縮小或腫瘤復發、進 展的關聯性，同時實時監控血液中CTC、ctDNA的含 量變化以及腫瘤新突變位點出現情況。通過數據的 積累，建立關聯PNA-T細胞體內動態變化規律與患 者療效的數學模型，從而實現醫生能對病情實時作 准確判定甚至預判的目標，以作出相應治療對策的 調整，使患者能更好地獲益。
增強精准細胞免疫治療體內療效的輔助技術
相對於體外培養條件，腫瘤部位存在抑制免疫 的微環境。例如，腫瘤細胞表面高表達的PDL1可 與T細胞表面PD1結合，使浸潤到腫瘤部位的T細 胞失能，這也是為何一些免疫檢查點（如PD1/PDL1、CTLA4）單抗能通過重新激活體內殘留的腫 瘤特異性T細胞，而在實體瘤的臨床治療中發揮良 好療效的基本原理。另一方面，T胞的持續發揮 作用，需要共刺激信號（T細胞第二信號）的輔助，回 輸后的T細胞如缺乏共刺激信號，將很快衰竭死 亡，這也是嵌合抗原受體（chimeric antigen receptor，CAR）T細胞技術將第一信號與第二信號偶聯在同 一分子上的根本原因。因而，為有效提高精准細胞 免疫治療的療效，一方面可以通過免疫檢查點單抗 的聯合使用，阻斷腫瘤微環境對回輸后腫瘤特異性T細胞的不良干擾效應；另一方面，可以借助基因轉 染技術，將相應的傳遞共刺激信號元件在體外導入 腫瘤特異性T細胞，從而延長回輸后腫瘤特異性T細胞在體內的存活時間與治療作用。在此方面，筆 者團隊已建立一系列核心技術，申請中國發明專利 5項（已授權1項），顯示出良好的應用價值。
在國際上另一種熱門的免疫細胞治療技術是CAR-T細胞治療技術。精准細胞免疫治療與它既 具有共性特征，也有其各自的特殊屬性，兩者具體比 較見表。
CAR是識別腫瘤細胞膜上腫瘤相關抗原（tumor associated antigen，TAA）的單鏈抗體和胞內信號域 “免疫受體酪氨酸活化基序（immunoreceptor tyrosine-based action motifs，ITAM；通常為CD3ζ或FcεRIγ）”通過鉸鏈區相連構成的嵌合基因。將CAR基因通過基因轉導/轉染的技術導入患者T細 胞后，使其表達CAR基因，獲得的CAR-T細胞具有識別並攻擊表達相應TAA的腫瘤細胞的能力。因 而，CAR-T技術本質是通過基因轉染手段快速獲得 腫瘤殺傷性T細胞的方法。由於CAR-T細胞所識 別的是腫瘤細胞表面的蛋白，而非與MHC分子結 合形成MHC-抗原復合物從而被提呈到細胞表面的 抗原，因而可繞過T細胞的MHC分子限制性。而 且，CAR-T技術通常將T細胞的第一信號與第二信 號偶聯到同一分子結構中，使CAR-T細胞具有更強 的自主性，基本無需其他類型免疫細胞的輔助即可 發揮治療作用。鑒於CAR-T技術的精妙設計，其具 有廣闊的應用前景。
精准細胞免疫治療與CAR-T技術所采用的效 應細胞均是患者自體的T淋巴細胞，不同之處在於 所針對的靶點類型的差別：精准細胞免疫治療瞄准 的是患者特有的新抗原（即腫瘤特異性抗原），這些 新抗原與MHC分子結合形成MHC-抗原復合物后 被提呈到細胞表面，在被提呈前，這些抗原可以分布 在細胞各個位置，包括細胞核、細胞質、細胞膜上，可 選擇范圍更廣。所以，不管是效應細胞本身還是所 針對的抗原，精准細胞免疫治療均是個性化的。CAR-T技術所針對的靶點是表達在腫瘤細胞膜上 的腫瘤相關抗原，可供選擇的范圍較窄，特異性相對 較差；但這一抗原可以是一類患者群體中普遍存在 的抗原靶點，如表達於B細胞淋巴瘤細胞表面的CD19蛋白。因而，CAR-T技術所選用的效應細胞 來源是個性化的，但治療靶點是非個性化的，無需通 過高通量的檢測手段配合。
通常情況下，精准細胞免疫治療策略是從患者 自體T細胞群體中尋找到天然存在的、能針對新抗 原的腫瘤特異性T細胞，歸根到底采用的是未經人 工改造的天然免疫細胞；而CAR-T技術涉及轉基因 過程，是人工改造的腫瘤特異性T細胞。目前，轉 基因修飾技術多采用慢病毒載體系統，慢病毒為RNA病毒，其規模化生產及病毒穩定性均面臨諸多 技術障礙。為解決該難題，筆者實驗室已研發出一 套高效的非病毒載體系統，該非病毒載體比慢病毒 載體具有更高的轉染率，具有易於規模化生產及穩 定性高的特點，將為CAR-T技術的廣泛臨床應用鋪 平道路。
精准T細胞（PNA-T）免疫治療與CAR-T免疫治療的比較
隨着人們對惡性腫瘤認識的不斷深刻，已經越 來越意識到實施腫瘤精准醫療的必要性與迫切性。 而相對於其他基因檢測指導下的個性化治療方式， 精准細胞免疫治療作為一種“活的葯物”，為快速制 定針對特定新抗原表位的治療方式提供足夠的廣度 與可行性。因而，有理由相信，精准醫學的“重中之 重”是腫瘤精准醫學，而中國的腫瘤精准醫學突破 口在於腫瘤精准細胞免疫治療。隨着精准細胞免疫 治療各項配套技術的日趨成熟與完善，它將在惡性 腫瘤治療中發揮越來越重要的作用。
Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing
Human tumours typically harbour a remarkable number of somatic mutations1. If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could potentially be immunogenic as they should be recognized as ‘non-self’ neo-antigens by the adaptive immune system. Recent work has confirmed that mutant peptides can serve as T-cell epitopes2, 3, 4, 5, 6, 7, 8, 9. However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing. We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties. We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. Of the >1,300 amino acid changes identified, ~13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry. The peptides were then structurally modelled bound to MHCI. Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic. Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses. The predictions also enabled the generation of peptide–MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination. These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.
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Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients
The development of human cancer is a multistep process characterized by the accumulation of genetic and epigenetic alterations that drive or reflect tumour progression. These changes distinguish cancer cells from their normal counterparts, allowing tumours to be recognized as foreign by the immune system1, 2, 3, 4. However, tumours are rarely rejected spontaneously, reflecting their ability to maintain an immunosuppressive microenvironment5. Programmed death-ligand 1 (PD-L1; also called B7-H1 or CD274), which is expressed on many cancer and immune cells, plays an important part in blocking the ‘cancer immunity cycle’ by binding programmed death-1 (PD-1) and B7.1 (CD80), both of which are negative regulators of T-lymphocyte activation. Binding of PD-L1 to its receptors suppresses T-cell migration, proliferation and secretion of cytotoxic mediators, and restricts tumour cell killing6, 7, 8, 9, 10. The PD-L1–PD-1 axis protects the host from overactive T-effector cells not only in cancer but also during microbial infections11. Blocking PD-L1 should therefore enhance anticancer immunity, but little is known about predictive factors of efficacy. This study was designed to evaluate the safety, activity and biomarkers of PD-L1 inhibition using the engineered humanized antibody MPDL3280A. Here we show that across multiple cancer types, responses (as evaluated by Response Evaluation Criteria in Solid Tumours, version 1.1) were observed in patients with tumours expressing high levels of PD-L1, especially when PD-L1 was expressed by tumour-infiltrating immune cells. Furthermore, responses were associated with T-helper type 1 (TH1) gene expression, CTLA4 expression and the absence of fractalkine (CX3CL1) in baseline tumour specimens. Together, these data suggest that MPDL3280A is most effective in patients in which pre-existing immunity is suppressed by PD-L1, and is re-invigorated on antibody treatment.
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The future of immune checkpoint therapy
Immune checkpoint therapy, which targets regulatory pathways in T cells to enhance antitumor immune responses, has led to important clinical advances and provided a new weapon against cancer. This therapy has elicited durable clinical responses and, in a fraction of patients, long-term remissions where patients exhibit no clinical signs of cancer for many years. The way forward for this class of novel agents lies in our ability to understand human immune responses in the tumor microenvironment. This will provide valuable information regarding the dynamic nature of the immune response and regulation of additional pathways that will need to be targeted through combination therapies to provide survival benefit for greater numbers of patients.
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Adoptive cell transfer as personalized immunotherapy for human cancer
Adoptive cell therapy (ACT) is a highly personalized cancer therapy that involves administration to the cancer-bearing host of immune cells with direct anticancer activity. ACT using naturally occurring tumor-reactive lymphocytes has mediated durable, complete regressions in patients with melanoma, probably by targeting somatic mutations exclusive to each cancer. These results have expanded the reach of ACT to the treatment of common epithelial cancers. In addition, the ability to genetically engineer lymphocytes to express conventional T cell receptors or chimeric antigen receptors has further extended the successful application of ACT for cancer treatment.
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Neoantigens in cancer immunotherapy
The clinical relevance of T cells in the control of a diverse set of human cancers is now beyond doubt. However, the nature of the antigens that allow the immune system to distinguish cancer cells from noncancer cells has long remained obscure. Recent technological innovations have made it possible to dissect the immune response to patient-specific neoantigens that arise as a consequence of tumor-specific mutations, and emerging data suggest that recognition of such neoantigens is a major factor in the activity of clinical immunotherapies. These observations indicate that neoantigen load may form a biomarker in cancer immunotherapy and provide an incentive for the development of novel therapeutic approaches that selectively enhance T cell reactivity against this class of antigens.
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Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells
Substantial regressions of metastatic lesions have been observed in up to 70% of patients with melanoma who received adoptively transferred autologous tumor-infiltrating lymphocytes (TILs) in phase 2 clinical trials1, 2. In addition, 40% of patients treated in a recent trial experienced complete regressions of all measurable lesions for at least 5 years following TIL treatment3. To evaluate the potential association between the ability of TILs to mediate durable regressions and their ability to recognize potent antigens that presumably include mutated gene products, we developed a new screening approach involving mining whole-exome sequence data to identify mutated proteins expressed in patient tumors. We then synthesized and evaluated candidate mutated T cell epitopes that were identified using a major histocompatibility complex–binding algorithm4 for recognition by TILs. Using this approach, we identified mutated antigens expressed on autologous tumor cells that were recognized by three bulk TIL lines from three individuals with melanoma that were associated with objective tumor regressions following adoptive transfer. This simplified approach for identifying mutated antigens recognized by T cells avoids the need to generate and laboriously screen cDNA libraries from tumors and may represent a generally applicable method for identifying mutated antigens expressed in a variety of tumor types.
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Cancer Immunotherapy Based on Mutation-Specific CD4+ T Cells in a Patient with Epithelial Cancer
Limited evidence exists that humans mount a mutation-specific T cell response to epithelial cancers. We used a whole-exomic-sequencing-based approach to demonstrate that tumor-infiltrating lymphocytes (TIL) from a patient with metastatic cholangiocarcinoma contained CD4+ T helper 1 (TH1) cells recognizing a mutation in erbb2 interacting protein (ERBB2IP) expressed by the cancer. After adoptive transfer of TIL containing about 25% mutation-specific polyfunctional TH1 cells, the patient achieved a decrease in target lesions with prolonged stabilization of disease. Upon disease progression, the patient was retreated with a >95% pure population of mutation-reactive TH1 cells and again experienced tumor regression. These results provide evidence that a CD4+ T cell response against a mutated antigen can be harnessed to mediate regression of a metastatic epithelial cancer.
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Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility
Circulating tumor cells (CTCs) are present at low concentrations in the peripheral blood of patients with solid tumors. It has been proposed that the isolation, ex vivo culture, and characterization of CTCs may provide an opportunity to noninvasively monitor the changing patterns of drug susceptibility in individual patients as their tumors acquire new mutations. In a proof-of-concept study, we established CTC cultures from six patients with estrogen receptor–positive breast cancer. Three of five CTC lines tested were tumorigenic in mice. Genome sequencing of the CTC lines revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new therapeutic targets. With optimization of CTC culture conditions, this strategy may help identify the best therapies for individual cancer patients over the course of their disease.
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Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies
The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction–based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.
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Cell-Based Therapeutics: The Next Pillar of Medicine
Two decades ago, the pharmaceutical industry—long dominated by small-molecule drugs—was revolutionized by the the advent of biologics. Today, biomedicine sits on the cusp of a new revolution: the use of microbial and human cells as versatile therapeutic engines. Here, we discuss the promise of this “third pillar” of therapeutics in the context of current scientific, regulatory, economic, and perceptual challenges. History suggests that the advent of cellular medicines will require the development of a foundational cellular engineering science that provides a systematic framework for safely and predictably altering and regulating cellular behaviors.
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Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens
The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity1, 2, 3, 4, 5, 6, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion1, 2, 7. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells8, 9. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits—including durable responses—to patients with different malignancies10, 11, 12, 13. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.
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PD-1 blockade induces responses by inhibiting adaptive immune resistance
Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types1, 2, 3, 4, 5. One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8+ T cells (termed adaptive immune resistance)6, 7. Here we show that pre-existing CD8+ T cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy. We analysed samples from 46 patients with metastatic melanoma obtained before and during anti-PD-1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next-generation sequencing for T-cell antigen receptors (TCRs). In serially sampled tumours, patients responding to treatment showed proliferation of intratumoral CD8+ T cells that directly correlated with radiographic reduction in tumour size. Pre-treatment samples obtained from responding patients showed higher numbers of CD8-, PD-1- and PD-L1-expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire. Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients. Our findings indicate that tumour regression after therapeutic PD-1 blockade requires pre-existing CD8+ T cells that are negatively regulated by PD-1/PD-L1-mediated adaptive immune resistance.
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MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer
There have been no major advances for the treatment of metastatic urothelial bladder cancer (UBC) in the last 30 years. Chemotherapy is still the