TW202126676A - Novel peptides and combination of peptides for use in immunotherapy against cll and other cancers - Google Patents

Abstract

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Description

用於抗CLL及其他癌症之免疫治療的新穎胜肽及胜肽組合物Novel peptides and peptide compositions for immunotherapy against CLL and other cancers

本發明涉及用於免疫治療方法的肽、蛋白質、核酸和細胞。特別是，本發明涉及癌症的免疫療法。本發明還涉及單獨使用或與其他腫瘤相關肽（刺激抗腫瘤免疫反應或體外刺激T細胞和轉入患者的疫苗複合物的活性藥物成分）聯合使用的腫瘤相關T細胞(CTL)肽表位。與主要組織相容性複合體(MHC)分子結合的肽或與此同類的肽也可能是抗體、可溶性T細胞受體和其他結合分子的靶標。The present invention relates to peptides, proteins, nucleic acids and cells used in immunotherapy methods. In particular, the present invention relates to immunotherapy of cancer. The present invention also relates to tumor-associated T cell (CTL) peptide epitopes used alone or in combination with other tumor-associated peptides (active pharmaceutical ingredients that stimulate anti-tumor immune responses or stimulate T cells in vitro and vaccine complexes transferred to patients). Peptides that bind to major histocompatibility complex (MHC) molecules or peptides of the same kind may also be targets of antibodies, soluble T cell receptors, and other binding molecules.

本發明涉及數種新型肽序列及其變體，它們源自人腫瘤細胞的HLA-I類分子，可用于引發抗腫瘤免疫反應的疫苗組合物中或作為開發藥物/免疫活性化合物和細胞的目標。The present invention relates to several novel peptide sequences and their variants, which are derived from HLA-I molecules of human tumor cells, which can be used in vaccine compositions for initiating anti-tumor immune responses or as targets for the development of drugs/immunologically active compounds and cells .

慢性淋巴細胞性白血病 (CLL) 是含有形態學成熟、免疫學上不完全成熟 B淋巴細胞的 B細胞腫瘤。當外周血中存在至少 5000 個 B淋巴細胞/µl、其中高達 55% 的為不成熟細胞時可確診 CLL。異常 B細胞顯示表達 CD19、dim CD20、dim CD5、CD23、CD79a 和 dim IgM 或 dim IgD 的特徵表型  (Gribben, 2010)。Chronic lymphocytic leukemia (CLL) is a B-cell tumor containing morphologically mature, immunologically incompletely mature B lymphocytes. CLL can be diagnosed when there are at least 5000 B lymphocytes/µl in the peripheral blood, and up to 55% of them are immature cells. Abnormal B cells show characteristic phenotypes expressing CD19, dim CD20, dim CD5, CD23, CD79a, and dim IgM or dim IgD (Gribben, 2010).

兩種不同分期系統可用於分類CLL：Rai系統和Binet系統。Rai分期系統包括根據異常細胞進行性累積將疾病分類為五個階段(0-IV)。Binet系統根據累及位置數量使用三個階段（A、B、C）對疾病進行分(http://www.cancer.gov/cancertopics/pdq/treatment/CLL/Patient)。Two different staging systems can be used to classify CLL: Rai system and Binet system. The Rai staging system includes classification of diseases into five stages (0-IV) based on the progressive accumulation of abnormal cells. The Binet system uses three stages (A, B, C) to classify the disease according to the number of involved locations (http://www.cancer.gov/cancertopics/pdq/treatment/CLL/Patient).

CLL主要是一種老年人疾病。診斷時平均年齡散發性病例為72歲，家族性病例為58歲。男性CLL發生率高於女性，男性與女性的比例約為2：1(Cartwright et al., 2002)。CLL is mainly a disease of the elderly. The average age at diagnosis was 72 years for sporadic cases and 58 years for familial cases. The incidence of CLL in men is higher than that in women, and the ratio of men to women is about 2:1 (Cartwright et al., 2002).

CLL是西方世界最常見的白血病，其中大約占全部白血病病例的1/3。美國和歐洲的發病率相似，估計每年新發病例約有16,000名。CLL在白種人中比非洲人更常見，在西班牙裔和本土美國人中較罕見，在亞洲人中很少見。在亞洲血統的人群中，CLL的發生率比白種人低3倍 (Gunawardana et al., 2008)。CLL is the most common leukemia in the western world, which accounts for about 1/3 of all leukemia cases. The incidence rates in the United States and Europe are similar, with an estimated 16,000 new cases each year. CLL is more common in whites than Africans, rare in Hispanics and Native Americans, and rare in Asians. In people of Asian descent, the incidence of CLL is three times lower than that of whites (Gunawardana et al., 2008).

CLL 患者的五年總生存率約為 79% (http://www.cancer.net/cancer-types/leukemia-chronic-lymphocytic-cll/statistics)。個體患者的預後取決於診斷時的疾病階段和若干預後因素的發生情況。按照Rai系統，0期患者的中位生存期為10年或更長，而I/II期患者的中位生存期為7年，III/IV期患者為0.75-4年 (Gribben, 2010)。數個因素與CLL預後不良相關。這些包括ZAP-70或CD38高水準表達、IgVH未突變狀態和細胞遺傳學畸變del 17p或del 11q (Gribben, 2010)。The five-year overall survival rate of CLL patients is approximately 79% (http://www.cancer.net/cancer-types/leukemia-chronic-lymphocytic-cll/statistics). The prognosis of an individual patient depends on the stage of the disease at the time of diagnosis and the occurrence of several prognostic factors. According to the Rai system, the median survival of stage 0 patients is 10 years or longer, while the median survival of stage I/II patients is 7 years, and that of stage III/IV patients is 0.75-4 years (Gribben, 2010). Several factors are associated with poor prognosis of CLL. These include high levels of ZAP-70 or CD38 expression, IgVH unmutated status, and cytogenetic aberrations del 17p or del 11q (Gribben, 2010).

雖然目前 CLL 不可治癒，但是很多患者只顯示緩慢的病情進展或症狀惡化。由於患者並沒有從早期治療中獲益，因此最初方法是「觀望和等待」(Richards et al., 1999)。對於有症狀或疾病進展迅速的患者，有幾種治療方案可供選擇。這些包括化學療法、靶向治療、基於免疫的療法（如單克隆抗體、嵌合抗原受體 (CAR) 和主動免疫療法）和幹細胞移植。Although CLL is currently incurable, many patients only show slow progression or worsening of symptoms. Since patients did not benefit from early treatment, the initial approach was "wait and see and wait" (Richards et al., 1999). For patients with symptoms or rapid disease progression, there are several treatment options. These include chemotherapy, targeted therapy, immune-based therapies (such as monoclonal antibodies, chimeric antigen receptor (CAR), and active immunotherapy), and stem cell transplantation.

用於 CLL 治療的化療藥物大多是烷化劑（如苯丁酸氮芥和環磷醯胺）或嘌呤類似物（如氟達拉濱）。德國 CLL 研究組 (GCLLSG) CCL4 表明，氟達拉濱/環磷醯胺組合療法優於氟達拉濱單一治療（完全緩解 (CR) 為 24% vs.7%）(Eichhorst et al., 2006)。Most of the chemotherapeutic drugs used in the treatment of CLL are alkylating agents (such as chlorambucil and cyclophosphamide) or purine analogs (such as fludarabine). The German CLL Study Group (GCLLSG) CCL4 showed that fludarabine/cyclophosphamide combination therapy is superior to fludarabine monotherapy (complete response (CR) 24% vs. 7%) (Eichhorst et al., 2006 ).

Ibrutinib 和 idelalisib 是靶向作用於 B細胞受體信號級聯中分子的激酶抑制劑。Ibrutinib 抑制 Bruton 酪氨酸激酶 (BTK)，這是一種對 B細胞成熟重要的 src 相關細胞質酪氨酸激酶，用於初始或二線治療 (Byrd et al., 2013; O'Brien et al., 2014)。Idelalisib 是與利妥昔單抗聯合使用於難治性 CLL 的一種 PI3K-δ抑制劑 (Furman et al., 2014)。Ibrutinib and idelalisib are kinase inhibitors that target molecules in the B cell receptor signaling cascade. Ibrutinib inhibits Bruton tyrosine kinase (BTK), a src-related cytoplasmic tyrosine kinase that is important for B cell maturation, and is used for initial or second-line treatment (Byrd et al., 2013; O'Brien et al., 2014). Idelalisib is a PI3K-δ inhibitor used in combination with rituximab for refractory CLL (Furman et al., 2014).

造血幹細胞移植 (HSCT) 可以考慮用於預後不良的患者，例如，有 del 17p 或 p53 突變的患者。HSCT 可以是同種異體形式，其中移植的細胞由 HLA 匹配人員捐贈，也可以是自體形式，其中在化療後再輸注患者自己的幹細胞 (Schetelig et al., 2008)。Hematopoietic stem cell transplantation (HSCT) may be considered for patients with poor prognosis, for example, patients with del 17p or p53 mutations. HSCT can be an allogeneic form, in which the transplanted cells are donated by HLA-matched personnel, or an autologous form, in which the patient's own stem cells are infused after chemotherapy (Schetelig et al., 2008).

單克隆抗體被廣泛應用於血液惡性疾病。這是由於基於免疫細胞表面分子良好表徵和血液或骨髓腫瘤細胞可取性瞭解了合適的抗原。CLL治療中所用的常見單克隆抗體靶向作用於CD20或CD52。利妥昔單抗是第一個被FDA批准用於治療NHL的單克隆抗CD20抗體，現已廣泛應用於CLL治療。與氟達拉濱/環磷醯胺組合治療相比，利妥昔單抗/氟達拉濱/環磷醯胺組合治療帶來了更高的CR率並提高了總生存率(OS)，後者已成為首選治療方案(GCLLSG CLL8)(Hallek et al., 2008)。Ofatumomab 靶向作用於CD20，用於治療難治性CLL患者(Wierda et al., 2011)。Obinutuzumab 是與苯丁酸氮芥組合的一線治療中使用的另一種單克隆抗CD20抗體(Goede et al., 2014)。Monoclonal antibodies are widely used in hematological malignancies. This is due to the knowledge of suitable antigens based on the good characterization of immune cell surface molecules and the desirability of blood or bone marrow tumor cells. Common monoclonal antibodies used in the treatment of CLL target CD20 or CD52. Rituximab is the first monoclonal anti-CD20 antibody approved by the FDA for the treatment of NHL and is now widely used in the treatment of CLL. Compared with fludarabine/cyclophosphamide combination therapy, rituximab/fludarabine/cyclophosphamide combination therapy brought a higher CR rate and improved overall survival (OS), The latter has become the preferred treatment option (GCLLSG CLL8) (Hallek et al., 2008). Ofatumomab targets CD20 and is used to treat patients with refractory CLL (Wierda et al., 2011). Obinutuzumab is another monoclonal anti-CD20 antibody used in first-line therapy in combination with chlorambucil (Goede et al., 2014).

阿侖單抗是一種抗CD52抗體，用於治療化療耐藥性疾病患者或存在不良預後因素（如 del 17p 或 p53 突變）的患者 (Parikh et al., 2011)。新型單克隆抗體靶向作用於 CD37（otlertuzumab、BI 836826、IMGN529和(177) Lu-tetulomab) 或 CD40（dacetuzumab 和 lucatumumab），並在臨床前環境中進行了測試（Robak and Robak, 2014）。Alemtuzumab is an anti-CD52 antibody used to treat patients with chemotherapy-resistant diseases or patients with poor prognostic factors (such as del 17p or p53 mutations) (Parikh et al., 2011). The new monoclonal antibodies target CD37 (otlertuzumab, BI 836826, IMGN529 and (177) Lu-tetulomab) or CD40 (dacetuzumab and lucatumumab) and have been tested in a preclinical setting (Robak and Robak, 2014).

幾項已完成和正在進行的試驗是基於具有 CD19 特異性的工程自體嵌合抗原受體 (CAR) 修飾的 T細胞 (Maus et al., 2014)。到目前為止，只有少數患者顯示出可檢測到或持續性的嵌合抗原受體。Porter 等人和 Kalos 等人在嵌合抗原受體 T細胞試驗中檢測到了一例部分反應 (PR) 和 2 例完全反應 (CR)(Kalos et al., 2011; Porter et al., 2011)。Several completed and ongoing trials are based on engineered auto-chimeric antigen receptor (CAR) modified T cells with CD19 specificity (Maus et al., 2014). So far, only a few patients have shown detectable or persistent chimeric antigen receptors. Porter et al. and Kalos et al. detected one partial response (PR) and two complete responses (CR) in the chimeric antigen receptor T cell test (Kalos et al., 2011; Porter et al., 2011).

主動免疫治療包括以下策略：基因治療、整體修飾的腫瘤細胞疫苗、基於 DC 的疫苗和腫瘤相關抗原 (TAA) 衍生肽疫苗。Active immunotherapy includes the following strategies: gene therapy, holistically modified tumor cell vaccines, DC-based vaccines, and tumor-associated antigen (TAA)-derived peptide vaccines.

基因治療方法利用自體基因修飾腫瘤細胞。這些 B-CLL 細胞用免疫（共）刺激基因（如 IL-2、IL-12、TNF-α、GM-CSF、CD80、CD40L、LFA-3 和 ICAM-1）轉染，以提高抗原表現和 T細胞活化 (Carballido et al., 2012)。雖然很容易觀察到腫瘤細胞中的特異性 T細胞應答和減少，但是免疫應答僅僅是短暫性的。Gene therapy methods use autologous genes to modify tumor cells. These B-CLL cells are transfected with immune (co)stimulatory genes (such as IL-2, IL-12, TNF-α, GM-CSF, CD80, CD40L, LFA-3 and ICAM-1) to improve antigen expression and T cell activation (Carballido et al., 2012). Although it is easy to observe the specific T cell response and decrease in tumor cells, the immune response is only transient.

幾項研究已經使用自體DC作為抗原表現細胞來引起抗腫瘤反應。DC已經在體外載有腫瘤相關肽、全腫瘤細胞裂解物、腫瘤衍生RNA或DNA。另一種策略採用全腫瘤細胞與DC融合並產生DC-B-CLL細胞雜合物。轉染的DC引發CD4+和CD8+ T細胞應答 (Muller et al., 2004)。載有腫瘤細胞裂解物或凋亡體的融合雜合體和DC增加腫瘤特異性CD8+ T細胞應答。顯示出臨床反應的患者IL-12血清水準增加，Tregs數量減少 (Palma et al., 2008)。Several studies have used autologous DC as antigen expressing cells to elicit an anti-tumor response. DCs have been loaded with tumor-associated peptides, whole tumor cell lysates, tumor-derived RNA or DNA in vitro. Another strategy uses the fusion of whole tumor cells with DCs to produce DC-B-CLL cell hybrids. The transfected DC triggers CD4+ and CD8+ T cell responses (Muller et al., 2004). Fusion hybrids and DCs loaded with tumor cell lysates or apoptotic bodies increase tumor-specific CD8+ T cell responses. The serum levels of IL-12 in patients who showed clinical response increased and the number of Tregs decreased (Palma et al., 2008).

不同的方法使用改變的腫瘤細胞來引發或增加 CLL 特異性免疫應答。本策略的一個實例是產生三瘤細胞：B-CLL 細胞融合至具有抗 APC 特異性的抗 Fc 受體表達雜交瘤細胞。三瘤細胞誘在體外誘導 CLL 特異性 T細胞應答 (Kronenberger et al., 2008)。Different methods use altered tumor cells to trigger or increase CLL-specific immune responses. An example of this strategy is to generate trioma cells: B-CLL cells are fused to anti-Fc receptor expressing hybridoma cells with anti-APC specificity. Trioma cells induce CLL-specific T cell responses in vitro (Kronenberger et al., 2008).

另一種策略利用以芽孢桿菌卡介苗作為佐劑的照射自體 CLL 細胞充當一種疫苗。數名患者顯示白細胞水準下降或病情穩定 (Hus et al., 2008)。除了分離的 CLL 細胞之外，在製備於血液治療單位後，使用來自 CLL 患者的全血作為疫苗。該疫苗引發 CLL 特異性 T細胞反應，並在幾名患者中帶來部分臨床反應或穩定病情 (Spaner et al., 2005)。Another strategy uses irradiated autologous CLL cells adjuvanted with Bacillus Calmette-Guerin as a vaccine. Several patients showed decreased white blood cell levels or stable disease (Hus et al., 2008). In addition to the isolated CLL cells, whole blood from CLL patients is used as a vaccine after being prepared in the blood treatment unit. The vaccine triggered a CLL-specific T-cell response, which resulted in partial clinical response or stable disease in several patients (Spaner et al., 2005).

幾種TAA在CLL中過度表達並適於接種。這些包括纖維調節素(Mayr et al., 2005)、RHAMM/CD168(Giannopoulos et al., 2006)、MDM2 (Mayr et al., 2006)、hTERT (Counter et al., 1995)、癌胚抗原未成熟層粘連蛋白受體蛋白質 (OFAiLRP) (Siegel et al., 2003)、脂肪分化相關蛋白 (Schmidt et al., 2004)、生存素 (Granziero et al., 2001)、KW1至KW14 (Krackhardt et al., 2002) 以及腫瘤衍生IgVHCDR3區域 (Harig et al., 2001; Carballido et al., 2012)。一項I期臨床試驗使用RHAMM衍生R3肽作為疫苗。6名患者中有5名可檢測到R3特異性CD8 + T細胞應答 (Giannopoulos et al., 2010)。Several TAAs are overexpressed in CLL and are suitable for vaccination. These include fibromodulin (Mayr et al., 2005), RHAMM/CD168 (Giannopoulos et al., 2006), MDM2 (Mayr et al., 2006), hTERT (Counter et al., 1995), carcinoembryonic antigen Mature laminin receptor protein (OFAiLRP) (Siegel et al., 2003), adipose differentiation-related protein (Schmidt et al., 2004), survivin (Granziero et al., 2001), KW1 to KW14 (Krackhardt et al. ., 2002) and tumor-derived IgVHCDR3 regions (Harig et al., 2001; Carballido et al., 2012). A phase I clinical trial used RHAMM-derived R3 peptide as a vaccine. Five of the six patients had detectable R3-specific CD8 + T cell responses (Giannopoulos et al., 2010).

考慮到治療癌症相關的嚴重副作用和費用，通常有必要確定可用於治療癌症的因子，尤其是CLL。通常也有必要確定代表癌症生物標誌物的因子，尤其是CLL，從而更好地診斷癌症、評估預後和預測治療成功性。Considering the serious side effects and costs associated with cancer treatment, it is often necessary to determine the factors that can be used to treat cancer, especially CLL. It is often necessary to identify factors that represent cancer biomarkers, especially CLL, to better diagnose cancer, assess prognosis, and predict the success of treatment.

癌症免疫治療代表了癌症細胞特異性靶向作用的一個選項，同時最大限度地減少副作用。癌症免疫療法利用存在的腫瘤相關抗原。Cancer immunotherapy represents an option for specific targeting of cancer cells while minimizing side effects. Cancer immunotherapy utilizes the presence of tumor-associated antigens.

腫瘤相關抗原(TAA)的目前分類主要包括以下幾組： a) 癌-睾丸抗原：T細胞能夠識別的最先確認的TAA屬於這一類抗原，由於其成員表達于組織學相異的人腫瘤中、正常組織中、僅在睾丸的***細胞/精原細胞中、偶爾在胎盤中，因此，它最初被稱為癌-睾丸(CT)抗原。由於睾丸細胞不表達HLAI類和II類分子，所以，在正常組織中，這些抗原不能被T細胞識別，因此在免疫學上可考慮為具有腫瘤特異性。CT抗原大家熟知的例子是MAGE家族成員和NY-ESO-1。 b) 分化抗原：腫瘤和正常組織（腫瘤源自該組織）都含有TAA。大多數已知的分化抗原發現於黑色素瘤和正常黑色素細胞中。許多此類黑色素細胞譜系相關蛋白參與黑色素的生物合成，因此這些蛋白不具有腫瘤特異性，但是仍然被廣泛用於癌症的免疫治療。例子包括，但不僅限於，黑色素瘤的酪氨酸酶和Melan-A/MART-1或攝護腺癌的PSA。 c) 過量表達的TAA：在組織學相異的腫瘤中以及許多正常組織中都檢測到了基因編碼被廣泛表達的TAA，一般表達水準較低。有可能許多由正常組織加工和潛在表現的表位低於T細胞識別的閾值水準，而它們在腫瘤細胞中的過量表達能夠透過打破先前確立的耐受性而引發抗癌反應。這類TAA的典型例子為Her-2/neu、生存素、端粒酶或WT1。 d) 腫瘤特異性抗原：這些獨特的TAA產生于正常基因（如β-catenin、CDK4等）的突變。這些分子變化中有一些與致瘤性轉化和/或進展相關。腫瘤特異性抗原一般可在不對正常組織帶來自體免疫反應風險的情況下誘導很強的免疫反應。另一方面，這些TAA在多數情況下只與其上確認了有TAA的確切腫瘤相關，並且通常在許多個體腫瘤之間並不都共用TAA。在含有腫瘤特定（相關）同種型蛋白的情況下，如果肽源自腫瘤（相關）外顯子也可能出現肽腫瘤特異性（或相關性）。 e) 由異常翻譯後修飾產生的TAA：此類TAA可能由腫瘤中既不具有特異性也不過量表達的蛋白產生，但其仍然具有腫瘤相關性（該相關性由主要對腫瘤具有活性的翻譯後加工所致）。此類TAA產生於變糖基化模式的改變，導致腫瘤產生針對MUC1的新型表位或在降解過程中導致諸如蛋白拼接的事件，這可能具有也可能不具有腫瘤特異性。 f) 腫瘤病毒蛋白：這些TTA是病毒蛋白，可在致癌過程中發揮關鍵作用，並且由於它們是外源蛋白（非人源蛋白），所以能夠激發T細胞反應。這類蛋白的例子有人乳頭狀瘤16型病毒蛋白、E6和E7，它們在宮頸癌中表達。The current classification of tumor-associated antigens (TAA) mainly includes the following groups: a) Cancer-testis antigen: The first confirmed TAA recognized by T cells belongs to this type of antigen, because its members are expressed in histologically different human tumors, normal tissues, and only spermatocytes/spermatogens in the testis In cells and occasionally in the placenta, it was originally called cancer-testis (CT) antigen. Since testicular cells do not express HLAI class and class II molecules, these antigens cannot be recognized by T cells in normal tissues, so they can be considered immunologically tumor-specific. Well-known examples of CT antigens are members of the MAGE family and NY-ESO-1. b) Differentiation antigen: both tumors and normal tissues (from which tumors originate) contain TAA. Most of the known differentiation antigens are found in melanoma and normal melanocytes. Many of these melanocyte lineage-related proteins are involved in the biosynthesis of melanin, so these proteins are not tumor-specific, but are still widely used in cancer immunotherapy. Examples include, but are not limited to, melanoma tyrosinase and Melan-A/MART-1 or prostate cancer PSA. c) Overexpressed TAA: TAA whose gene coding is widely expressed has been detected in histologically different tumors and many normal tissues, and the level of expression is generally low. It is possible that many epitopes processed and latently expressed by normal tissues are below the threshold level recognized by T cells, and their overexpression in tumor cells can trigger an anti-cancer response by breaking the previously established tolerance. Typical examples of this type of TAA are Her-2/neu, Survivin, Telomerase or WT1. d) Tumor-specific antigens: These unique TAAs are produced by mutations in normal genes (such as β-catenin, CDK4, etc.). Some of these molecular changes are related to tumorigenic transformation and/or progression. Tumor-specific antigens can generally induce a strong immune response without risking an autoimmune response to normal tissues. On the other hand, in most cases, these TAAs are only related to the exact tumor on which TAA is confirmed, and TAA is usually not shared among many individual tumors. In the case of a tumor-specific (related) isotype protein, the peptide may also be tumor-specific (or related) if the peptide is derived from a tumor (related) exon. e) TAA produced by abnormal post-translational modification: This type of TAA may be produced by a protein that is neither specific nor overexpressed in the tumor, but it is still tumor-related (this correlation is determined by translations that are mainly active on the tumor). After processing). This type of TAA results from changes in glycosylation patterns, leading to new epitopes targeting MUC1 in tumors or events such as protein splicing during degradation, which may or may not be tumor-specific. f) Tumor virus proteins: These TTAs are viral proteins that can play a key role in the process of carcinogenesis, and because they are foreign proteins (non-human proteins), they can stimulate T cell responses. Examples of such proteins are human papilloma type 16 virus proteins, E6 and E7, which are expressed in cervical cancer.

基於T細胞的免疫治療靶向作用于主要組織相容性複合體(MHC)分子表現的來源於腫瘤相關蛋白或腫瘤特異性蛋白的肽表位。腫瘤特異性T淋巴細胞所識別的抗原，即其表位，可以是源自所有蛋白類型的分子，如酶、受體、轉錄因子等，它們在相應腫瘤的細胞中被表達，並且與同源未變的細胞相比，其表達通常上調。T cell-based immunotherapy targets peptide epitopes derived from tumor-associated proteins or tumor-specific proteins expressed by major histocompatibility complex (MHC) molecules. The antigens recognized by tumor-specific T lymphocytes, that is, their epitopes, can be molecules derived from all protein types, such as enzymes, receptors, transcription factors, etc., which are expressed in the cells of the corresponding tumor and are homologous Compared with unaltered cells, its expression is usually up-regulated.

MHC分子有兩類：MHC I類和MHC II類。MHC I類分子由一條α重鏈和β-2-微球蛋白，MHC II類分子由一條α和一條β鏈組成。其三位構造形成一個結合槽，用於與肽進行非共價相互作用。There are two types of MHC molecules: MHC class I and MHC class II. MHC class I molecules are composed of an alpha heavy chain and beta-2-microglobulin, and MHC class II molecules are composed of an alpha and a beta chain. Its three-position structure forms a binding groove for non-covalent interaction with peptides.

大部分有核細胞上都可發現 MHC-I類分子。他們表現主要為內源性的蛋白、缺陷核糖體產物 (DRIP) 和較大肽裂解生成的肽。然而，源自內體結構或外源性來源的肽也經常在 MHC-I類分子上發現。這種 I-類分子非經典表現方式在文獻中被稱為交叉表現  (Brossart and Bevan, 1997; Rock et al., 1990)。MHC II類分子主要發現于專業抗原表現細胞 (APC) 上，並且主要表現，例如，在內吞作用過程中由 APC 佔據並且隨後被加工的外源性或跨膜蛋白的肽。MHC class I molecules can be found on most nucleated cells. They appear mainly as endogenous proteins, defective ribosomal products (DRIP), and peptides produced by cleavage of larger peptides. However, peptides derived from endosomal structures or exogenous sources are also often found on MHC class I molecules. This non-classical expression of class I molecules is called cross-expression in the literature (Brossart and Bevan, 1997; Rock et al., 1990). MHC class II molecules are mainly found on specialized antigen expressing cells (APC) and mainly express, for example, peptides of exogenous or transmembrane proteins that are occupied by APC during endocytosis and subsequently processed.

肽和 MHC I類的複合體由負載相應 T細胞受體 (TCR) 的 CD8 陽性 T細胞進行識別，而肽和 MHC II類分子的複合體由負載相應 TCR 的 CD4 陽性輔助 T細胞進行識別。因此，TCR、肽和 MHC 按照 1:1:1 的化學計量呈現，這一點已是共識。The complex of peptide and MHC class I is recognized by CD8-positive T cells carrying the corresponding T cell receptor (TCR), and the complex of peptide and MHC class II molecule is recognized by the CD4-positive helper T cells carrying the corresponding TCR. Therefore, it is a consensus that TCR, peptide, and MHC are presented in a 1:1:1 stoichiometry.

CD4陽性輔助T細胞在誘導和維持CD8陽性細胞毒性T細胞的有效反應中發揮重要作用。腫瘤相關抗原(TAA)衍生的CD4陽性T細胞表位的識別對開發能引發抗腫瘤免疫反應的藥物產品可能非常重要 (Gnjatic et al., 2003)。在腫瘤部位，T輔助細胞維持著對細胞毒性T細胞(CTL)友好的細胞因子環境 (Mortara et al., 2006) 並吸引效應細胞，如CTL、天然殺傷(NK)細胞、巨噬細胞和粒細胞 (Hwang et al., 2007)。CD4-positive helper T cells play an important role in inducing and maintaining the effective response of CD8-positive cytotoxic T cells. The recognition of tumor-associated antigen (TAA)-derived CD4-positive T cell epitopes may be very important for the development of drug products that can trigger an anti-tumor immune response (Gnjatic et al., 2003). At the tumor site, T helper cells maintain a cytokine environment that is friendly to cytotoxic T cells (CTL) (Mortara et al., 2006) and attract effector cells such as CTL, natural killer (NK) cells, macrophages, and granulocytes. Cells (Hwang et al., 2007).

在沒有炎症的情況下，MHC II類分子的表達主要局限於免疫系統細胞，尤其是專業抗原表現細胞 (APC)，例如，單核細胞、單核細胞源性細胞、巨噬細胞、樹突狀細胞。在癌症患者的腫瘤細胞中發現有 MHC II  類分子的表達 (Dengjel et al., 2006)。In the absence of inflammation, the expression of MHC class II molecules is mainly limited to immune system cells, especially professional antigen expressing cells (APC), such as monocytes, monocyte-derived cells, macrophages, dendrites cell. The expression of MHC class II molecules is found in tumor cells of cancer patients (Dengjel et al., 2006).

本發明的拉長（較長）肽可作為 MHC-II類活性表位。The elongated (longer) peptides of the present invention can be used as MHC-II active epitopes.

MHC-II類表位活化的輔助 T細胞在編排抗腫瘤免疫的CTL效應子功能中發揮著重要作用。觸發TH1細胞反應的輔助T細胞表位支援CD8陽性殺傷T細胞的效應子功能，其中包括直接作用於腫瘤細胞的細胞毒性功能（該類腫瘤細胞表面顯示有腫瘤相關肽/MHC複合體）。這樣，腫瘤相關T輔助細胞表位單獨使用或與其他腫瘤相關肽結合使用可作為刺激抗腫瘤免疫反應的疫苗化合物的活性藥物成分。Helper T cells activated by MHC-II epitopes play an important role in orchestrating the CTL effector functions of anti-tumor immunity. The helper T cell epitope that triggers the response of TH1 cells supports the effector functions of CD8-positive killer T cells, including the cytotoxic function that directly acts on tumor cells (the surface of such tumor cells shows tumor-related peptide/MHC complexes). In this way, tumor-associated T helper cell epitopes used alone or in combination with other tumor-associated peptides can be used as active pharmaceutical ingredients for vaccine compounds that stimulate anti-tumor immune responses.

哺乳動物（如小鼠）模型顯示，即使沒有CD8陽性T淋巴細胞，CD4陽性T細胞也能透過分泌干擾素-γ (IFNγ)抑制血管生成而足以抑制腫瘤的表現 (Beatty and Paterson, 2001; Mumberg et al., 1999)。沒有CD4T細胞作為直接抗腫瘤效應因子的證據 (Braumuller et al., 2013; Tran et al., 2014)。Mammalian (such as mouse) models have shown that even without CD8-positive T lymphocytes, CD4-positive T cells can inhibit angiogenesis by secreting interferon-γ (IFNγ), which is sufficient to inhibit tumor performance (Beatty and Paterson, 2001; Mumberg) et al., 1999). There is no evidence of CD4 T cells as a direct anti-tumor effector (Braumuller et al., 2013; Tran et al., 2014).

由於HLA II類分子的組成性表達通常僅限於免疫細胞，因此，直接從原發腫瘤中分離II類肽之前被認為是不可能的事。然而，Dengjel等人成功地在腫瘤中直接識別了多個MHC II類表位 (WO 2007/028574, EP 1 760 088 B1)。Since the constitutive expression of HLA class II molecules is usually limited to immune cells, it was previously considered impossible to isolate class II peptides directly from the primary tumor. However, Dengjel et al. succeeded in directly identifying multiple MHC class II epitopes in tumors (WO 2007/028574, EP 1 760 088 B1).

由於CD8依賴型和CD4依賴型這兩種反應共同並協同地促進抗腫瘤作用，因此，確定和表徵由CD8+ T細胞（配體：MHC I類分子 + 肽表位）或CD4陽性T輔助細胞（配體：MHC II類分子）識別的腫瘤相關抗原對開發腫瘤疫苗非常重要。Since the two reactions, CD8-dependent and CD4-dependent, jointly and synergistically promote anti-tumor effects, it is determined and characterized by CD8+ T cells (ligand: MHC class I molecules + peptide epitopes) or CD4-positive T helper cells ( Ligand: tumor-associated antigens recognized by MHC class II molecules are very important for the development of tumor vaccines.

對於MHC I類肽觸發（引發）細胞免疫反應的肽，它也必須與 MHC 分子結合。這一過程依賴於 MHC 分子的等位基因以及肽氨基酸序列的特異性多態性。MHC-I類-結合肽的長度通常為 8-12 個氨基酸殘基，並且在其與 MHC 分子相應結合溝槽相互作用的序列中通常包含兩個保守殘基（「錨」）。這樣，每個 MHC 的等位基因都有「結合基序」，從而確定哪些肽能與結合溝槽特異性結合 。For MHC class I peptides to trigger (trigger) cellular immune responses, they must also be combined with MHC molecules. This process relies on the alleles of the MHC molecule and the specific polymorphism of the peptide amino acid sequence. MHC class I-binding peptides are usually 8-12 amino acid residues in length, and usually contain two conserved residues ("anchors") in the sequence that interacts with the corresponding binding groove of the MHC molecule. In this way, each MHC allele has a "binding motif" to determine which peptides can specifically bind to the binding groove.

在MHC-I類依賴性免疫反應中，肽不僅能與腫瘤細胞表達的某些MHC-I類分子結合，而且它們之後還必須能被T細胞負載的特異性T細胞受體(TCR)識別。In the MHC-I-dependent immune response, peptides can not only bind to certain MHC-I molecules expressed by tumor cells, but they must then be recognized by the specific T cell receptor (TCR) carried by T cells.

對於被T淋巴細胞識別為腫瘤特異性抗原或相關性抗原以及用於治療的蛋白質，必須具備特殊的條件。該抗原應主要由腫瘤細胞表達，而不由正常健康組織表達，或表達數量相對較少。在一個優選的實施方案中，與正常健康組織相比，所述肽應在腫瘤細胞中過度表現。更為適宜的情況是，該相應抗原不僅出現於一種腫瘤中，而且濃度（即每個細胞的相應肽拷貝數目）高。腫瘤特異性抗原和腫瘤相關抗原往往是源自直接參與因細胞週期控制或凋亡抑制中的其功能而發生的正常細胞向腫瘤細胞轉化的蛋白。另外，這些直接導致轉化事件的蛋白的下游靶標可能會被上調，因此可能與腫瘤間接相關。這些間接腫瘤相關抗原也可能是預防接種方法的靶標(Singh-Jasuja et al., 2004)。至關重要的是，表位存在於抗原氨基酸序列中，以確保這種來自腫瘤相關抗原的肽（「免疫原性肽」）可導致體外或體內 T細胞反應。For T lymphocytes to be recognized as tumor-specific antigens or related antigens and proteins used for treatment, special conditions must be met. The antigen should be mainly expressed by tumor cells, not by normal healthy tissues, or expressed in a relatively small amount. In a preferred embodiment, the peptide should be overexpressed in tumor cells compared to normal healthy tissues. A more appropriate situation is that the corresponding antigen not only appears in a tumor, but also has a high concentration (that is, the number of copies of the corresponding peptide per cell). Tumor-specific antigens and tumor-associated antigens are often derived from proteins directly involved in the conversion of normal cells to tumor cells due to their functions in cell cycle control or apoptosis inhibition. In addition, the downstream targets of these proteins that directly cause transformation events may be up-regulated, and therefore may be indirectly related to tumors. These indirect tumor-associated antigens may also be targets of vaccination methods (Singh-Jasuja et al., 2004). It is essential that the epitope is present in the amino acid sequence of the antigen to ensure that this peptide derived from the tumor-associated antigen ("immunogenic peptide") can cause a T cell response in vitro or in vivo.

基本上，任何能與MHC分子結合的肽都可能充當一個T細胞表位。誘導體外或體內T細胞反應的前提是存在具有相應TCR的T細胞並且不存在對該特定表位的免疫耐受性。Basically, any peptide that can bind to MHC molecules can act as a T cell epitope. The prerequisite for inducing T cell response in vitro or in vivo is the presence of T cells with corresponding TCR and the absence of immune tolerance to the specific epitope.

因此，TAA是基於T細胞療法（包括但不限於腫瘤疫苗）研發的起點。識別和表徵TAA的方法通常基於對患者或健康受試者T細胞的使用情況，或基於腫瘤與正常組織肽之間差別轉錄特性或差別表達模式的產生。然而，對腫瘤組織或人腫瘤細胞株中過量表達或選擇性表達的基因的識別並不提供在免疫療法中使用這些基因所轉錄抗原的準確資訊。這是因為，有著相應TCR的T細胞必須要存在而且對這個特定表位的免疫耐受性必須不存在或為最低水準，因此，這些抗原的表位只有一部分適合這種應用。因此，在本發明的一非常優選的實施例中，只選擇那些針對可發現功能性和/或增殖性T細胞情況的過量表現或選擇性表現肽，這一點非常重要。這種功能性T細胞被定義為在以特異性抗原刺激後能夠克隆地擴展並能夠執行效應子功能（「效應子T細胞」）的T細胞。Therefore, TAA is the starting point for the development of T cell therapies (including but not limited to tumor vaccines). The method of identifying and characterizing TAA is usually based on the use of T cells in patients or healthy subjects, or on the generation of differential transcription characteristics or differential expression patterns between tumor and normal tissue peptides. However, the identification of over-expressed or selectively expressed genes in tumor tissues or human tumor cell lines does not provide accurate information about the antigens transcribed from these genes in immunotherapy. This is because T cells with corresponding TCR must exist and the immune tolerance to this specific epitope must not exist or be at a minimum level. Therefore, only a part of these antigen epitopes are suitable for this application. Therefore, in a very preferred embodiment of the present invention, it is very important to select only those peptides that are overexpressed or selectively expressed for the condition where functional and/or proliferative T cells can be found. Such functional T cells are defined as T cells that can clonely expand and perform effector functions ("effector T cells") after stimulation with specific antigens.

在透過根據本發明的特定 TCR（例如可溶性 TCR）和抗體或其他結合分子（支架）靶向作用於肽-MHC 的情況下，潛在肽的免疫原性是次要的。在這些情況下，表現是決定因素。In the case of targeting peptide-MHC through a specific TCR (such as soluble TCR) and antibodies or other binding molecules (scaffolds) according to the present invention, the immunogenicity of the potential peptide is of secondary importance. In these cases, performance is the deciding factor.

在本發明的第一方面，本發明涉及一種肽，包含選自包括SEQ ID NO:1至SEQ ID NO:385的組的一個氨基酸序列、或該序列的與SEQ ID NO:1至SEQ ID NO:385具有至少77%，優選至少88%同源（優選至少77%或至少88%相同）的一種變體序列（其中所述變體與MHC結合和/或誘導T細胞與所述肽發生交叉反應），或其藥用鹽（其中所述肽不是潛在全長多肽）。In the first aspect of the present invention, the present invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 385, or the same sequence as SEQ ID NO: 1 to SEQ ID NO. : 385 has at least 77%, preferably at least 88% homology (preferably at least 77% or at least 88% identical) of a variant sequence (wherein the variant binds to MHC and/or induces T cells to cross with the peptide Reaction), or a pharmaceutically acceptable salt thereof (wherein the peptide is not a potential full-length polypeptide).

本發明進一步涉及本發明的一種肽，包含選自包括SEQ ID NO:1至SEQ ID NO:385的組的一個序列、或與SEQ ID NO:1至SEQ ID NO:385具有至少77%、優選至少88%同源性（優選為至少77%或至少88%相同）的一種變體，其中所述肽或其變體的總長度為8至100個、優選為8至30個、最優選為8至14個氨基酸。The present invention further relates to a peptide of the present invention, comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 385, or having at least 77% of SEQ ID NO: 1 to SEQ ID NO: 385, preferably A variant with at least 88% homology (preferably at least 77% or at least 88% identical), wherein the total length of the peptide or variant thereof is 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids.

下表顯示了根據本發明的肽、它們各自的 SEQ ID NO、以及這些肽的可能源（潛在）基因。表 1 和表 2 中的所有肽均與 HLA-A*02 結合。表 2 中的肽之前在大型列表中披露，作為高通量篩查結果，錯誤率高，或使用演算法計算出，但之前與癌症毫無關聯。表 3 中的肽是可與本發明其他肽組合使用的其他肽。表 4 中的肽還可用於診斷和/或治療各種其他惡性疾病，這些疾病涉及過量表達或過度表現各潛在多肽。 表 1：本發明中的肽 J =磷酸絲氨酸 序列ID號 序列 基因ID 正式基因符號 1 AIPPSFASIFL 3507 IGHM 2 ALHRPDVYL 3507 IGHM 3 VIAELPPKV 3507 IGHM 4 VIAELPPKVSV 3507 IGHM 5 ALIFKIASA 4214 MAP3K1 6 ALDTLEDDMTI 2222 FDFT1 7 ALLERTGYTL 10236,10492 HNRNPR, SYNCRIP 8 ALAASALPALV 6124 RPL4 9 ALCDTLITV 27033 ZBTB32 10 FVYGESVEL 27033 ZBTB32 11 ALFTFJPLTV 54665 RSBN1 12 ALGEDEITL 25909,285116 AHCTF1, AHCTF1P1 13 VVDGMPPGV 2969,2970 GTF2I, GTF2IP1 14 ALLRLLPGL 25920 COBRA1 15 ALPEVSVEA 79649 MAP7D3 16 ALPGGAAVAAV 79697 C14orf169 17 ALTKTNLQL 9750 FAM65B 18 LLGEFSIKM 9750 FAM65B 19 QVMEKLAAV 9750 FAM65B 20 ALVDPGPDFVV 546 ATRX 21 ALWAGLLTL 2208 FCER2 22 ALWDPVIEL 27340 UTP20 23 ALYLTEVFL 55024 BANK1 24 AMAGDVVYA 160365 CLECL1 25 ATYSGLESQSV 3185 HNRNPF 26 AVLLVLPLV 11322 TMC6 27 AVLGLVWLL 11322 TMC6 28 AVLHLLLSV 1535 CYBA 29 AVLQAVTAV 58155 PTBP2 30 ELLEGSEIYL 147007,23098 TMEM199, SARM1 31 ELMHGVAGV 165631 PARP15 32 FIDKFTPPV 3117,3122 HLA-DQA1, HLA-DRA 33 FIINSSNIFL 253959 RALGAPA1 34 YLPYIFPNI 253959 RALGAPA1 35 FILPSSLYL 81539 SLC38A1 36 FILTHVDQL 257106 ARHGAP30 37 FIMEGGAMVL 4174 MCM5 38 FIMEGGAMV 4174 MCM5 39 FLDALLTDV 79915 ATAD5 40 FLDEDDMSL 23141 ANKLE2 41 FLDPSLDPLL 58508,85318 MLL3, BAGE3 42 FLEEGGVVTV 5810 RAD1 43 FLLGPEALSFA 55183 RIF1 44 FLLSINDFL 157680 VPS13B 45 FLPELPADLEA 11064 CNTRL 46 YIIDSAQAV 11064 CNTRL 47 FLSDQPEPYL 160518,23258 DENND5B, DENND5A 48 FLSPQQPPLL 8621 CDK13 49 FLTDLFAQL 55671 SMEK1 50 FLFEPVVKAFL 55671 SMEK1 51 FLVEAPHDWDL 9790 BMS1 52 FLVETGFLHV 100268168,114987, 4602 WDR31, MYB 53 FLWQHVELVL 55181 SMG8 54 FLYPFPLALF 440026 TMEM41B 55 FMEPTLLML 23 ABCF1 56 FVFEAPYTL 139818 DOCK11 57 GLSEISLRL 139818 DOCK11 58 YIQQGIFSV 139818 DOCK11 59 FVFGDENGTVSL 79084 WDR77 60 FVLDHEDGLNL 4926 NUMA1 61 FVYFIVREV 285527 FRYL 62 GIIDGSPRL 80183 KIAA0226L 63 SLAHVAGCEL 80183 KIAA0226L 64 KLLESVASA 80183 KIAA0226L 65 GLDDMKANL 5079 PAX5 66 SLAGGLDDMKA 5079 PAX5 67 GLDDVTVEV 51019 CCDC53 68 GLDQQFAGLDL 1654 DDX3X 69 GLHQREIFL 9922 IQSEC1 70 FVPDTPVGV 9922 IQSEC1 71 GLKHDIARV 388512 CLEC17A 72 GLLDAGKMYV 911 CD1C 73 GLLEVISALQL 9816 URB2 74 GLLRASFLL 11344,54106 TWF2, TLR9 75 GLSIFAQDLRL 11344,54106 TWF2, TLR9 76 GLLRIIPYL 23352 UBR4 77 GLLRLTWFL 11214 AKAP13 78 GLPSFLTEV 100532732,4439 MSH5-SAPCD1, MSH5 79 GLQAKIQEA 23224 SYNE2 80 VLIEDELEEL 23224 SYNE2 81 WLVGQEFEL 23224 SYNE2 82 GLQSGVDIGV 1265 CNN2 83 GQGEVLVYV 2316 FLNA 84 GVMDVNTAL 391370,6206 RPS12P4, RPS12 85 HLMLHTAAL 80012 PHC3 86 HLYPGAVTI 55103 RALGPS2 87 HQIEAVDGEEL 10567 RABAC1 88 ILDEIGADVQA 29920 PYCR2 89 ILDFGTFQL 54832 VPS13C 90 VIADLGLIRV 54832 VPS13C 91 ILDLNTYNV 5336 PLCG2 92 ILEPLNPLL 9330 GTF3C3 93 ILFNTQINI 64801 ARV1 94 ILFPLRFTL 9675 TTI1 95 ILGYMAHEHKV 6530 SLC6A2 96 ILIDKTSFV 951 CD37 97 LLFATQITL 951 CD37 98 SLIKYFLFV 951 CD37 99 ILIFHSVAL 84636 GPR174 100 ILNNEVFAI 26999 CYFIP2 101 ILVVIEPLL 23451 SF3B1 102 IQDRAVPSL 3930 LBR 103 KLGGTPAPA 5218 CDK14 104 KLILLDTPLFL 253190,94009 SERHL2, SERHL 105 KLMNDIADI 472 ATM 106 FMASHLDYL 472 ATM 107 ILYNLYDLL 472 ATM 108 VIYTLIHYI 472 ATM 109 KLWEGLTELV 10235 RASGRP2 110 LLFDHLEPMEL 10235 RASGRP2 111 KLWNVAAPLYL 92105 INTS4 112 KTLDVDATYEI 3978 LIG1 113 KVPAEEVLVAV 91782 CHMP7 114 LIPEGPPQV 64224 HERPUD2 115 LLFDKLYLL 25885 POLR1A 116 LLIGATMQV 23225 NUP210 117 LLILENILL 23225 NUP210 118 RLLILENILL 23225 NUP210 119 VLPAEFFEV 23225 NUP210 120 AIDAALTSV 23225 NUP210 121 RLYEITIEV 23225 NUP210 122 LLIPVVPGV 55178 RNMTL1 123 LLLAEAELLTL 57724 EPG5 124 LLLEETEKQAV 92154,9788 MTSS1L, MTSS1 125 LLLEIGEVGKLFV 23158 TBC1D9 126 LLPEGGITAI 9904 RBM19 127 LLPTAPTTV 55729 ATF7IP 128 LLSEEEYHL 9793 CKAP5 129 LLVGTLDVV 23061,23158 TBC1D9B, TBC1D9 130 LLVLIPVYL 23344 ESYT1 131 LQALEVLKI 27304 MOCS3 132 LVYEAIIMV 27250 PDCD4 133 YLLSGDISEA 27250 PDCD4 134 MLLEHGITLV 200576 PIKFYVE 135 MTAGFSTIAGSV 64078 SLC28A3 136 NLDKLWTLV 389435,6157 RPL27AP6, RPL27A 137 NLIKTVIKL 25816 TNFAIP8 138 NLLDIDAPVTV 5530 PPP3CA 139 NLTDVVEKL 120892 LRRK2 140 QIAELPATSV 401145 CCSER1 141 QILSEIVEA 10724 MGEA5 142 QLDEPAPQV 387032,80317 ZKSCAN4, ZKSCAN3 143 QLLDTYFTL 7405 UVRAG 144 QLPPFPREL 8498 RANBP3 145 SALDTITTV 3832 KIF11 146 SIIEGPIIKL 23347 SMCHD1 147 SILETVATL 253714 MMS22L 148 SIVASLITV 221188 GPR114 149 SLDNGGYYI 4067 LYN 150 SLFDQPLSII 117289 TAGAP 151 SLFDSAYGA 2313 FLI1 152 SLIRILQTI 160518 DENND5B 153 SLLAELHVL 115352 FCRL3 154 SLLAELHVLTV 115352 FCRL3 155 SLMLEVPAL 1756 DMD 156 SLNIGDVQL 973 CD79A 157 SLNIRDFTM 147694,7565 ZNF548, ZNF17 158 SLPEAPLDV 29123 ANKRD11 159 SLQEEKLIYV 84668 FAM126A 160 SLSFLVPSL 9631 NUP155 161 SMDDGMINV 7011 TEP1 162 SMKDDLENV 3688 ITGB1 163 SQLDISEPYKV 3394 IRF8 164 SVHKGFAFV 101060301,3183,343069,440563,649330 HNRNPC, HNRNPCL1 165 TLDDDLDTV 51092 SIDT2 166 TLDPNQVSL 85464 SSH2 167 TLDTSKLYV 5923 RASGRF1 168 YLLDQSFVM 5923 RASGRF1 169 TLLLGLTEV 55423 SIRPG 170 TLTFRVETV 51379 CRLF3 171 TLVPPAALISI 6907 TBL1X 172 TLYDMLASI 10973 ASCC3 173 TVIENIHTI 23358 USP24 174 VLAELPIIVV 8295 TRRAP 175 FTVPRVVAV 8295 TRRAP 176 VLAEQNIIPSA 54855 FAM46C 177 VLDDRELLL 23178 PASK 178 VLFFNVQEV 57536 KIAA1328 179 VLLGLEMTL 1606 DGKA 180 LLKDGPEIGL 1606 DGKA 181 VLLSIPFVSV 94103 ORMDL3 182 VLLSVPGPPV 2264 FGFR4 183 VLMPTVYQQGV 54472 TOLLIP 184 VLSHNLYTV 50619 DEF6 185 VMDDQRDLI 972 CD74 186 VMDPTKILI 255967 PAN3 187 VMDTHLVNI 9779 TBC1D5 188 VMGDIPAAV 4678 NASP 189 VMLEMTPEL 3112,6891 HLA-DOB, TAP2 190 VVMGTVPRL 1729 DIAPH1 191 YIFDGSDGGV 5287 PIK3C2B 192 YIQEYLTLL 57674 RNF213 193 YLDLSNNRL 81793 TLR10 194 YLDNVLAEL 1840,23220 DTX1, DTX4 195 YLGGFALSV 6850 SYK 196 YLLLQTYVL 51259 TMEM216 197 YLQEVPILTL 1633 DCK 198 YLTFLPAEV 64135 IFIH1 199 YLVELSSLL 80279 CDK5RAP3 200 YMFEEVPIVI 8667 EIF3H 201 YQLELHGIEL 5698 PSMB9 202 YVDDVFLRV 10347 ABCA7 203 ALLSSQLAL 8925 HERC1 204 GLLQINDKIAL 5000 ORC4 205 GLSQANFTL 10288,10859,10990,11024,11025,11026,11027,23547,79166,79168 LILRB2, LILRB1, LILRB5, LILRA1, LILRB3, LILRA3, LILRA2, LILRA4, LILRP2, LILRA6 206 HMQDVRVLL 84824 FCRLA 207 IIADLDTTIMFA 7094,83660 TLN1, TLN2 208 ILLKTEGINL 166379 BBS12 209 ILQAELPSL 57508 INTS2 210 KLLVQDFFL 8564 KMO 211 LIDVKPLGV 134510 UBLCP1 212 NIIEAINELLV 8892 EIF2B2 213 RLLYQLVFL 3566 IL4R 214 RLQELTEKL 255394 TCP11L2 215 VMQDIVYKL 84759 PCGF1 216 WLAGDVPAA 24148 PRPF6 217 ALDEPPYLTV 51742 ARID4B 218 ALGEEWKGYVV 6194,728131 RPS6 219 ALLNLLESA 5788 PTPRC 220 ALPEILFAKV 643 CXCR5 221 ALVSTIIMV 80228 ORAI2 222 ALWELSLKI 2889 RAPGEF1 223 ALWVSQPPEI 259197 NCR3 224 AMEALVVEV 1660 DHX9 225 ILQERELLPV 1660 DHX9 226 AMNISVPQV 23387 SIK3 227 FLAEASVMTQL 1445 CSK 228 FLGGLSPGV 2968 GTF2H4 229 FLLNLQNCHL 55608 ANKRD10 230 FLQDSKVIFV 4772 NFATC1 231 FLYIRQLAI 26155,401010 NOC2L 232 FMHQIIDQV 79789 CLMN 233 GIIDINVRL 51163 DBR1 234 GLDDAEYAL 7376 NR1H2 235 GLDDLLLFL 8914 TIMELESS 236 GLLESGRHYL 9744 ACAP1 237 GLQENLDVVV 8881 CDC16 238 GLVETELQL 55690 PACS1 239 ILAGEMLSV 7203 CCT3 240 ILARDILEI 5714 PSMD8 241 ILGDILLKV 79831 KDM8 242 ILLGIQELL 7329 UBE2I 243 ILPTLEKELFL 79886 CAAP1 244 ILQALAVHL 54936 ADPRHL2 245 KIMDYSLLLGV 23396 PIP5K1C 246 KLDETGVAL 7155 TOP2B 247 KLKDRLPSI 81606 LBH 248 KTVEPPISQV 57466 SCAF4 249 LLPTGVFQV 51131 PHF11 250 LLVQEPDGLMV 404734,54882 ANKHD1-EIF4EBP3, ANKHD1 251 LLYDNVPGA 11262 SP140 252 NLLDPGSSYLL 5074 PAWR 253 NLWSVDGEVTV 92017 SNX29 254 QLIPKLIFL 57705 WDFY4 255 YLFEEAISM 57705 WDFY4 256 QLLPVSNVVSV 55374 TMCO6 257 RIINGIIISV 84329 HVCN1 258 RLDYITAEI 10471 PFDN6 259 RLLDEQFAVL 9026 HIP1R 260 SLDDVEGMSV 80196 RNF34 261 SLVEAQGWLV 161176 SYNE3 262 SLWNAGTSV 3495 IGHD 263 SQWEDIHVV 55843 ARHGAP15 264 TILDYINVV 89790,89858 SIGLEC10, SIGLEC12 265 TLLADDLEIKL 2124 EVI2B 266 TLLDQLDTQL 8678 BECN1 267 TLLDWQDSL 8289 ARID1A 268 TLLQVFHLL 63892 THADA 269 TLTDEQFLV 738 VPS51 270 TVLPVPPLSV 5430 POLR2A 271 VIRNIVEAA 100128168,100996747,389472,441502,6231,643003,728937,729188 RPS26P39, RPS26P32, RPS26P11, RPS26, RPS26P28, RPS26P25, RPS26P58 272 VLDELPPLI 55832,91689 CAND1, C22orf32 273 VLGEYSYLL 23431 AP4E1 274 VLLEYHIAYL 8087 FXR1 275 VLLFIEHSV 134265,7805 AFAP1L1, LAPTM5 276 VLNDGAPNV 117246 FTSJ3 277 VMILKLPFL 25929 GEMIN5 278 YLDDLLPKL 89910 UBE3B 279 YMAPEVVEA 2872 MKNK2 280 YTLDSLYWSV 81887 LAS1L The following table shows the peptides according to the invention, their respective SEQ ID NOs, and the possible source (potential) genes of these peptides. All peptides in Table 1 and Table 2 bind to HLA-A*02. The peptides in Table 2 were previously disclosed in a large list. As a result of high-throughput screening, the error rate was high, or they were calculated using algorithms, but they had nothing to do with cancer before. The peptides in Table 3 are other peptides that can be used in combination with other peptides of the present invention. The peptides in Table 4 can also be used to diagnose and/or treat various other malignant diseases, which involve overexpression or overexpression of each potential polypeptide. Table 1: Peptide J = phosphoserine in the present invention Serial ID number sequence Gene ID Official gene symbol 1 AIPPSFASIFL 3507 IGHM 2 ALHRPDVYL 3507 IGHM 3 VIAELPPKV 3507 IGHM 4 VIAELPPKVSV 3507 IGHM 5 ALIFKIASA 4214 MAP3K1 6 ALDTLEDDMTI 2222 FDFT1 7 ALLERTGYTL 10236,10492 HNRNPR, SYNCRIP 8 ALAASALPALV 6124 RPL4 9 ALCDTLITV 27033 ZBTB32 10 FVYGESVEL 27033 ZBTB32 11 ALFTFJPLTV 54665 RSBN1 12 ALGEDEITL 25909,285116 AHCTF1, AHCTF1P1 13 VVDGMPPGV 2969,2970 GTF2I, GTF2IP1 14 ALLRLLPGL 25920 COBRA1 15 ALPEVSVEA 79649 MAP7D3 16 ALPGGAAVAAV 79697 C14orf169 17 ALTKTNLQL 9750 FAM65B 18 LLGEFSIKM 9750 FAM65B 19 QVMEKLAAV 9750 FAM65B 20 ALVDPGPDFVV 546 ATRX twenty one ALWAGLLTL 2208 FCER2 twenty two ALWDPVIEL 27340 UTP20 twenty three ALYLTEVFL 55024 BANK1 twenty four AMAGDVVYA 160365 CLECL1 25 ATYSGLESQSV 3185 HNRNPF 26 AVLLVLPLV 11322 TMC6 27 AVLGLVWLL 11322 TMC6 28 AVLHLLLSV 1535 CYBA 29 AVLQAVTAV 58155 PTBP2 30 ELLEGSEIYL 147007,23098 TMEM199, SARM1 31 ELMHGVAGV 165631 PARP15 32 FIDKFTPPV 3117,3122 HLA-DQA1, HLA-DRA 33 FIINSSNIFL 253959 RALGAPA1 34 YLPYIFPNI 253959 RALGAPA1 35 FILPSSLYL 81539 SLC38A1 36 FILTHVDQL 257106 ARHGAP30 37 FIMEGGAMVL 4174 MCM5 38 FIMEGGAMV 4174 MCM5 39 FLDALLTDV 79915 ATAD5 40 FLDEDDMSL 23141 ANKLE2 41 FLDPSLDPLL 58508,85318 MLL3, BAGE3 42 FLEEGGVVTV 5810 RAD1 43 FLLGPEALSFA 55183 RIF1 44 FLLSINDFL 157680 VPS13B 45 FLPELPADLEA 11064 CNTRL 46 YIIDSAQAV 11064 CNTRL 47 FLSDQPEPYL 160518,23258 DENND5B, DENND5A 48 FLSPQQPPLL 8621 CDK13 49 FLTDLFAQL 55671 SMEK1 50 FLFEPVVKAFL 55671 SMEK1 51 FLVEAPHDWDL 9790 BMS1 52 FLVETGFLHV 100268168,114987, 4602 WDR31, MYB 53 FLWQHVELVL 55181 SMG8 54 FLYPFPLALF 440026 TMEM41B 55 FMEPTLLML twenty three ABCF1 56 FVFEAPYTL 139818 DOCK11 57 GLSEISLRL 139818 DOCK11 58 YIQQGIFSV 139818 DOCK11 59 FVFGDENGTVSL 79084 WDR77 60 FVLDHEDGLNL 4926 NUMA1 61 FVYFIVREV 285527 FRYL 62 GIIDGSPRL 80183 KIAA0226L 63 SLAHVAGCEL 80183 KIAA0226L 64 KLLESVASA 80183 KIAA0226L 65 GLDDMKANL 5079 PAX5 66 SLAGGLDDMKA 5079 PAX5 67 GLDDVTVEV 51019 CCDC53 68 GLDQQFAGLDL 1654 DDX3X 69 GLHQREIFL 9922 IQSEC1 70 FVPDTPVGV 9922 IQSEC1 71 GLKHDIARV 388512 CLEC17A 72 GLLDAGKMYV 911 CD1C 73 GLLEVISALQL 9816 URB2 74 GLLRASFLL 11344,54106 TWF2, TLR9 75 GLSIFAQDLRL 11344,54106 TWF2, TLR9 76 GLLRIIPYL 23352 UBR4 77 GLLRLTWFL 11214 AKAP13 78 GLPSFLTEV 100532732,4439 MSH5-SAPCD1, MSH5 79 GLQAKIQEA 23224 SYNE2 80 VLIEDELEEL 23224 SYNE2 81 WLVGQEFEL 23224 SYNE2 82 GLQSGVDIGV 1265 CNN2 83 GQGEVLVYV 2316 FLNA 84 GVMDVNTAL 391370,6206 RPS12P4, RPS12 85 HLMLHTAAL 80012 PHC3 86 HLYPGAVTI 55103 RALGPS2 87 HQIEAVDGEEL 10567 RABAC1 88 ILDEIGADVQA 29920 PYCR2 89 ILDFGTFQL 54832 VPS13C 90 VIADLGLIRV 54832 VPS13C 91 ILDLNTYNV 5336 PLCG2 92 ILEPLNPLL 9330 GTF3C3 93 ILFNTQINI 64801 ARV1 94 ILFPLRFTL 9675 TTI1 95 ILGYMAHEHKV 6530 SLC6A2 96 ILIDKTSFV 951 CD37 97 LLFATQITL 951 CD37 98 SLIKYFLFV 951 CD37 99 ILIFHSVAL 84636 GPR174 100 ILNNEVFAI 26999 CYFIP2 101 ILVVIEPLL 23451 SF3B1 102 IQDRAVPSL 3930 LBR 103 KLGGTPAPA 5218 CDK14 104 KLILLDTPLFL 253190,94009 SERHL2, SERHL 105 KLMNDIADI 472 ATM 106 FMASHLDYL 472 ATM 107 ILYNLYDLL 472 ATM 108 VIYTLIHYI 472 ATM 109 KLWEGLTELV 10235 RASGRP2 110 LLFDHLEPMEL 10235 RASGRP2 111 KLWNVAAPLYL 92105 INTS4 112 KTLDVDATYEI 3978 LIG1 113 KVPAEEVLVAV 91782 CHMP7 114 LIPEGPPQV 64224 HERPUD2 115 LLFDKLYLL 25885 POLR1A 116 LLIGATMQV 23225 NUP210 117 LLILENILL 23225 NUP210 118 RLLILENILL 23225 NUP210 119 VLPAEFFEV 23225 NUP210 120 AIDAALTSV 23225 NUP210 121 RLYEITIEV 23225 NUP210 122 LLIPVVPGV 55178 RNMTL1 123 LLLAEAELLTL 57724 EPG5 124 LLLEETEKQAV 92154,9788 MTSS1L, MTSS1 125 LLLEIGEVGKLFV 23158 TBC1D9 126 LLPEGGITAI 9904 RBM19 127 LLPTAPTTV 55729 ATF7IP 128 LLSEEEYHL 9793 CKAP5 129 LLVGTLDVV 23061,23158 TBC1D9B, TBC1D9 130 LLVLIPVYL 23344 ESYT1 131 LQALEVLKI 27304 MOCS3 132 LVYEAIIMV 27250 PDCD4 133 YLLSGDISEA 27250 PDCD4 134 MLLEHGITLV 200576 PIKFYVE 135 MTAGFSTIAGSV 64078 SLC28A3 136 NLDKLWTLV 389435,6157 RPL27AP6, RPL27A 137 NLIKTVIKL 25816 TNFAIP8 138 NLLDIDAPVTV 5530 PPP3CA 139 NLTDVVEKL 120892 LRRK2 140 QIAELPATSV 401145 CCSER1 141 QILSEIVEA 10724 MGEA5 142 QLDEPAPQV 387032,80317 ZKSCAN4, ZKSCAN3 143 QLLDTYFTL 7405 UVRAG 144 QLPPFPREL 8498 RANBP3 145 SALDTITTV 3832 KIF11 146 SIIEGPIIKL 23347 SMCHD1 147 SILETVATL 253714 MMS22L 148 SIVASLITV 221188 GPR114 149 SLDNGGYYI 4067 LYN 150 SLFDQPLSII 117289 TAGAP 151 SLFDSAYGA 2313 FLI1 152 SLIRILQTI 160518 DENND5B 153 SLLAELHVL 115352 FCRL3 154 SLLAELHVLTV 115352 FCRL3 155 SLMLEVPAL 1756 DMD 156 SLNIGDVQL 973 CD79A 157 SLNIRDFTM 147694,7565 ZNF548, ZNF17 158 SLPEAPLDV 29123 ANKRD11 159 SLQEEKLIYV 84668 FAM126A 160 SLSFLVPSL 9631 NUP155 161 SMDDGMINV 7011 TEP1 162 SMKDDLENV 3688 ITGB1 163 SQLDISEPYKV 3394 IRF8 164 SVHKGFAFV 101060301,3183,343069,440563,649330 HNRNPC, HNRNPCL1 165 TLDDDLDTV 51092 SIDT2 166 TLDPNQVSL 85464 SSH2 167 TLDTSKLYV 5923 RASGRF1 168 YLLDQSFVM 5923 RASGRF1 169 TLLLGLTEV 55423 SIRPG 170 TLTFRVETV 51379 CRLF3 171 TLVPPAALISI 6907 TBL1X 172 TLYDMLASI 10973 ASCC3 173 TVIENIHTI 23358 USP24 174 VLAELPIIVV 8295 TRRAP 175 FTVPRVVAV 8295 TRRAP 176 VLAEQNIIPSA 54855 FAM46C 177 VLDDRELLL 23178 PASK 178 VLFFNVQEV 57536 KIAA1328 179 VLLGLEMTL 1606 DGKA 180 LLKDGPEIGL 1606 DGKA 181 VLLSIPFVSV 94103 ORMDL3 182 VLLSVPGPPV 2264 FGFR4 183 VLMPTVYQQGV 54472 TOLLIP 184 VLSHNLYTV 50619 DEF6 185 VMDDQRDLI 972 CD74 186 VMDPTKILI 255967 PAN3 187 VMDTHLVNI 9779 TBC1D5 188 VMGDIPAAV 4678 NASP 189 VMLEMTPEL 3112,6891 HLA-DOB, TAP2 190 VVMGTVPRL 1729 DIAPH1 191 YIFDGSDGGV 5287 PIK3C2B 192 YIQEYLTLL 57674 RNF213 193 YLDLSNNRL 81793 TLR10 194 YLDNVLAEL 1840,23220 DTX1, DTX4 195 YLGGFALSV 6850 SYK 196 YLLLQTYVL 51259 TMEM216 197 YLQEVPILTL 1633 DCK 198 YLTFLPAEV 64135 IFIH1 199 YLVELSSLL 80279 CDK5RAP3 200 YMFEEVPIVI 8667 EIF3H 201 YQLELHGIEL 5698 PSMB9 202 YVDDVFLRV 10347 ABCA7 203 ALLSSQLAL 8925 HERC1 204 GLLQINDKIAL 5000 ORC4 205 GLSQANFTL 10288,10859,10990,11024,11025,11026,11027,23547,79166,79168 LILRB2, LILRB1, LILRB5, LILRA1, LILRB3, LILRA3, LILRA2, LILRA4, LILRP2, LILRA6 206 HMQDVRVLL 84824 FCRLA 207 IIADLDTTIMFA 7094,83660 TLN1, TLN2 208 ILLKTEGINL 166379 BBS12 209 ILQAELPSL 57508 INTS2 210 KLLVQDFFL 8564 KMO 211 LIDVKPLGV 134510 UBLCP1 212 NIIEAINELLV 8892 EIF2B2 213 RLLYQLVFL 3566 IL4R 214 RLQELTEKL 255394 TCP11L2 215 VMQDIVYKL 84759 PCGF1 216 WLAGDVPAA 24148 PRPF6 217 ALDEPPYLTV 51742 ARID4B 218 ALGEEWKGYVV 6194,728131 RPS6 219 ALLNLLESA 5788 PTPRC 220 ALPEILFAKV 643 CXCR5 221 ALVSTIIMV 80228 ORAI2 222 ALWELSLKI 2889 RAPGEF1 223 ALWVSQPPEI 259197 NCR3 224 AMEALVVEV 1660 DHX9 225 ILQERELLPV 1660 DHX9 226 AMNISVPQV 23387 SIK3 227 FLAEASVMTQL 1445 CSK 228 FLGGLSPGV 2968 GTF2H4 229 FLLNLQNCHL 55608 ANKRD10 230 FLQDSKVIFV 4772 NFATC1 231 FLYIRQLAI 26155,401010 NOC2L 232 FMHQIIDQV 79789 CLMN 233 GIIDINVRL 51163 DBR1 234 GLDDAEYAL 7376 NR1H2 235 GLDDLLLFL 8914 TIMELESS 236 GLLESGRHYL 9744 ACAP1 237 GLQENLDVVV 8881 CDC16 238 GLVETELQL 55690 PACS1 239 ILAGEMLSV 7203 CCT3 240 ILARDILEI 5714 PSMD8 241 ILGDILLKV 79831 KDM8 242 ILLGIQELL 7329 UBE2I 243 ILPTLEKELFL 79886 CAAP1 244 ILQALAVHL 54936 ADPRHL2 245 KIMDYSLLLGV 23396 PIP5K1C 246 KLDETGVAL 7155 TOP2B 247 KLKDRLPSI 81606 LBH 248 KTVEPPISQV 57466 SCAF4 249 LLPTGVFQV 51131 PHF11 250 LLVQEPDGLMV 404734,54882 ANKHD1-EIF4EBP3, ANKHD1 251 LLYDNVPGA 11262 SP140 252 NLLDPGSSYLL 5074 PAWR 253 NLWSVDGEVTV 92017 SNX29 254 QLIPKLIFL 57705 WDFY4 255 YLFEEAISM 57705 WDFY4 256 QLLPVSNVVSV 55374 TMCO6 257 RIINGIIISV 84329 HVCN1 258 RLDYITAEI 10471 PFDN6 259 RLLDEQFAVL 9026 HIP1R 260 SLDDVEGMSV 80196 RNF34 261 SLVEAQGWLV 161176 SYNE3 262 SLWNAGTSV 3495 IGHD 263 SQWEDIHVV 55843 ARHGAP15 264 TILDYINVV 89790,89858 SIGLEC10, SIGLEC12 265 TLLADDLEIKL 2124 EVI2B 266 TLLDQLDTQL 8678 BECN1 267 TLLDWQDSL 8289 ARID1A 268 TLLQVFHLL 63892 THADA 269 TLTDEQFLV 738 VPS51 270 TVLPVPPLSV 5430 POLR2A 271 VIRNIVEAA 100128168,100996747,389472,441502,6231,643003,728937,729188 RPS26P39, RPS26P32, RPS26P11, RPS26, RPS26P28, RPS26P25, RPS26P58 272 VLDELPPLI 55832,91689 CAND1, C22orf32 273 VLGEYSYLL 23431 AP4E1 274 VLLEYHIAYL 8087 FXR1 275 VLLFIEHSV 134265,7805 AFAP1L1, LAPTM5 276 VLNDGAPNV 117246 FTSJ3 277 VMILKLPFL 25929 GEMIN5 278 YLDDLLPKL 89910 UBE3B 279 YMAPEVVEA 2872 MKNK2 280 YTLDSLYWSV 81887 LAS1L

KIAA0226L（也稱作 C13orf18）編碼 KIAA0226 樣基因，位於染色體 13q14.13 上 (RefSeq, 2002)。KIAA0226L 被認為是一種腫瘤抑制基因，在宮頸癌中超甲基化。KIAA0226L 重新活化導致細胞生長、活力和集落形成下降 (Huisman et al., 2015; Eijsink et al., 2012; Huisman et al., 2013)。KIAA0226L 的甲基化模式可用於分辨前體病變和正常宮頸癌 (Milutin et al., 2015)。KIAA0226L 的甲基化模式不可用作宮頸癌的特定生物標誌物 (Sohrabi et al., 2014)。KIAA0226L 重新活化部分去甲基化其啟動子區域，也降低壓制性組蛋白甲基化 (Huisman et al., 2013)。KIAA0226L (also called C13orf18) encodes a KIAA0226-like gene located on chromosome 13q14.13 (RefSeq, 2002). KIAA0226L is considered to be a tumor suppressor gene and is hypermethylated in cervical cancer. Reactivation of KIAA0226L leads to decreased cell growth, viability and colony formation (Huisman et al., 2015; Eijsink et al., 2012; Huisman et al., 2013). The methylation pattern of KIAA0226L can be used to distinguish between precursor lesions and normal cervical cancer (Milutin et al., 2015). The methylation pattern of KIAA0226L cannot be used as a specific biomarker for cervical cancer (Sohrabi et al., 2014). KIAA0226L reactivates partially demethylated its promoter region and also reduces repressive histone methylation (Huisman et al., 2013).

FCRL3，也稱為 FCRH3 或 IRTA3，編碼 Fc 受體樣 3，這是免疫球蛋白受體超家族的幾種 FC 受體樣糖蛋白之一，其可能在免疫系統調節中起作用 (RefSeq, 2002)。FCRL3 在高腫瘤負荷的 Sézary 綜合征患者中的 CD4 + CD26–T細胞上上調，這表明FCRL3 表達與高迴圈腫瘤負荷相關 (Wysocka et al., 2014)。FCRL3 在慢性淋巴細胞白血病中上調  (Polson et al., 2006)。FCRL3是慢性淋巴細胞白血病中具有預後相關性的潛在標誌物 (Zucchetto et al., 2011)。 表 2：本發明中的其他 肽，之前與癌症無已知的關聯。 序列ID號 序列 基因 ID 正式基因符號 281 NLLDDRGMTAL 146206 RLTPR 282 LLRDGIELV 152926 PPM1K 283 ILQPMDIHV 54832 VPS13C 284 LLSAAEPVPA 974 CD79B 285 GVATAGCVNEV 3394 IRF8 286 FLLEDLSQKL 2177 FANCD2 287 FLWEEKFNSL 10750 GRAP 288 GLAESTGLLAV 50855,84552 PARD6A, PARD6G 289 ILEEQPMDMLL 84301 DDI2 290 LANPHELSL 84301 DDI2 291 ILLNEDDLVTI 57724 EPG5 292 AAALIIHHV 25942 SIN3A 293 ALDIMIPMV 4217 MAP3K5 294 ALLDQLHTLL 8202 NCOA3 295 ALLQKLQQL 122618 PLD4 296 FIAPTGHSL 23157,55752 SEPT6, SEPT11 297 FLVEPQEDTRL 8237 USP11 298 IILPVEVEV 23505 TMEM131 299 ILEENIPVL 55653 BCAS4 300 ILLNPAYDVYL 23225 NUP210 301 AASPIITLV 23225 NUP210 302 ILQDLTFVHL 2889 RAPGEF1 303 ILSQPTPSL 29028 ATAD2 304 LAIVPVNTL 23132 RAD54L2 305 LLFPQIEGIKI 26263 FBXO22 306 VVAEELENV 26263 FBXO22 307 LLLTKPTEA 5336 PLCG2 308 SLYDVSRMYV 5336 PLCG2 309 ILYGTQFVL 5336 PLCG2 310 LLSTLHLLV 64098 PARVG 311 LLVDVEPKV 100129492,6632 SNRPD1 312 LLYNSTDPTL 64005 MYO1G 313 LMADLEGLHL 8546 AP3B1 314 LMKDCEAEV 5518 PPP2R1A 315 LVYEAPETV 57448 BIRC6 316 MLLEHGITL 200576 PIKFYVE 317 NLLAHIWAL 58513 EPS15L1 318 NLQVTQPTV 100505503,402057,442216,6218 RPS17L, RPS17P16, RPS17P5, RPS17 319 QVIPQLQTV 6671 SP4 320 TIAPVTVAV 6671 SP4 321 RLLEFELAQL 6093 ROCK1 322 SLASIHVPL 23163 GGA3 323 SLDLFNCEV 10541,23520,646791,647020,723972,8125 ANP32B, ANP32C, ANP32BP1, ANP32AP1, ANP32A 324 SLYSALQQA 11190 CEP250 325 TLENGVPCV 23240 KIAA0922 326 VLAFLVHEL 11176 BAZ2A 327 VLIKWFPEV 54509 RHOF 328 VLLPQETAEIHL 5292 PIM1 329 VLMDGSVKL 11184 MAP4K1 330 VLMWEIYSL 695 BTK 331 VLWELAHLPTL 23358 USP24 332 VMIQHVENL 4033 LRMP 333 VTLEFPQLIRV 126382 NR2C2AP 334 YLLEEKIASL 51199 NIN 335 YLYQEQYFI 10668 CGRRF1 336 YMAVTTQEV 79840 NHEJ1 337 YMYEKESEL 22806 IKZF3 338 FLDMTNWNL 64771 C6orf106 339 GLWGTVVNI 25914 RTTN 340 KLLEEICNL 10075 HUWE1 341 LLAELPASVHA 2319 FLOT2 342 SLITPLQAV 5591 PRKDC 343 TLLEALDCI 158078,1915 EEF1A1P5, EEF1A1 344 VLAFENPQV 25852 ARMC8 345 YLIEPDVELQRI 25852 ARMC8 346 VLVQVSPSL 23404 EXOSC2 347 YLGPVSPSL 4154 MBNL1 348 ALAKPPVVSV 57634 EP400 349 ALATHILSL 11215 AKAP11 350 ALEDRVWEL 285025 CCDC141 351 ALSEKLARL 115106 HAUS1 352 ALVFELHYV 57705 WDFY4 353 ATPMPTPSV 55206 SBNO1 354 FIMDDPAGNSYL 8882 ZNF259 355 FIWPMLIHI 11325 DDX42 356 FLHDHQAEL 259197 NCR3 357 FLIQEIKTL 23352 UBR4 358 FLTDYLNDL 54880 BCOR 359 FMQDPMEVFV 10212 DDX39A 360 HLIDTNKIQL 55619 DOCK10 361 ILQEFESKL 2074,267004 ERCC6, PGBD3 362 ILTELGGFEV 51366 UBR5 363 ITTEVVNELYV 57222 ERGIC1 364 KMDWIFHTI 5290 PIK3CA 365 LISPLLLPV 144132 DNHD1 366 LLSETCVTI 23469 PHF3 367 NLWSLVAKV 9889 ZBED4 368 QLQPTDALLCV 125950 RAVER1 369 RLLDLENSLLGL 54899 PXK 370 SIFASPESV 10285 SMNDC1 371 SLADDSVLERL 23195 MDN1 372 SLFGPLPGPGPALV 9595 CYTIP 373 TLLADQGEIRV 9219 MTA2 374 VLSVITEEL 55621 TRMT1 375 VLWFKPVEL 55720 TSR1 376 VLYNQRVEEI 6730 SRP68 377 VVDGTCVAV 57589 KIAA1432 378 YILGKFFAL 8914 TIMELESS 379 YLAELVTPIL 1981 EIF4G1 380 YLDRKLLTL 6850 SYK 381 YLLEENKIKL 139818 DOCK11 382 YLLPLLQRL 55794 DDX28 383 YLLREWVNL 23019 CNOT1 384 YMIGSEVGNYL 6598 SMARCB1 385 YTIPLAIKL 128869 PIGU 表 3：用於例如個性化癌症療法的肽 序列ID號 序列 基因 ID 正式基因符號 386 FLGDYVENL 54832 VPS13C 387 YLILSSHQL 1269 CNR2 388 GLLSALENV 1269 CNR2 389 GLAALAVHL 2175 FANCA 390 GLEERLYTA 29933 GPR132 391 FLLEREQLL 165055 CCDC138 392 FLWERPTLLV 79922 MRM1 393 FVMEGEPPKL 348654 GEN1 394 ILSTEIFGV 79703 C11orf80 395 ALYGKLLKL 157680 VPS13B 396 TLLGKQVTL 157680 VPS13B 397 FLPPEHTIVYI 9896 FIG4 398 FLAELPGSLSL 5326 PLAGL2 399 ALAAPDIVPAL 79886 CAAP1 400 ALFQPHLINV 10097 ACTR2 401 AMADKMDMSL 10189 ALYREF 402 LLVSNLDFGV 10189 ALYREF 403 FIMPATVADATAV 23352 UBR4 404 FLQPDLDSL 10514 MYBBP1A 405 FLVEKQPPQV 6778 STAT6 406 FLWPKEVEL 146206 RLTPR 407 FMIDASVHPTL 221960,51622 CCZ1B, CCZ1 408 FMVDNEAIYDI 10376,112714,113457,51807,7277,7278,7846,84790 TUBA1B, TUBA3E, TUBA3D, TUBA8, TUBA4A, TUBA3C, TUBA1A, TUBA1C 409 GLDAATATV 9869 SETDB1 410 GLFDGVPTTA 122618 PLD4 411 GLFEDVTQPGILL 23140 ZZEF1 412 ILGTEDLIVEV 79719 AAGAB 413 ILLEHGADPNL 22852 ANKRD26 414 ILSVVNSQL 80183 KIAA0226L 415 ILVTSIFFL 643 CXCR5 416 IMEDIILTL 1656 DDX6 417 IQIGEETVITV 2316 FLNA 418 IVTEIISEI 64151 NCAPG 419 LLNEILEQV 64151 NCAPG 420 KLIDDQDISISL 80208 SPG11 421 KLWTGGLDNTV 7088,7090,7091 TLE1, TLE3, TLE4 422 KQFEGTVEI 675 BRCA2 423 LLIGTDVSL 9730 VPRBP 424 LLPPLESLATV 5518 PPP2R1A 425 LLSDVRFVL 53339 BTBD1 426 LLWGNLPEI 653820,729533 FAM72B, FAM72A 427 LLYDAVHIV 2899 GRIK3 428 LMYPYIYHV 54954 FAM120C 429 NLLETKLQL 219988 PATL1 430 QLIEKNWLL 56992 KIF15 431 RMVAEIQNV 11262 SP140 432 SAVDFIRTL 9263 STK17A 433 SILDRDDIFV 8237 USP11 434 SIQQSIERLLV 4809 NHP2L1 435 SLFNQEVQI 100528032,22914,8302 KLRK1, KLRC4 436 SLFSSLEPQIQPV 23029 RBM34 437 SLEPQIQPV 23029 RBM34 438 SLFIGEKAVLL 23029 RBM34 439 SLLDLHTKV 27340 UTP20 440 SLLEQGKEPWMV 147949,163087,342892,374899, 84503 ZNF583, ZNF383, ZNF850, ZNF829, ZNF527 441 SLLEVNEASSV 149628 PYHIN1 442 SLNDLEKDVMLL 6597 SMARCA4 443 SLTIDGIYYV 1659 DHX8 444 SMSGYDQVL 3187,3188 HNRNPH1, HNRNPH2 445 VIIKGLEEITV 3832 KIF11 446 VILTSSPFL 10800 CYSLTR1 447 VLDDSIYLV 57565 KLHL14 448 VMNDRLYAI 57565 KLHL14 449 LLDAMNYHL 57565 KLHL14 450 VLGPGPPPL 254359 ZDHHC24 451 VLIEYNFSI 55215 FANCI 452 VLLSLLEKV 1130 LYST 453 SLLPLVWKI 1130 LYST 454 VLMDEGAVLTL 54596 L1TD1 455 VLPDPEVLEAV 57326 PBXIP1 456 YAAPGGLIGV 1968,255308 EIF2S3 457 YIMEPSIFNTL 51497 TH1L 458 YIQEHLLQI 10625 IVNS1ABP 459 YLDFSNNRL 51284 TLR7 460 YLEDGFAYV 5558 PRIM2 461 YLLNLNHLGL 23471 TRAM1 462 YVLPKLYVKL 100128168,100996747,441502,6231,643003,644166,644928,644934,646753,728937, 729188 RPS26P39, RPS26P11, RPS26, RPS26P28, RPS26P20, RPS26P15, RPS26P50, RPS26P2, RPS26P25, RPS26P58 463 ALMAVVSGL 55103 RALGPS2 464 ALSDETKNNWEV 5591 PRKDC 465 FLYDEIEAEVNL 23191,26999 CYFIP1, CYFIP2 466 FLYDEIEAEV 23191,26999 CYFIP1, CYFIP2 467 IMQDFPAEIFL 25914 RTTN 468 KIQEILTQV 10643 IGF2BP3 469 SLDGTELQL 284114 TMEM102 470 TLTNIIHNL 94101 ORMDL1 471 VLLAAGPSAA 23225 NUP210 472 YLFESEGLVL 283431 GAS2L3 473 ALADDDFLTV 4173 MCM4 474 ALADLIEKELSV 4850 CNOT4 475 ALDDMISTL 7203 CCT3 476 ALITEVVRL 26005 C2CD3 477 ALLDQTKTLAESAL 7094 TLN1 478 AMFESSQNVLL 64328 XPO4 479 YLAHFIEGL 64328 XPO4 480 FLAEDPKVTL 60489 APOBEC3G 481 FLIDTSASM 203522,26512 DDX26B, INTS6 482 FLTDLEDLTL 26151 NAT9 483 FLTEMVHFI 93517 SDR42E1 484 FLVDGPRVQL 90204 ZSWIM1 485 GLLDCPIFL 2177 FANCD2 486 GLLDLPFRVGV 23347 SMCHD1 487 GLSDGNPSL 79684 MSANTD2 488 GVYDGEEHSV 4113 MAGEB2 489 HLANIVERL 117854,445372,53840 TRIM6, TRIM6-TRIM34, TRIM34 490 ILDEKPVII 23460 ABCA6 491 ILFSEDSTKLFV 84916 CIRH1A 492 ILLDDNMQIRL 5261 PHKG2 493 LIIDQADIYL 27042 DIEXF 494 LLGDSSFFL 283254 HARBI1 495 LLLDEEGTFSL 27013 CNPPD1 496 LLVEQPPLAGV 4773 NFATC2 497 LLWDVPAPSL 1388,7148 ATF6B, TNXB 498 LTAPPEALLMV 79050 NOC4L 499 NLMEMVAQL 55636 CHD7 500 QIITSVVSV 5378 PMS1 501 QLALKVEGV 25896 INTS7 502 QLLDETSAITL 10915 TCERG1 503 QLYEEPDTKL 10270 AKAP8 504 RLYSGISGLEL 84172 POLR1B 505 RVLEVGALQAV 25885 POLR1A 506 SLLPLDDIVRV 3708,3709 ITPR1, ITPR2 507 SLLTEQDLWTV 90806 ANGEL2 508 SVDSAPAAV 10635 RAD51AP1 509 TLQEVVTGV 55750 AGK 510 TVDVATPSV 8924 HERC2 511 VLAYFLPEA 4171 MCM2 512 VLISVLQAI 26999 CYFIP2 513 YLIPFTGIVGL 26001 RNF167 514 YLLDDGTLVV 8872 CDC123 515 YLMPGFIHL 168400,55510 DDX53, DDX43 516 YLPDIIKDQKA 5496 PPM1G 517 YLQLTQSEL 283237 TTC9C 518 YLTEVFLHVV 55024 BANK1 519 YLVEGRFSV 55125 CEP192 520 YLVYILNEL 51202 DDX47 521 YLYGQTTTYL 7153 TOP2A 522 YVLTQPPSV 28796,28815,28831,3537,3538 IGLV3-21, IGLV2-14, IGLJ3, IGLC1, IGLC2 FCRL3, also known as FCRH3 or IRTA3, encodes Fc receptor-like 3, which is one of several FC receptor-like glycoproteins of the immunoglobulin receptor superfamily, which may play a role in immune system regulation (RefSeq, 2002 ). FCRL3 is up-regulated on CD4 + CD26–T cells in Sézary syndrome patients with high tumor burden, which indicates that FCRL3 expression is associated with high loop tumor burden (Wysocka et al., 2014). FCRL3 is upregulated in chronic lymphocytic leukemia (Polson et al., 2006). FCRL3 is a potential marker with prognostic relevance in chronic lymphocytic leukemia (Zucchetto et al., 2011). Table 2: The other peptides of the present invention have no known association with cancer before. Serial ID number sequence Gene ID Official gene symbol 281 NLLDDRGMTAL 146206 RLTPR 282 LLRDGIELV 152926 PPM1K 283 ILQPMDIHV 54832 VPS13C 284 LLSAAEPVPA 974 CD79B 285 GVATAGCVNEV 3394 IRF8 286 FLLEDLSQKL 2177 FANCD2 287 FLWEEKFNSL 10750 GRAP 288 GLAESTGLLAV 50855,84552 PARD6A, PARD6G 289 ILEEQPMDMLL 84301 DDI2 290 LANPHELSL 84301 DDI2 291 ILLNEDDLVTI 57724 EPG5 292 AAALIIHHV 25942 SIN3A 293 ALDIMIPMV 4217 MAP3K5 294 ALLDQLHTLL 8202 NCOA3 295 ALLQKLQQL 122618 PLD4 296 FIAPTGHSL 23157,55752 SEPT6, SEPT11 297 FLVEPQEDTRL 8237 USP11 298 IILPVEVEV 23505 TMEM131 299 ILEENIPVL 55653 BCAS4 300 ILLNPAYDVYL 23225 NUP210 301 AASPIITLV 23225 NUP210 302 ILQDLTFVHL 2889 RAPGEF1 303 ILSQPTPSL 29028 ATAD2 304 LAIVPVNTL 23132 RAD54L2 305 LLFPQIEGIKI 26263 FBXO22 306 VVAEELENV 26263 FBXO22 307 LLLTKPTEA 5336 PLCG2 308 SLYDVSRMYV 5336 PLCG2 309 ILYGTQFVL 5336 PLCG2 310 LLSTLHLLV 64098 PARVG 311 LLVDVEPKV 100129492,6632 SNRPD1 312 LLYNSTDPTL 64005 MYO1G 313 LMADLEGLHL 8546 AP3B1 314 LMKDCEAEV 5518 PPP2R1A 315 LVYEAPETV 57448 BIRC6 316 MLLEHGITL 200576 PIKFYVE 317 NLLAHIWAL 58513 EPS15L1 318 NLQVTQPTV 100505503,402057,442216,6218 RPS17L, RPS17P16, RPS17P5, RPS17 319 QVIPQLQTV 6671 SP4 320 TIAPVTVAV 6671 SP4 321 RLLEFELAQL 6093 ROCK1 322 SLASIHVPL 23163 GGA3 323 SLDLFNCEV 10541,23520,646791,647020,723972,8125 ANP32B, ANP32C, ANP32BP1, ANP32AP1, ANP32A 324 SLYSALQQA 11190 CEP250 325 TLENGVPCV 23240 KIAA0922 326 VLAFLVHEL 11176 BAZ2A 327 VLIKWFPEV 54509 RHOF 328 VLLPQETAEIHL 5292 PIM1 329 VLMDGSVKL 11184 MAP4K1 330 VLMWEIYSL 695 BTK 331 VLWELAHLPTL 23358 USP24 332 VMIQHVENL 4033 LRMP 333 VTLEFPQLIRV 126382 NR2C2AP 334 YLLEEKIASL 51199 NIN 335 YLYQEQYFI 10668 CGRRF1 336 YMAVTTQEV 79840 NHEJ1 337 YMYEKESEL 22806 IKZF3 338 FLDMTNWNL 64771 C6orf106 339 GLWGTVVNI 25914 RTTN 340 KLLEEICNL 10075 HUWE1 341 LLAELPASVHA 2319 FLOT2 342 SLITPLQAV 5591 PRKDC 343 TLLEALDCI 158078,1915 EEF1A1P5, EEF1A1 344 VLAFENPQV 25852 ARMC8 345 YLIEPDVELQRI 25852 ARMC8 346 VLVQVSPSL 23404 EXOSC2 347 YLGPVSPSL 4154 MBNL1 348 ALAKPPVVSV 57634 EP400 349 ALATHILSL 11215 AKAP11 350 ALEDRVWEL 285025 CCDC141 351 ALSEKLARL 115106 HAUS1 352 ALVFELHYV 57705 WDFY4 353 ATPMPTPSV 55206 SBNO1 354 FIMDDPAGNSYL 8882 ZNF259 355 FIWPMLIHI 11325 DDX42 356 FLHDHQAEL 259197 NCR3 357 FLIQEIKTL 23352 UBR4 358 FLTDYLNDL 54880 BCOR 359 FMQDPMEVFV 10212 DDX39A 360 HLIDTNKIQL 55619 DOCK10 361 ILQEFESKL 2074,267004 ERCC6, PGBD3 362 ILTELGGFEV 51366 UBR5 363 ITTEVVNELYV 57222 ERGIC1 364 KMDWIFHTI 5290 PIK3CA 365 LISPLLLPV 144132 DNHD1 366 LLSETCVTI 23469 PHF3 367 NLWSLVAKV 9889 ZBED4 368 QLQPTDALLCV 125950 RAVER1 369 RLLDLENSLLGL 54899 PXK 370 SIFASPESV 10285 SMNDC1 371 SLADDSVLERL 23195 MDN1 372 SLFGPLPGPGPALV 9595 CYTIP 373 TLLADQGEIRV 9219 MTA2 374 VLSVITEEL 55621 TRMT1 375 VLWFKPVEL 55720 TSR1 376 VLYNQRVEEI 6730 SRP68 377 VVDGTCVAV 57589 KIAA1432 378 YILGKFFAL 8914 TIMELESS 379 YLAELVTPIL 1981 EIF4G1 380 YLDRKLLTL 6850 SYK 381 YLLEENKIKL 139818 DOCK11 382 YLLPLLQRL 55794 DDX28 383 YLLREWVNL 23019 CNOT1 384 YMIGSEVGNYL 6598 SMARCB1 385 YTIPLAIKL 128869 PIGU Table 3: Peptides for eg personalized cancer therapy Serial ID number sequence Gene ID Official gene symbol 386 FLGDYVENL 54832 VPS13C 387 YLILSSHQL 1269 CNR2 388 GLLSALENV 1269 CNR2 389 GLAALAVHL 2175 FANCA 390 GLEERLYTA 29933 GPR132 391 FLLEREQLL 165055 CCDC138 392 FLWERPTLLV 79922 MRM1 393 FVMEGEPPKL 348654 GEN1 394 ILSTEIFGV 79703 C11orf80 395 ALYGKLLKL 157680 VPS13B 396 TLLGKQVTL 157680 VPS13B 397 FLPPEHTIVYI 9896 FIG4 398 FLAELPGSLSL 5326 PLAGL2 399 ALAAPDIVPAL 79886 CAAP1 400 ALFQPHLINV 10097 ACTR2 401 AMADKMDMSL 10189 ALYREF 402 LLVSNLDFGV 10189 ALYREF 403 FIMPATVADATAV 23352 UBR4 404 FLQPDLDSL 10514 MYBBP1A 405 FLVEKQPPQV 6778 STAT6 406 FLWPKEVEL 146206 RLTPR 407 FMIDASVHPTL 221960,51622 CCZ1B, CCZ1 408 FMVDNEAIYDI 10376,112714,113457,51807,7277,7278,7846,84790 TUBA1B, TUBA3E, TUBA3D, TUBA8, TUBA4A, TUBA3C, TUBA1A, TUBA1C 409 GLDAATATV 9869 SETDB1 410 GLFDGVPTTA 122618 PLD4 411 GLFEDVTQPGILL 23140 ZZEF1 412 ILGTEDLIVEV 79719 AAGAB 413 ILLEHGADPNL 22852 ANKRD26 414 ILSVVNSQL 80183 KIAA0226L 415 ILVTSIFFL 643 CXCR5 416 IMEDIILTL 1656 DDX6 417 IQIGEETVITV 2316 FLNA 418 IVTEIISEI 64151 NCAPG 419 LLNEILEQV 64151 NCAPG 420 KLIDDQDISISL 80208 SPG11 421 KLWTGGLDNTV 7088,7090,7091 TLE1, TLE3, TLE4 422 KQFEGTVEI 675 BRCA2 423 LLIGTDVSL 9730 VPRBP 424 LLPPLESLATV 5518 PPP2R1A 425 LLSDVRFVL 53339 BTBD1 426 LLWGNLPEI 653820,729533 FAM72B, FAM72A 427 LLYDAVHIV 2899 GRIK3 428 LMYPYIYHV 54954 FAM120C 429 NLLETKLQL 219988 PATL1 430 QLIEKNWLL 56992 KIF15 431 RMVAEIQNV 11262 SP140 432 SAVDFIRTL 9263 STK17A 433 SILDRDDIFV 8237 USP11 434 SIQQSIERLLV 4809 NHP2L1 435 SLFNQEVQI 100528032,22914,8302 KLRK1, KLRC4 436 SLFSSLEPQIQPV 23029 RBM34 437 SLEPQIQPV 23029 RBM34 438 SLFIGEKAVLL 23029 RBM34 439 SLLDLHTKV 27340 UTP20 440 SLLEQGKEPWMV 147949,163087,342892,374899, 84503 ZNF583, ZNF383, ZNF850, ZNF829, ZNF527 441 SLLEVNEASSV 149628 PYHIN1 442 SLNDLEKDVMLL 6597 SMARCA4 443 SLTIDGIYYV 1659 DHX8 444 SMSGYDQVL 3187,3188 HNRNPH1, HNRNPH2 445 VIIKGLEEITV 3832 KIF11 446 VILTSSPFL 10800 CYSLTR1 447 VLDDSIYLV 57565 KLHL14 448 VMNDRLYAI 57565 KLHL14 449 LLDAMNYHL 57565 KLHL14 450 VLGPGPPPL 254359 ZDHHC24 451 VLIEYNFSI 55215 FANCI 452 VLLSLLEKV 1130 LYST 453 SLLPLVWKI 1130 LYST 454 VLMDEGAVLTL 54596 L1TD1 455 VLPDPEVLEAV 57326 PBXIP1 456 YAAPGGLIGV 1968,255308 EIF2S3 457 YIMEPSIFNTL 51497 TH1L 458 YIQEHLLQI 10625 IVNS1ABP 459 YLDFSNNRL 51284 TLR7 460 YLEDGFAYV 5558 PRIM2 461 YLLNLNHLGL 23471 TRAM1 462 YVLPKLYVKL 100128168,100996747,441502,6231,643003,644166,644928,644934,646753,728937, 729188 RPS26P39, RPS26P11, RPS26, RPS26P28, RPS26P20, RPS26P15, RPS26P50, RPS26P2, RPS26P25, RPS26P58 463 ALMAVVSGL 55103 RALGPS2 464 ALSDETKNNWEV 5591 PRKDC 465 FLYDEIEAEVNL 23191,26999 CYFIP1, CYFIP2 466 FLYDEIEAEV 23191,26999 CYFIP1, CYFIP2 467 IMQDFPAEIFL 25914 RTTN 468 KIQEILTQV 10643 IGF2BP3 469 SLDGTELQL 284114 TMEM102 470 TLTNIIHNL 94101 ORMDL1 471 VLLAAGPSAA 23225 NUP210 472 YLFESEGLVL 283431 GAS2L3 473 ALADDDFLTV 4173 MCM4 474 ALADLIEKELSV 4850 CNOT4 475 ALDDMISTL 7203 CCT3 476 ALITEVVRL 26005 C2CD3 477 ALLDQTKTLAESAL 7094 TLN1 478 AMFESSQNVLL 64328 XPO4 479 YLAHFIEGL 64328 XPO4 480 FLAEDPKVTL 60489 APOBEC3G 481 FLIDTSASM 203522,26512 DDX26B, INTS6 482 FLTDLEDLTL 26151 NAT9 483 FLTEMVHFI 93517 SDR42E1 484 FLVDGPRVQL 90204 ZSWIM1 485 GLLDCPIFL 2177 FANCD2 486 GLLDLPFRVGV 23347 SMCHD1 487 GLSDGNPSL 79684 MSANTD2 488 GVYDGEEHSV 4113 MAGEB2 489 HLANIVERL 117854,445372,53840 TRIM6, TRIM6-TRIM34, TRIM34 490 ILDEKPVII 23460 ABCA6 491 ILFSEDSTKLFV 84916 CIRH1A 492 ILLDDNMQIRL 5261 PHKG2 493 LIIDQADIYL 27042 DIEXF 494 LLGDSSFFL 283254 HARBI1 495 LLLDEEGTFSL 27013 CNPPD1 496 LLVEQPPLAGV 4773 NFATC2 497 LLWDVPAPSL 1388,7148 ATF6B, TNXB 498 LTAPPEALLMV 79050 NOC4L 499 NLMEMVAQL 55636 CHD7 500 QIITSVVSV 5378 PMS1 501 QLALKVEGV 25896 INTS7 502 QLLDETSAITL 10915 TCERG1 503 QLYEEPDTKL 10270 AKAP8 504 RLYSGISGLEL 84172 POLR1B 505 RVLEVGALQAV 25885 POLR1A 506 SLLPLDDIVRV 3708,3709 ITPR1, ITPR2 507 SLLTEQDLWTV 90806 ANGEL2 508 SVDSAPAAV 10635 RAD51AP1 509 TLQEVVTGV 55750 AGK 510 TVDVATPSV 8924 HERC2 511 VLAYFLPEA 4171 MCM2 512 VLISVLQAI 26999 CYFIP2 513 YLIPFTGIVGL 26001 RNF167 514 YLLDDGTLVV 8872 CDC123 515 YLMPGFIHL 168400,55510 DDX53, DDX43 516 YLPDIIKDQKA 5496 PPM1G 517 YLQLTQSEL 283237 TTC9C 518 YLTEVFLHVV 55024 BANK1 519 YLVEGRFSV 55125 CEP192 520 YLVYILNEL 51202 DDX47 521 YLYGQTTTYL 7153 TOP2A 522 YVLTQPPSV 28796,28815,28831,3537,3538 IGLV3-21, IGLV2-14, IGLJ3, IGLC1, IGLC2

本發明還一般涉及本發明的肽用於治療增殖性疾病，例如，急性骨髓性白血病、膽管癌、腦癌、乳腺癌、結直腸癌、食管癌、膽囊癌、胃癌、肝細胞癌、梅克爾細胞癌、黑色素瘤、非霍奇金淋巴瘤、非小細胞肺癌、卵巢癌、胰腺癌、攝護腺癌、腎細胞癌、小細胞肺癌、膀胱癌和子宮癌。The present invention also generally relates to the use of the peptides of the present invention in the treatment of proliferative diseases, for example, acute myelogenous leukemia, cholangiocarcinoma, brain cancer, breast cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, hepatocellular carcinoma, Merkel Cell carcinoma, melanoma, non-Hodgkin’s lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and uterine cancer.

特別優選的是本發明的肽（單獨或組合），其選自包括SEQ ID NO:1至 SEQ ID NO:385組成的組。更優選的是所述肽（單獨或組合）選自包括SEQ ID NO:1至 SEQ ID NO:202 組成的組（見表 1）並且其用於免疫治療 CLL、急性骨髓性白血病、膽管癌、腦癌、乳腺癌、結直腸癌、食管癌、膽囊癌、胃癌、肝細胞癌、梅克爾細胞癌、黑色素瘤、非霍奇金淋巴瘤、非小細胞肺癌、卵巢癌、胰腺癌、攝護腺癌、腎細胞癌、小細胞肺癌、膀胱癌和子宮癌，優選為CLL。Particularly preferred are the peptides of the present invention (alone or in combination), which are selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:385. More preferably, the peptide (alone or in combination) is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 202 (see Table 1) and is used for immunotherapy of CLL, acute myeloid leukemia, cholangiocarcinoma, Brain cancer, breast cancer, colorectal cancer, esophageal cancer, gallbladder cancer, stomach cancer, hepatocellular carcinoma, Merkel cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, nursing care Adenocarcinoma, renal cell carcinoma, small cell lung cancer, bladder cancer and uterine cancer, preferably CLL.

如示下面的表 4A 和 B 所示，其中本發明的許多肽也發現於其他腫瘤中，因此也可用於其他適應症的免疫治療。另請參閱圖1和實施例 1。As shown in Tables 4A and B below, many of the peptides of the present invention are also found in other tumors, so they can also be used in immunotherapy for other indications. Please also refer to Figure 1 and Example 1.

表4A：本發明的肽及其在其他增殖性疾病（特別是其他癌性疾病）中的特定用途。該表顯示，對於其他腫瘤類型的選定肽，發現他們過量表現（特定表現）於 5% 以上測定的腫瘤樣本，或表現於 5% 以上測定的腫瘤樣本且幾何學平均值腫瘤與正常組織的比值大於 3。過度表現定義為與最高表現的正常樣本相比在腫瘤樣本上表現更高。經測試相比過度表現的正常組織有：脂肪組織、腎上腺、血細胞、血管、骨髓、腦、乳腺、食道、眼、膽囊、心臟、腎、大腸、肝、肺、淋巴結、神經、胰腺、甲狀旁腺、腹膜、腦垂體、胸膜、唾液腺、骨骼肌、皮膚、小腸、脾、胃、甲狀腺、氣管、輸尿管、膀胱。 序列ID號： 序列 其他相關器官/疾病 1 AIPPSFASIFL NHL 2 ALHRPDVYL NHL 3 VIAELPPKV NHL 5 ALIFKIASA AML、BRCA 7 ALLERTGYTL HCC、NHL 8 ALAASALPALV AML 9 ALCDTLITV NHL 10 FVYGESVEL NHL 12 ALGEDEITL NHL、AML、黑色素瘤 13 VVDGMPPGV NHL 14 ALLRLLPGL NHL、AML 15 ALPEVSVEA NHL、AML、黑色素瘤 19 QVMEKLAAV NHL 20 ALVDPGPDFVV NHL 21 ALWAGLLTL NHL 22 ALWDPVIEL NHL 24 AMAGDVVYA NHL 25 ATYSGLESQSV 黑色素瘤 26 AVLLVLPLV NHL 27 AVLGLVWLL NSCLC、NHL、子宮癌 28 AVLHLLLSV NHL、BRCA 29 AVLQAVTAV SCLC、NHL、AML、黑色素瘤、OC、膽囊癌、膽管癌 30 ELLEGSEIYL NHL 32 FIDKFTPPV NHL 33 FIINSSNIFL NHL 35 FILPSSLYL RCC、NHL、AML、膽囊癌、膽管癌 37 FIMEGGAMVL AML 38 FIMEGGAMV NHL 39 FLDALLTDV SCLC、NHL、AML、黑色素瘤 40 FLDEDDMSL SCLC、NHL、膀胱癌 42 FLEEGGVVTV NHL、黑色素瘤 43 FLLGPEALSFA NHL、AML 44 FLLSINDFL 黑色素瘤 45 FLPELPADLEA NHL、黑色素瘤 46 YIIDSAQAV NHL 48 FLSPQQPPLL NHL 49 FLTDLFAQL NHL 52 FLVETGFLHV 黑色素瘤 55 FMEPTLLML NHL、膀胱癌 56 FVFEAPYTL NHL、AML 57 GLSEISLRL NHL、AML、黑色素瘤 58 YIQQGIFSV NHL 59 FVFGDENGTVSL NHL、食管癌、膀胱癌、子宮癌 60 FVLDHEDGLNL 黑色素瘤、食管癌 62 GIIDGSPRL NHL 63 SLAHVAGCEL NHL 64 KLLESVASA NHL 66 SLAGGLDDMKA NHL 67 GLDDVTVEV HCC、NHL、黑色素瘤、食管癌、OC、膽囊癌、膽管癌 68 GLDQQFAGLDL RCC、HCC、NHL、黑色素瘤 69 GLHQREIFL NHL 70 FVPDTPVGV NHL 71 GLKHDIARV NHL 72 GLLDAGKMYV NHL、黑色素瘤 74 GLLRASFLL NHL、AML、BRCA 75 GLSIFAQDLRL NHL 76 GLLRIIPYL NHL 78 GLPSFLTEV SCLC、NHL、AML、黑色素瘤 79 GLQAKIQEA NHL、食管癌、子宮癌 80 VLIEDELEEL NHL 81 WLVGQEFEL NHL 82 GLQSGVDIGV NHL 83 GQGEVLVYV SCLC、GC、食管癌 86 HLYPGAVTI HCC、NHL 87 HQIEAVDGEEL GC、NHL、食管癌 88 ILDEIGADVQA PC、NHL 89 ILDFGTFQL NHL 91 ILDLNTYNV NHL 92 ILEPLNPLL NHL 93 ILFNTQINI SCLC、NHL、AML、黑色素瘤、OC 94 ILFPLRFTL NSCLC、RCC、CRC、NHL、黑色素瘤、OC 96 ILIDKTSFV NHL 97 LLFATQITL NHL 98 SLIKYFLFV NHL 99 ILIFHSVAL NHL、AML 100 ILNNEVFAI NHL、AML 101 ILVVIEPLL NHL 102 IQDRAVPSL AML 103 KLGGTPAPA NHL 105 KLMNDIADI NHL 109 KLWEGLTELV AML 110 LLFDHLEPMEL NHL 111 KLWNVAAPLYL OC 112 KTLDVDATYEI HCC、NHL、黑色素瘤、食管癌、OC 113 KVPAEEVLVAV NHL 116 LLIGATMQV SCLC、NHL、AML 117 LLILENILL NHL、AML、黑色素瘤 118 RLLILENILL NHL 119 VLPAEFFEV NHL、AML、膀胱癌、子宮癌 120 AIDAALTSV NHL、AML 121 RLYEITIEV SCLC、NHL、AML、BRCA、OC 122 LLIPVVPGV NSCLC、NHL 124 LLLEETEKQAV RCC、HCC 125 LLLEIGEVGKLFV NHL 126 LLPEGGITAI NHL 127 LLPTAPTTV NHL 130 LLVLIPVYL NHL、黑色素瘤 133 YLLSGDISEA NHL 136 NLDKLWTLV GC、食管癌 137 NLIKTVIKL NHL 138 NLLDIDAPVTV SCLC、NHL 139 NLTDVVEKL RCC、NHL 141 QILSEIVEA NHL、黑色素瘤 145 SALDTITTV NHL 146 SIIEGPIIKL NHL、膀胱癌 147 SILETVATL NHL、AML 148 SIVASLITV NHL、AML 149 SLDNGGYYI NHL 150 SLFDQPLSII NHL 151 SLFDSAYGA AML 152 SLIRILQTI HCC 153 SLLAELHVL NHL 154 SLLAELHVLTV NHL 155 SLMLEVPAL HCC、NHL 156 SLNIGDVQL NHL 157 SLNIRDFTM AML 158 SLPEAPLDV NHL、AML、子宮癌 159 SLQEEKLIYV 腦癌、NHL、MCC、黑色素瘤、食管癌、膀胱癌 160 SLSFLVPSL NHL、AML、膀胱癌 161 SMDDGMINV CRC、NHL、膽囊癌、膽管癌 162 SMKDDLENV NSCLC、SCLC、RCC、HCC、NHL、BRCA、黑色素瘤、食管癌、子宮癌、膽囊癌、膽管癌 163 SQLDISEPYKV NHL 164 SVHKGFAFV NSCLC、RCC、腦癌、GC、AML、食管癌、膀胱癌、子宮癌 165 TLDDDLDTV NHL 166 TLDPNQVSL AML 167 TLDTSKLYV NHL 168 YLLDQSFVM NHL 169 TLLLGLTEV NHL、黑色素瘤 170 TLTFRVETV NHL、黑色素瘤 172 TLYDMLASI CRC、NHL、AML、黑色素瘤 175 FTVPRVVAV NHL 177 VLDDRELLL NHL 178 VLFFNVQEV SCLC、NHL 179 VLLGLEMTL NHL、食管癌 180 LLKDGPEIGL NHL 181 VLLSIPFVSV HCC、NHL 183 VLMPTVYQQGV AML 185 VMDDQRDLI AML 186 VMDPTKILI AML、膀胱癌 187 VMDTHLVNI NHL、膀胱癌 188 VMGDIPAAV NHL、AML、膽囊癌、膽管癌 189 VMLEMTPEL SCLC、PC、NHL、BRCA、黑色素瘤、膽囊癌、膽管癌 190 VVMGTVPRL NHL、AML 192 YIQEYLTLL NHL 193 YLDLSNNRL NHL 194 YLDNVLAEL NHL 195 YLGGFALSV NHL、AML 196 YLLLQTYVL NHL 197 YLQEVPILTL NHL、黑色素瘤 199 YLVELSSLL HCC、NHL、AML 200 YMFEEVPIVI NSCLC、SCLC、CRC、HCC、NHL、黑色素瘤、食管癌、OC、膀胱癌、膽囊癌、膽管癌 201 YQLELHGIEL 食管癌 202 YVDDVFLRV 膀胱癌 203 ALLSSQLAL NHL 204 GLLQINDKIAL SCLC、RCC、HCC、黑色素瘤、OC、膀胱癌 205 GLSQANFTL NHL 207 IIADLDTTIMFA SCLC、NHL、黑色素瘤、OC 208 ILLKTEGINL NHL、BRCA、OC、膀胱癌 209 ILQAELPSL NHL 210 KLLVQDFFL HCC、NHL 211 LIDVKPLGV NHL、BRCA、黑色素瘤 212 NIIEAINELLV NHL、BRCA、黑色素瘤 213 RLLYQLVFL NHL、食管癌、膀胱癌、膽囊癌、膽管癌 214 RLQELTEKL NHL、AML 215 VMQDIVYKL NHL、食管癌、膀胱癌 216 WLAGDVPAA SCLC、NHL 217 ALDEPPYLTV NHL、黑色素瘤 218 ALGEEWKGYVV 食管癌 219 ALLNLLESA NHL、AML、OC 220 ALPEILFAKV NHL 221 ALVSTIIMV NHL、AML 222 ALWELSLKI NHL 223 ALWVSQPPEI NHL 224 AMEALVVEV NHL 225 ILQERELLPV NHL、黑色素瘤、OC、膀胱癌 227 FLAEASVMTQL NHL 228 FLGGLSPGV NHL、食管癌 229 FLLNLQNCHL NHL 230 FLQDSKVIFV NHL 231 FLYIRQLAI AML 232 FMHQIIDQV RCC、CRC、黑色素瘤 233 GIIDINVRL PC、NHL、黑色素瘤、食管癌、膀胱癌、子宮癌、膽囊癌、膽管癌 234 GLDDAEYAL NHL、AML、黑色素瘤、膀胱癌 235 GLDDLLLFL SCLC、PC、NHL、AML、膀胱癌、子宮癌、膽囊癌、膽管癌 236 GLLESGRHYL NHL、AML 237 GLQENLDVVV SCLC、NHL、黑色素瘤、子宮癌 238 GLVETELQL NHL、AML 239 ILAGEMLSV NHL、AML、黑色素瘤、膽囊癌、膽管癌 240 ILARDILEI NHL、膀胱癌 241 ILGDILLKV AML 242 ILLGIQELL NHL、AML 243 ILPTLEKELFL AML 244 ILQALAVHL NHL、AML、OC 245 KIMDYSLLLGV SCLC、RCC、OC 246 KLDETGVAL SCLC、NHL、AML、黑色素瘤 247 KLKDRLPSI NHL 248 KTVEPPISQV NHL 249 LLPTGVFQV 膀胱癌 250 LLVQEPDGLMV SCLC、HCC、黑色素瘤、膀胱癌 251 LLYDNVPGA NHL 252 NLLDPGSSYLL 食管癌 253 NLWSVDGEVTV NHL 254 QLIPKLIFL NHL、黑色素瘤 255 YLFEEAISM NHL 257 RIINGIIISV NHL 258 RLDYITAEI RCC、NHL、AML、黑色素瘤 260 SLDDVEGMSV NHL、膽囊癌、膽管癌 261 SLVEAQGWLV NHL 263 SQWEDIHVV NHL、AML 264 TILDYINVV 腦癌、NHL、AML、BRCA、OC、子宮癌 265 TLLADDLEIKL 食管癌 266 TLLDQLDTQL NHL、OC、膀胱癌 267 TLLDWQDSL NHL、AML、黑色素瘤 268 TLLQVFHLL NHL 269 TLTDEQFLV NHL、AML 271 VIRNIVEAA GC、CRC、PC、AML、食管癌 272 VLDELPPLI SCLC、NHL 273 VLGEYSYLL NHL 274 VLLEYHIAYL NSCLC、SCLC、HCC、NHL、膀胱癌 275 VLLFIEHSV NHL 276 VLNDGAPNV NSCLC、SCLC、NHL、黑色素瘤、子宮癌、膽囊癌、膽管癌 278 YLDDLLPKL PC、NHL 280 YTLDSLYWSV NHL 281 NLLDDRGMTAL NHL、AML 282 LLRDGIELV NHL、黑色素瘤、OC 283 ILQPMDIHV NHL 284 LLSAAEPVPA NHL 286 FLLEDLSQKL NHL 288 GLAESTGLLAV NHL 290 LANPHELSL AML 291 ILLNEDDLVTI SCLC、NHL、黑色素瘤 292 AAALIIHHV 膀胱癌 294 ALLDQLHTLL NHL 295 ALLQKLQQL NHL 296 FIAPTGHSL SCLC、NHL、AML、BRCA 297 FLVEPQEDTRL NHL、黑色素瘤 298 IILPVEVEV SCLC、NHL、AML、膀胱癌 299 ILEENIPVL NHL 300 ILLNPAYDVYL NHL、黑色素瘤 303 ILSQPTPSL NHL、AML、黑色素瘤、OC 304 LAIVPVNTL AML 305 LLFPQIEGIKI NHL、黑色素瘤 306 VVAEELENV NHL、黑色素瘤、OC 307 LLLTKPTEA NHL 308 SLYDVSRMYV NHL 309 ILYGTQFVL NHL 310 LLSTLHLLV NHL 311 LLVDVEPKV NSCLC、SCLC、RCC、HCC、PC、NHL、AML、黑色素瘤 312 LLYNSTDPTL NHL 313 LMADLEGLHL 黑素瘤、膀胱癌 314 LMKDCEAEV NHL 316 MLLEHGITL NHL 317 NLLAHIWAL AML 320 TIAPVTVAV NHL 321 RLLEFELAQL SCLC、CRC、NHL、OC 322 SLASIHVPL NHL 323 SLDLFNCEV NHL 324 SLYSALQQA NHL 325 TLENGVPCV NHL 326 VLAFLVHEL 食管癌 327 VLIKWFPEV NHL 328 VLLPQETAEIHL NHL、BRCA、黑色素瘤、OC、膀胱癌 329 VLMDGSVKL NHL 330 VLMWEIYSL NHL 332 VMIQHVENL NHL 333 VTLEFPQLIRV SCLC、HCC、NHL 334 YLLEEKIASL NHL 335 YLYQEQYFI SCLC、NHL、AML、膀胱癌 337 YMYEKESEL NHL 338 FLDMTNWNL HCC、NHL、黑色素瘤、OC、膀胱癌 339 GLWGTVVNI NHL、AML、黑色素瘤、子宮癌 341 LLAELPASVHA HCC、NHL、膀胱癌 342 SLITPLQAV NHL、AML、膀胱癌、膽囊癌、膽管癌 343 TLLEALDCI NHL、AML、黑色素瘤、食管癌 344 VLAFENPQV 食管癌 346 VLVQVSPSL RCC、NHL、AML、膽囊癌、膽管癌 347 YLGPVSPSL NHL 348 ALAKPPVVSV SCLC、HCC、黑色素瘤、食管癌 349 ALATHILSL NHL、膀胱癌 350 ALEDRVWEL NHL、食管癌 351 ALSEKLARL HCC、NHL、AML、膀胱癌 352 ALVFELHYV NHL 353 ATPMPTPSV SCLC、NHL 354 FIMDDPAGNSYL CRC、NHL、AML、黑色素瘤、食管癌、膀胱癌、膽囊癌、膽管癌 357 FLIQEIKTL NHL、OC、膀胱癌 358 FLTDYLNDL NHL 359 FMQDPMEVFV 黑色素瘤 360 HLIDTNKIQL NHL 362 ILTELGGFEV NHL 363 ITTEVVNELYV NHL、黑色素瘤 364 KMDWIFHTI 膀胱癌 365 LISPLLLPV NHL、AML、黑色素瘤、膀胱癌 367 NLWSLVAKV NSCLC、NHL、AML、黑色素瘤、OC 369 RLLDLENSLLGL AML 370 SIFASPESV 黑色素瘤、食管癌、膀胱癌 371 SLADDSVLERL RCC、HCC、NHL、AML、食管癌、膀胱癌 373 TLLADQGEIRV NHL、黑色素瘤 374 VLSVITEEL NHL、AML 375 VLWFKPVEL NSCLC、HCC、NHL、黑色素瘤、食管癌 376 VLYNQRVEEI 膀胱癌 377 VVDGTCVAV NHL、黑色素瘤 378 YILGKFFAL NHL、AML 379 YLAELVTPIL NSCLC、SCLC、NHL、黑色素瘤 380 YLDRKLLTL NHL 381 YLLEENKIKL NHL 382 YLLPLLQRL NHL、AML 383 YLLREWVNL HCC、NHL 384 YMIGSEVGNYL NHL、膀胱癌 385 YTIPLAIKL NHL、AML、BRCA、食管癌 Table 4A: The peptides of the present invention and their specific uses in other proliferative diseases (especially other cancerous diseases). This table shows that for selected peptides of other tumor types, they are found to be overexpressed (specifically) in more than 5% of the tumor samples measured, or in more than 5% of the tumor samples measured and the ratio of the geometric mean tumor to normal tissue Greater than 3. Overperformance is defined as higher performance on tumor samples compared to the highest performing normal samples. The normal tissues that have been tested for overexpression are: adipose tissue, adrenal glands, blood cells, blood vessels, bone marrow, brain, breast, esophagus, eyes, gallbladder, heart, kidney, large intestine, liver, lung, lymph nodes, nerves, pancreas, thyroid Parathyroid glands, peritoneum, pituitary gland, pleura, salivary glands, skeletal muscle, skin, small intestine, spleen, stomach, thyroid, trachea, ureter, bladder. Serial ID number: sequence Other related organs/diseases 1 AIPPSFASIFL NHL 2 ALHRPDVYL NHL 3 VIAELPPKV NHL 5 ALIFKIASA AML, BRCA 7 ALLERTGYTL HCC, NHL 8 ALAASALPALV AML 9 ALCDTLITV NHL 10 FVYGESVEL NHL 12 ALGEDEITL NHL, AML, melanoma 13 VVDGMPPGV NHL 14 ALLRLLPGL NHL, AML 15 ALPEVSVEA NHL, AML, melanoma 19 QVMEKLAAV NHL 20 ALVDPGPDFVV NHL twenty one ALWAGLLTL NHL twenty two ALWDPVIEL NHL twenty four AMAGDVVYA NHL 25 ATYSGLESQSV Melanoma 26 AVLLVLPLV NHL 27 AVLGLVWLL NSCLC, NHL, uterine cancer 28 AVLHLLLSV NHL, BRCA 29 AVLQAVTAV SCLC, NHL, AML, melanoma, OC, gallbladder cancer, cholangiocarcinoma 30 ELLEGSEIYL NHL 32 FIDKFTPPV NHL 33 FIINSSNIFL NHL 35 FILPSSLYL RCC, NHL, AML, gallbladder cancer, cholangiocarcinoma 37 FIMEGGAMVL AML 38 FIMEGGAMV NHL 39 FLDALLTDV SCLC, NHL, AML, melanoma 40 FLDEDDMSL SCLC, NHL, bladder cancer 42 FLEEGGVVTV NHL, melanoma 43 FLLGPEALSFA NHL, AML 44 FLLSINDFL Melanoma 45 FLPELPADLEA NHL, melanoma 46 YIIDSAQAV NHL 48 FLSPQQPPLL NHL 49 FLTDLFAQL NHL 52 FLVETGFLHV Melanoma 55 FMEPTLLML NHL, bladder cancer 56 FVFEAPYTL NHL, AML 57 GLSEISLRL NHL, AML, melanoma 58 YIQQGIFSV NHL 59 FVFGDENGTVSL NHL, esophageal cancer, bladder cancer, uterine cancer 60 FVLDHEDGLNL Melanoma, esophageal cancer 62 GIIDGSPRL NHL 63 SLAHVAGCEL NHL 64 KLLESVASA NHL 66 SLAGGLDDMKA NHL 67 GLDDVTVEV HCC, NHL, melanoma, esophageal cancer, OC, gallbladder cancer, cholangiocarcinoma 68 GLDQQFAGLDL RCC, HCC, NHL, melanoma 69 GLHQREIFL NHL 70 FVPDTPVGV NHL 71 GLKHDIARV NHL 72 GLLDAGKMYV NHL, melanoma 74 GLLRASFLL NHL, AML, BRCA 75 GLSIFAQDLRL NHL 76 GLLRIIPYL NHL 78 GLPSFLTEV SCLC, NHL, AML, melanoma 79 GLQAKIQEA NHL, esophageal cancer, uterine cancer 80 VLIEDELEEL NHL 81 WLVGQEFEL NHL 82 GLQSGVDIGV NHL 83 GQGEVLVYV SCLC, GC, esophageal cancer 86 HLYPGAVTI HCC, NHL 87 HQIEAVDGEEL GC, NHL, esophageal cancer 88 ILDEIGADVQA PC, NHL 89 ILDFGTFQL NHL 91 ILDLNTYNV NHL 92 ILEPLNPLL NHL 93 ILFNTQINI SCLC, NHL, AML, melanoma, OC 94 ILFPLRFTL NSCLC, RCC, CRC, NHL, melanoma, OC 96 ILIDKTSFV NHL 97 LLFATQITL NHL 98 SLIKYFLFV NHL 99 ILIFHSVAL NHL, AML 100 ILNNEVFAI NHL, AML 101 ILVVIEPLL NHL 102 IQDRAVPSL AML 103 KLGGTPAPA NHL 105 KLMNDIADI NHL 109 KLWEGLTELV AML 110 LLFDHLEPMEL NHL 111 KLWNVAAPLYL OC 112 KTLDVDATYEI HCC, NHL, melanoma, esophageal cancer, OC 113 KVPAEEVLVAV NHL 116 LLIGATMQV SCLC, NHL, AML 117 LLILENILL NHL, AML, melanoma 118 RLLILENILL NHL 119 VLPAEFFEV NHL, AML, bladder cancer, uterine cancer 120 AIDAALTSV NHL, AML 121 RLYEITIEV SCLC, NHL, AML, BRCA, OC 122 LLIPVVPGV NSCLC, NHL 124 LLLEETEKQAV RCC, HCC 125 LLLEIGEVGKLFV NHL 126 LLPEGGITAI NHL 127 LLPTAPTTV NHL 130 LLVLIPVYL NHL, melanoma 133 YLLSGDISEA NHL 136 NLDKLWTLV GC, esophageal cancer 137 NLIKTVIKL NHL 138 NLLDIDAPVTV SCLC, NHL 139 NLTDVVEKL RCC, NHL 141 QILSEIVEA NHL, melanoma 145 SALDTITTV NHL 146 SIIEGPIIKL NHL, bladder cancer 147 SILETVATL NHL, AML 148 SIVASLITV NHL, AML 149 SLDNGGYYI NHL 150 SLFDQPLSII NHL 151 SLFDSAYGA AML 152 SLIRILQTI HCC 153 SLLAELHVL NHL 154 SLLAELHVLTV NHL 155 SLMLEVPAL HCC, NHL 156 SLNIGDVQL NHL 157 SLNIRDFTM AML 158 SLPEAPLDV NHL, AML, uterine cancer 159 SLQEEKLIYV Brain cancer, NHL, MCC, melanoma, esophageal cancer, bladder cancer 160 SLSFLVPSL NHL, AML, bladder cancer 161 SMDDGMINV CRC, NHL, gallbladder cancer, cholangiocarcinoma 162 SMKDDLENV NSCLC, SCLC, RCC, HCC, NHL, BRCA, melanoma, esophageal cancer, uterine cancer, gallbladder cancer, cholangiocarcinoma 163 SQLDISEPYKV NHL 164 SVHKGFAFV NSCLC, RCC, brain cancer, GC, AML, esophageal cancer, bladder cancer, uterine cancer 165 TLDDDLDTV NHL 166 TLDPNQVSL AML 167 TLDTSKLYV NHL 168 YLLDQSFVM NHL 169 TLLLGLTEV NHL, melanoma 170 TLTFRVETV NHL, melanoma 172 TLYDMLASI CRC, NHL, AML, melanoma 175 FTVPRVVAV NHL 177 VLDDRELLL NHL 178 VLFFNVQEV SCLC, NHL 179 VLLGLEMTL NHL, esophageal cancer 180 LLKDGPEIGL NHL 181 VLLSIPFVSV HCC, NHL 183 VLMPTVYQQGV AML 185 VMDDQRDLI AML 186 VMDPTKILI AML, bladder cancer 187 VMDTHLVNI NHL, bladder cancer 188 VMGDIPAAV NHL, AML, gallbladder cancer, cholangiocarcinoma 189 VMLEMTPEL SCLC, PC, NHL, BRCA, melanoma, gallbladder cancer, cholangiocarcinoma 190 VVMGTVPRL NHL, AML 192 YIQEYLTLL NHL 193 YLDLSNNRL NHL 194 YLDNVLAEL NHL 195 YLGGFALSV NHL, AML 196 YLLLQTYVL NHL 197 YLQEVPILTL NHL, melanoma 199 YLVELSSLL HCC, NHL, AML 200 YMFEEVPIVI NSCLC, SCLC, CRC, HCC, NHL, melanoma, esophageal cancer, OC, bladder cancer, gallbladder cancer, cholangiocarcinoma 201 YQLELHGIEL Esophageal cancer 202 YVDDVFLRV Bladder Cancer 203 ALLSSQLAL NHL 204 GLLQINDKIAL SCLC, RCC, HCC, melanoma, OC, bladder cancer 205 GLSQANFTL NHL 207 IIADLDTTIMFA SCLC, NHL, melanoma, OC 208 ILLKTEGINL NHL, BRCA, OC, bladder cancer 209 ILQAELPSL NHL 210 KLLVQDFFL HCC, NHL 211 LIDVKPLGV NHL, BRCA, melanoma 212 NIIEAINELLV NHL, BRCA, melanoma 213 RLLYQLVFL NHL, esophageal cancer, bladder cancer, gallbladder cancer, cholangiocarcinoma 214 RLQELTEKL NHL, AML 215 VMQDIVYKL NHL, esophageal cancer, bladder cancer 216 WLAGDVPAA SCLC, NHL 217 ALDEPPYLTV NHL, melanoma 218 ALGEEWKGYVV Esophageal cancer 219 ALLNLLESA NHL, AML, OC 220 ALPEILFAKV NHL 221 ALVSTIIMV NHL, AML 222 ALWELSLKI NHL 223 ALWVSQPPEI NHL 224 AMEALVVEV NHL 225 ILQERELLPV NHL, melanoma, OC, bladder cancer 227 FLAEASVMTQL NHL 228 FLGGLSPGV NHL, esophageal cancer 229 FLLNLQNCHL NHL 230 FLQDSKVIFV NHL 231 FLYIRQLAI AML 232 FMHQIIDQV RCC, CRC, melanoma 233 GIIDINVRL PC, NHL, melanoma, esophageal cancer, bladder cancer, uterine cancer, gallbladder cancer, cholangiocarcinoma 234 GLDDAEYAL NHL, AML, melanoma, bladder cancer 235 GLDDLLLFL SCLC, PC, NHL, AML, bladder cancer, uterine cancer, gallbladder cancer, cholangiocarcinoma 236 GLLESGRHYL NHL, AML 237 GLQENLDVVV SCLC, NHL, melanoma, uterine cancer 238 GLVETELQL NHL, AML 239 ILAGEMLSV NHL, AML, melanoma, gallbladder cancer, cholangiocarcinoma 240 ILARDILEI NHL, bladder cancer 241 ILGDILLKV AML 242 ILLGIQELL NHL, AML 243 ILPTLEKELFL AML 244 ILQALAVHL NHL, AML, OC 245 KIMDYSLLLGV SCLC, RCC, OC 246 KLDETGVAL SCLC, NHL, AML, melanoma 247 KLKDRLPSI NHL 248 KTVEPPISQV NHL 249 LLPTGVFQV Bladder Cancer 250 LLVQEPDGLMV SCLC, HCC, melanoma, bladder cancer 251 LLYDNVPGA NHL 252 NLLDPGSSYLL Esophageal cancer 253 NLWSVDGEVTV NHL 254 QLIPKLIFL NHL, melanoma 255 YLFEEAISM NHL 257 RIINGIIISV NHL 258 RLDYITAEI RCC, NHL, AML, melanoma 260 SLDDVEGMSV NHL, gallbladder cancer, cholangiocarcinoma 261 SLVEAQGWLV NHL 263 SQWEDIHVV NHL, AML 264 TILDYINVV Brain cancer, NHL, AML, BRCA, OC, uterine cancer 265 TLLADDLEIKL Esophageal cancer 266 TLLDQLDTQL NHL, OC, bladder cancer 267 TLLDWQDSL NHL, AML, melanoma 268 TLLQVFHLL NHL 269 TLTDEQFLV NHL, AML 271 VIRNIVEAA GC, CRC, PC, AML, esophageal cancer 272 VLDELPPLI SCLC, NHL 273 VLGEYSYLL NHL 274 VLLEYHIAYL NSCLC, SCLC, HCC, NHL, bladder cancer 275 VLLFIEHSV NHL 276 VLNDGAPNV NSCLC, SCLC, NHL, melanoma, uterine cancer, gallbladder cancer, cholangiocarcinoma 278 YLDDLLPKL PC, NHL 280 YTLDSLYWSV NHL 281 NLLDDRGMTAL NHL, AML 282 LLRDGIELV NHL, melanoma, OC 283 ILQPMDIHV NHL 284 LLSAAEPVPA NHL 286 FLLEDLSQKL NHL 288 GLAESTGLLAV NHL 290 LANPHELSL AML 291 ILLNEDDLVTI SCLC, NHL, melanoma 292 AAALIIHHV Bladder Cancer 294 ALLDQLHTLL NHL 295 ALLQKLQQL NHL 296 FIAPTGHSL SCLC, NHL, AML, BRCA 297 FLVEPQEDTRL NHL, melanoma 298 IILPVEVEV SCLC, NHL, AML, bladder cancer 299 ILEENIPVL NHL 300 ILLNPAYDVYL NHL, melanoma 303 ILSQPTPSL NHL, AML, melanoma, OC 304 LAIVPVNTL AML 305 LLFPQIEGIKI NHL, melanoma 306 VVAEELENV NHL, melanoma, OC 307 LLLTKPTEA NHL 308 SLYDVSRMYV NHL 309 ILYGTQFVL NHL 310 LLSTLHLLV NHL 311 LLVDVEPKV NSCLC, SCLC, RCC, HCC, PC, NHL, AML, melanoma 312 LLYNSTDPTL NHL 313 LMADLEGLHL Melanoma, bladder cancer 314 LMKDCEAEV NHL 316 MLLEHGITL NHL 317 NLLAHIWAL AML 320 TIAPVTVAV NHL 321 RLLEFELAQL SCLC, CRC, NHL, OC 322 SLASIHVPL NHL 323 SLDLFNCEV NHL 324 SLYSALQQA NHL 325 TLENGVPCV NHL 326 VLAFLVHEL Esophageal cancer 327 VLIKWFPEV NHL 328 VLLPQETAEIHL NHL, BRCA, melanoma, OC, bladder cancer 329 VLMDGSVKL NHL 330 VLMWEIYSL NHL 332 VMIQHVENL NHL 333 VTLEFPQLIRV SCLC, HCC, NHL 334 YLLEEKIASL NHL 335 YLYQEQYFI SCLC, NHL, AML, bladder cancer 337 YMYEKESEL NHL 338 FLDMTNWNL HCC, NHL, melanoma, OC, bladder cancer 339 GLWGTVVNI NHL, AML, melanoma, uterine cancer 341 LLAELPASVHA HCC, NHL, bladder cancer 342 SLITPLQAV NHL, AML, bladder cancer, gallbladder cancer, cholangiocarcinoma 343 TLLEALDCI NHL, AML, melanoma, esophageal cancer 344 VLAFENPQV Esophageal cancer 346 VLVQVSPSL RCC, NHL, AML, gallbladder cancer, cholangiocarcinoma 347 YLGPVSPSL NHL 348 ALAKPPVVSV SCLC, HCC, melanoma, esophageal cancer 349 ALATHILSL NHL, bladder cancer 350 ALEDRVWEL NHL, esophageal cancer 351 ALSEKLARL HCC, NHL, AML, bladder cancer 352 ALVFELHYV NHL 353 ATPMPTPSV SCLC, NHL 354 FIMDDPAGNSYL CRC, NHL, AML, melanoma, esophageal cancer, bladder cancer, gallbladder cancer, cholangiocarcinoma 357 FLIQEIKTL NHL, OC, bladder cancer 358 FLTDYLNDL NHL 359 FMQDPMEVFV Melanoma 360 HLIDTNKIQL NHL 362 ILTELGGFEV NHL 363 ITTEVVNELYV NHL, melanoma 364 KMDWIFHTI Bladder Cancer 365 LISPLLLPV NHL, AML, melanoma, bladder cancer 367 NLWSLVAKV NSCLC, NHL, AML, melanoma, OC 369 RLLDLENSLLGL AML 370 SIFASPESV Melanoma, esophageal cancer, bladder cancer 371 SLADDSVLERL RCC, HCC, NHL, AML, esophageal cancer, bladder cancer 373 TLLADQGEIRV NHL, melanoma 374 VLSVITEEL NHL, AML 375 VLWFKPVEL NSCLC, HCC, NHL, melanoma, esophageal cancer 376 VLYNQRVEEI Bladder Cancer 377 VVDGTCVAV NHL, melanoma 378 YILGKFFAL NHL, AML 379 YLAELVTPIL NSCLC, SCLC, NHL, melanoma 380 YLDRKLLTL NHL 381 YLLEENKIKL NHL 382 YLLPLLQRL NHL, AML 383 YLLREWVNL HCC, NHL 384 YMIGSEVGNYL NHL, bladder cancer 385 YTIPLAIKL NHL, AML, BRCA, esophageal cancer

NSCLC=非小細胞肺癌，SCLC=小細胞肺癌，RCC=腎癌，CRC =結腸或直腸癌，GC =胃癌， HCC = 肝癌，PC =胰腺癌，PrC=攝護腺癌，白血病，BRCA=乳腺癌，MCC = 梅克爾細胞癌，NHL=非霍奇金淋巴瘤NSCLC=non-small cell lung cancer, SCLC=small cell lung cancer, RCC=kidney cancer, CRC=colon or rectal cancer, GC=gastric cancer, HCC=liver cancer, PC=pancreatic cancer, PrC=prostate cancer, leukemia, BRCA=breast Carcinoma, MCC = Merkel cell carcinoma, NHL = non-Hodgkin's lymphoma

因此，本發明的另一個方面涉及根據SEQ ID No. 1、2、3、7、9、10、13、14、15、19、20、21、22、24、26、27、28、29、30、32、33、35、38、39、40、42、43、45、46、48、49、55、56、57、58、59、62、63、64、66、67、68、69、70、71、72、74、75、76、78、79、80、81、82、86、87、88、89、91、92、93、94、96、97、98、99、100、101、103、105、110、112、113、116、117、118、119、120、121、122、125、126、127、130、133、137、138、139、141、145、146、147、148、149、150、153、154、155、156、158、159、160、161、162、163、165、167、168、169、170、172、175、177、178、179、180、181、187、188、189、190、192、193、194、195、196、197、199、200、203、205、207、208、209、210、211、212、213、214、215、216、217、219、220、221、222、223、224、225、227、228、229、230、233、234、235、236、237、238、239、240、242、244、246、247、248、251、253、254、255、257、258、260、261、263、264、266、267、268、269、272、273、274、275、276、278、280、281、282、283、284、286、288、290、291、294、295、296、297、298、299、300、303、305、306、307、308、309、310、311、312、314、316、320、321、322、323、324、325、327、328、329、330、332、333、334、335、337、338、339、341、342、343、346、347、349、350、351、352、353、354、357、358、360、362、363、365、367、371、373、374、375、377、378、379、380、381、382、383、384 和 385 中任一項所述的本發明的至少一種肽在一種優選實施方案中聯合用於治療 NHL。Therefore, another aspect of the present invention relates to according to SEQ ID No. 1, 2, 3, 7, 9, 10, 13, 14, 15, 19, 20, 21, 22, 24, 26, 27, 28, 29, 30, 32, 33, 35, 38, 39, 40, 42, 43, 45, 46, 48, 49, 55, 56, 57, 58, 59, 62, 63, 64, 66, 67, 68, 69, 70, 71, 72, 74, 75, 76, 78, 79, 80, 81, 82, 86, 87, 88, 89, 91, 92, 93, 94, 96, 97, 98, 99, 100, 101, 103, 105, 110, 112, 113, 116, 117, 118, 119, 120, 121, 122, 125, 126, 127, 130, 133, 137, 138, 139, 141, 145, 146, 147, 148, 149, 150, 153, 154, 155, 156, 158, 159, 160, 161, 162, 163, 165, 167, 168, 169, 170, 172, 175, 177, 178, 179, 180, 181, 187, 188, 189, 190, 192, 193, 194, 195, 196, 197, 199, 200, 203, 205, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 219, 220, 221, 222, 223, 224, 225, 227, 228, 229, 230, 233, 234, 235, 236, 237, 238, 239, 240, 242, 244, 246, 247, 248, 251, 253, 254, 255, 257, 258, 260, 261, 263, 264, 266, 267, 268, 269, 272, 273, 274, 275, 276, 278, 280, 281, 282, 283, 284, 286, 288, 290, 291, 294, 295, 296, 297, 298, 299, 300, 303, 305, 306, 307, 308, 309, 310, 311, 312, 314, 316, 320, 321, 322, 323, 324, 325, 327, 328, 329, 330, 332, 333, 334, 335, 337, 338, 339, 341, 342, 343, 346, 347, 349, 350, 351, 352, 353, 354, 357, 358, 360, 362, 363, 365, 367, 371, 373, 374, 375, 377, 378, 379, 380, 381, 382, 383, 384, and 38 At least one peptide of the invention described in any one of 5 is combined in a preferred embodiment for the treatment of NHL.

因此，本發明的另一個方面涉及根據SEQ ID No. 5、8、12、14、15、29、35、37、39、43、56、57、74、78、93、99、100、102、109、116、117、119、120、121、147、148、151、157、158、160、164、166、172、183、185、186、188、190、195、199、214、219、221、231、234、235、236、238、239、241、242、243、244、246、258、263、264、267、269、271、281、290、296、298、303、304、311、317、335、339、342、343、346、351、354、365、367、369、374、378、382 和 385 中任一項所述的本發明的至少一種肽在一種優選實施方案中聯合用於治療 AML。Therefore, another aspect of the present invention relates to according to SEQ ID No. 5, 8, 12, 14, 15, 29, 35, 37, 39, 43, 56, 57, 74, 78, 93, 99, 100, 102, 109, 116, 117, 119, 120, 121, 147, 148, 151, 157, 158, 160, 164, 166, 172, 183, 185, 186, 188, 190, 195, 199, 214, 219, 221, 231, 234, 235, 236, 238, 239, 241, 242, 243, 244, 246, 258, 263, 264, 267, 269, 271, 281, 290, 296, 298, 303, 304, 311, 317, At least one peptide of the present invention according to any one of 335, 339, 342, 343, 346, 351, 354, 365, 367, 369, 374, 378, 382 and 385 is used in combination for treatment in a preferred embodiment AML.

因此，本發明的另一個方面涉及根據SEQ ID No. 5、28、74、121、162、208、211、212、264、296、328 和 385 中任一項所述的本發明的至少一種肽在一種優選實施方案中聯合用於治療 BRCA。Therefore, another aspect of the present invention relates to at least one peptide of the present invention according to any one of SEQ ID No. 5, 28, 74, 121, 162, 208, 211, 212, 264, 296, 328 and 385 In a preferred embodiment the combination is used to treat BRCA.

因此，本發明的另一個方面涉及根據SEQ ID No. 7、67、68、78、86、112、124、152、155、162、181、199、200、204、210、246、250、274、311、333、338、341、348、351、371、375 和 383 中任一項所述的本發明的至少一種肽在一種優選實施方案中聯合用於治療 HCC。Therefore, another aspect of the present invention relates to according to SEQ ID No. 7, 67, 68, 78, 86, 112, 124, 152, 155, 162, 181, 199, 200, 204, 210, 246, 250, 274, At least one peptide of the present invention described in any one of 311, 333, 338, 341, 348, 351, 371, 375 and 383 is used in combination in a preferred embodiment for the treatment of HCC.

因此，本發明的另一個方面涉及根據SEQ ID No. 12、15、25、29、39、42、44、45、52、57、60、67、68、72、78、93、94、112、117、130、141、159、162、169、170、172、189、197、200、204、207、211、212、217、225、232、233、234、237、239、246、250、254、258、267、276、282、291、297、300、303、305、306、311、313、328、338、339、343、348、354、359、363、365、367、370、373、375、377 和 379 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療黑色素瘤。Therefore, another aspect of the present invention relates to according to SEQ ID No. 12, 15, 25, 29, 39, 42, 44, 45, 52, 57, 60, 67, 68, 72, 78, 93, 94, 112, 117, 130, 141, 159, 162, 169, 170, 172, 189, 197, 200, 204, 207, 211, 212, 217, 225, 232, 233, 234, 237, 239, 246, 250, 254, 258, 267, 276, 282, 291, 297, 300, 303, 305, 306, 311, 313, 328, 338, 339, 343, 348, 354, 359, 363, 365, 367, 370, 373, 375, At least one peptide of the present invention (in combination in a preferred embodiment) of any one of 377 and 379 is used for the treatment of melanoma.

因此，本發明的另一個方面涉及根據SEQ ID No. 27、94、122、162、164、200、274、276、311、367、375 和 379 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療 NSCLC。Therefore, another aspect of the present invention relates to at least one peptide of the present invention according to any one of SEQ ID No. 27, 94, 122, 162, 164, 200, 274, 276, 311, 367, 375 and 379 (In combination in a preferred embodiment) for the treatment of NSCLC.

因此，本發明的另一個方面涉及根據SEQ ID No. 27、59、79、119、158、162、164、233、235、237、264、276 和 339 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療子宮癌。Therefore, another aspect of the present invention relates to at least one of the present invention according to any one of SEQ ID No. 27, 59, 79, 119, 158, 162, 164, 233, 235, 237, 264, 276 and 339 A peptide (in combination in a preferred embodiment) is used to treat uterine cancer.

因此，本發明的另一個方面涉及根據SEQ ID No. 29、67、93、94、111、112、200、204、207、208、219、225、244、245、264、266、282、303、306、321、328、338、357 和 367 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療卵巢癌 (OC)。Therefore, another aspect of the present invention relates to according to SEQ ID No. 29, 67, 93, 94, 111, 112, 200, 204, 207, 208, 219, 225, 244, 245, 264, 266, 282, 303, At least one peptide of the present invention described in any one of 306, 321, 328, 338, 357 and 367 (in combination in a preferred embodiment) is used for the treatment of ovarian cancer (OC).

因此，本發明的另一個方面涉及根據SEQ ID No. 29、35、67、161、162、188、189、200、213、233、235、239、260、276、342、346 和 354 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療膽囊癌和/或膽管癌。Therefore, another aspect of the present invention relates to any one of SEQ ID No. 29, 35, 67, 161, 162, 188, 189, 200, 213, 233, 235, 239, 260, 276, 342, 346 and 354 At least one peptide of the present invention (in combination in a preferred embodiment) described in the item is used for the treatment of gallbladder cancer and/or cholangiocarcinoma.

因此，本發明的另一個方面涉及根據SEQ ID No. 35、68、94、124、139、162、164、197、204、232、245、258、311、346 和 371 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療RCC。Therefore, another aspect of the present invention relates to according to any one of SEQ ID No. 35, 68, 94, 124, 139, 162, 164, 197, 204, 232, 245, 258, 311, 346 and 371 At least one peptide of the invention (in combination in a preferred embodiment) is used to treat RCC.

因此，本發明的另一個方面涉及根據SEQ ID No. 39、40、83、93、121、138、162、178、189、200、204、207、216、235、237、245、250、272、274、276、291、296、298、311、321、333、335、348、353 和 379 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療 SCLC。Therefore, another aspect of the present invention relates to according to SEQ ID No. 39, 40, 83, 93, 121, 138, 162, 178, 189, 200, 204, 207, 216, 235, 237, 245, 250, 272, At least one peptide (in combination in a preferred embodiment) of the present invention described in any one of 274, 276, 291, 296, 298, 311, 321, 333, 335, 348, 353 and 379 is used for the treatment of SCLC.

因此，本發明的另一個方面涉及根據SEQ ID No. 40、55、59、119、146、159、160、164、186、187、200、202、204、208、213、215、225、233、234、235、240、249、250、266、274、292、298、313、328、335、338、341、342、349、351、354、357、364、365、370、371、376 和 384 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療膀胱癌。Therefore, another aspect of the present invention relates to according to SEQ ID No. 40, 55, 59, 119, 146, 159, 160, 164, 186, 187, 200, 202, 204, 208, 213, 215, 225, 233, 234, 235, 240, 249, 250, 266, 274, 292, 298, 313, 328, 335, 338, 341, 342, 349, 351, 354, 357, 364, 365, 370, 371, 376 and 384 At least one of the peptides of the invention (in a preferred embodiment) according to any one of them is used for the treatment of bladder cancer.

因此，本發明的另一個方面涉及根據SEQ ID No. 59、60、67、79、83、87、112、136、159、162、164、179、200、201、213、215、218、228、233、252、265、271、326、343、344、348、350、354、370、371、375 和 385 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療食管癌。Therefore, another aspect of the present invention relates to according to SEQ ID No. 59, 60, 67, 79, 83, 87, 112, 136, 159, 162, 164, 179, 200, 201, 213, 215, 218, 228, At least one peptide of the present invention according to any one of 233, 252, 265, 271, 326, 343, 344, 348, 350, 354, 370, 371, 375 and 385 (in combination in a preferred embodiment) For the treatment of esophageal cancer.

因此，本發明的另一個方面涉及根據SEQ ID No. 83、87、136、164 和 271 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療胃癌 (GC)。Therefore, another aspect of the present invention relates to at least one peptide of the present invention (in combination in a preferred embodiment) according to any one of SEQ ID No. 83, 87, 136, 164 and 271 for the treatment of gastric cancer ( GC).

因此，本發明的另一個方面涉及根據SEQ ID No. 88、189、233、235、271、278 和 311 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療胰腺癌 (PC)。Therefore, another aspect of the present invention relates to the use of at least one peptide (in combination in a preferred embodiment) of the present invention according to any one of SEQ ID No. 88, 189, 233, 235, 271, 278 and 311 For the treatment of pancreatic cancer (PC).

因此，本發明的另一個方面涉及根據SEQ ID No. 94、161、172、200、232、271、321 和 354 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療結直腸癌 (CRC)。Therefore, another aspect of the present invention relates to at least one peptide of the present invention according to any one of SEQ ID No. 94, 161, 172, 200, 232, 271, 321 and 354 (in a preferred embodiment combined ) Is used to treat colorectal cancer (CRC).

因此，本發明的另一個方面涉及根據SEQ ID No. 159 所述的本發明的肽用於治療 MCC。Therefore, another aspect of the present invention relates to the use of the peptide of the present invention according to SEQ ID No. 159 for the treatment of MCC.

因此，本發明的另一個方面涉及本發明中肽的用途（優選聯合 ）用於治療選自 CLL、急性骨髓性白血病、膽管癌、腦癌、乳腺癌、結直腸癌、食管癌、膽囊癌、胃癌、肝細胞癌、梅克爾細胞癌、黑色素瘤、非霍奇金淋巴瘤、非小細胞肺癌、卵巢癌、胰腺癌、攝護腺癌、腎細胞癌、小細胞肺癌、膀胱癌和子宮癌組中的增殖性疾病。Therefore, another aspect of the present invention relates to the use of the peptide of the present invention (preferably in combination) for the treatment of CLL, acute myeloid leukemia, cholangiocarcinoma, brain cancer, breast cancer, colorectal cancer, esophageal cancer, gallbladder cancer, Gastric cancer, hepatocellular carcinoma, Merkel cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and uterine cancer Proliferative diseases in the group.

表 4B：本發明的肽及其在其他增殖性疾病（特別是其他癌性疾病）中的特定用途。與表 4A 一樣，該表顯示，對於其他腫瘤類型的選定肽，發現他們顯示過量表現（包括特定表現）於 5% 以上測定的腫瘤樣本，或表現於 5% 以上測定的腫瘤樣本且幾何學平均值腫瘤與正常組織的比值大於 3。過度表現定義為與最高表現的正常樣本相比在腫瘤樣本上表現更高。經測試過度表現的正常組織有：脂肪組織、腎上腺、動脈、骨髓、腦、中樞神經、結腸、十二指腸、食道、眼、膽囊、心臟、腎、肝、肺、淋巴結、單核白細胞、胰腺、甲狀旁腺、外周神經、腹膜、垂體、胸膜、直腸、唾液腺、骨骼肌、皮膚、小腸、脾、胃、甲狀腺、氣管、輸尿管、膀胱、靜脈。 序列ID號 序列 其他實體 5 ALIFKIASA 子宮癌 7 ALLERTGYTL 子宮癌、AML 8 ALAASALPALV AML、HNSCC 11 ALFTFSPLTV AML、HNSCC、NHL、NSCLC、OC、SCLC 13 VVDGMPPGV BRCA、子宮癌 14 ALLRLLPGL BRCA、黑色素瘤、HNSCC 15 ALPEVSVEA 食管癌 25 ATYSGLESQSV AML 26 AVLLVLPLV NHL、NSCLC 27 AVLGLVWLL HNSCC 29 AVLQAVTAV 子宮癌 30 ELLEGSEIYL CRC、BRCA、黑色素瘤、子宮癌 35 FILPSSLYL SCLC、子宮癌 37 FIMEGGAMVL NHL 38 FIMEGGAMV HNSCC 40 FLDEDDMSL HNSCC 41 FLDPSLDPLL 子宮癌 42 FLEEGGVVTV SCLC、AML 44 FLLSINDFL RCC、子宮癌、AML、NHL 45 FLPELPADLEA NSCLC、子宮癌、OC 48 FLSPQQPPLL HNSCC 49 FLTDLFAQL NHL、黑色素瘤 51 FLVEAPHDWDL 子宮癌 55 FMEPTLLML HNSCC 58 YIQQGIFSV NHL、NSCLC 59 FVFGDENGTVSL SCLC、HNSCC 67 GLDDVTVEV AML 68 GLDQQFAGLDL BRCA、OC、子宮癌、膽囊癌、膽管癌、HNSCC 76 GLLRIIPYL RCC 82 GLQSGVDIGV AML、食管癌、PC 83 GQGEVLVYV NHL 84 GVMDVNTAL AML、HCC、HNSCC、NHL、NSCLC、GC、RCC 86 HLYPGAVTI AML 87 HQIEAVDGEEL PC 89 ILDFGTFQL BRCA 90 VIADLGLIRV 黑色素瘤 91 ILDLNTYNV AML 93 ILFNTQINI 子宮癌、HNSCC 94 ILFPLRFTL 膽囊癌、膽管癌、AML、HNSCC 101 ILVVIEPLL 子宮癌 107 ILYNLYDLL 黑色素瘤 108 VIYTLIHYI BRCA、黑色素瘤 111 KLWNVAAPLYL 黑色素瘤 112 KTLDVDATYEI 子宮癌 113 KVPAEEVLVAV NHL、NSCLC 114 LIPEGPPQV AML、HNSCC 115 LLFDKLYLL 子宮癌 118 RLLILENILL HNSCC 119 VLPAEFFEV SCLC 124 LLLEETEKQAV HNSCC 125 LLLEIGEVGKLFV BRCA 126 LLPEGGITAI SCLC、HCC、黑色素瘤、子宮癌、HNSCC 127 LLPTAPTTV SCLC 128 LLSEEEYHL 膀胱癌、NHL、HNSCC 130 LLVLIPVYL 子宮癌 136 NLDKLWTLV AML、CRC、黑色素瘤、NSCLC、腦癌 138 NLLDIDAPVTV 子宮癌、AML 141 QILSEIVEA 子宮癌 145 SALDTITTV NSCLC、RCC、GC、HCC、AML 146 SIIEGPIIKL HNSCC 147 SILETVATL 黑色素瘤 152 SLIRILQTI SCLC 156 SLNIGDVQL AML 159 SLQEEKLIYV HNSCC 160 SLSFLVPSL 子宮癌 161 SMDDGMINV SCLC、黑色素瘤、子宮癌、AML、HNSCC 162 SMKDDLENV PC、OC、HNSCC 164 SVHKGFAFV CRC、黑色素瘤、HNSCC 172 TLYDMLASI SCLC、膀胱癌、子宮癌 173 TVIENIHTI CRC、HCC、黑色素瘤、NHL、NSCLC、OC、食管癌、GC、RCC 174 VLAELPIIVV NSCLC、OC 176 VLAEQNIIPSA NSCLC、SCLC、GC、黑色素瘤、NHL、HNSCC 179 VLLGLEMTL HNSCC 181 VLLSIPFVSV 黑色素瘤、膽囊癌、膽管癌 184 VLSHNLYTV NHL 187 VMDTHLVNI CRC、黑色素瘤、子宮癌 188 VMGDIPAAV 膀胱癌、HNSCC 189 VMLEMTPEL HNSCC 190 VVMGTVPRL 黑色素瘤 191 YIFDGSDGGV 子宮癌 192 YIQEYLTLL SCLC、膀胱癌、AML、HNSCC 197 YLQEVPILTL 子宮癌 198 YLTFLPAEV BRCA、子宮癌 199 YLVELSSLL 黑色素瘤 200 YMFEEVPIVI BRCA、HNSCC 201 YQLELHGIEL NHL 202 YVDDVFLRV AML、HNSCC 203 ALLSSQLAL NHL、AML、NSCLC 204 GLLQINDKIAL BRCA、子宮癌、AML 208 ILLKTEGINL 黑色素瘤、HNSCC 211 LIDVKPLGV RCC、腦癌、HCC、食管癌、HNSCC 212 NIIEAINELLV 子宮癌、AML、HNSCC 215 VMQDIVYKL HNSCC 216 WLAGDVPAA AML、HNSCC 217 ALDEPPYLTV AML 218 ALGEEWKGYVV AML 221 ALVSTIIMV 腦癌 224 AMEALVVEV AML 226 AMNISVPQV AML、PrC、腦癌C_CCC、HNSCC、NHL、NSCLC、OC、SCLC、子宮癌 228 FLGGLSPGV SCLC、BRCA、子宮癌、AML、HNSCC 231 FLYIRQLAI 子宮癌 232 FMHQIIDQV 子宮癌 234 GLDDAEYAL 子宮癌、HNSCC 235 GLDDLLLFL BRCA、HNSCC 237 GLQENLDVVV AML 239 ILAGEMLSV SCLC、HCC 240 ILARDILEI 黑色素瘤 242 ILLGIQELL BRCA 247 KLKDRLPSI HCC 257 RIINGIIISV AML 258 RLDYITAEI GC、子宮癌 260 SLDDVEGMSV SCLC、HNSCC 261 SLVEAQGWLV AML 264 TILDYINVV HNSCC 266 TLLDQLDTQL CRC、BRCA、子宮癌 268 TLLQVFHLL 子宮癌 269 TLTDEQFLV BRCA 272 VLDELPPLI BRCA、子宮癌、HNSCC 273 VLGEYSYLL NHL、HNSCC 274 VLLEYHIAYL 黑色素瘤、HNSCC 275 VLLFIEHSV 黑色素瘤 279 YMAPEVVEA HNSCC、NHL、NSCLC、SCLC、子宮癌 280 YTLDSLYWSV SCLC、BRCA、黑色素瘤、HNSCC 286 FLLEDLSQKL 子宮癌 289 ILEEQPMDMLL 子宮癌 293 ALDIMIPMV AML、CRC、腦癌C_CCC、HNSCC、黑色素瘤、NHL、NSCLC、OC、腦癌、RCC、SCLC、膀胱癌、子宮癌 294 ALLDQLHTLL AML 298 IILPVEVEV CRC、HNSCC 299 ILEENIPVL 子宮癌、AML 303 ILSQPTPSL 子宮癌 305 LLFPQIEGIKI 膀胱癌、HNSCC 306 VVAEELENV RCC 311 LLVDVEPKV 子宮癌、HNSCC 312 LLYNSTDPTL 膀胱癌 313 LMADLEGLHL 子宮癌、AML 317 NLLAHIWAL NHL 321 RLLEFELAQL 腦癌、PrC、子宮癌、AML、HNSCC 323 SLDLFNCEV 子宮癌、AML 324 SLYSALQQA SCLC、子宮癌、AML 326 VLAFLVHEL 食管癌、NSCLC、RCC 327 VLIKWFPEV 子宮癌 333 VTLEFPQLIRV BRCA 334 YLLEEKIASL AML 335 YLYQEQYFI CRC、腦癌C_CCC、NSCLC、子宮癌 336 YMAVTTQEV 子宮癌 339 GLWGTVVNI HNSCC 340 KLLEEICNL 黑色素瘤、子宮癌、AML、NHL 342 SLITPLQAV HNSCC 343 TLLEALDCI GC、子宮癌、HNSCC 344 VLAFENPQV HNSCC 346 VLVQVSPSL HNSCC 348 ALAKPPVVSV NSCLC、膀胱癌、NHL、HNSCC 349 ALATHILSL HCC、黑色素瘤 351 ALSEKLARL CRC、HNSCC、黑色素瘤、NSCLC、OC、PC、腦癌、RCC、子宮癌 353 ATPMPTPSV BRCA、子宮癌、膽囊癌、膽管癌、AML、HNSCC 354 FIMDDPAGNSYL 腦癌、HNSCC 355 FIWPMLIHI 黑色素瘤、子宮癌 357 FLIQEIKTL HCC 358 FLTDYLNDL AML 359 FMQDPMEVFV 黑色素瘤、NHL、NSCLC、OC 360 HLIDTNKIQL NHL、NSCLC 361 ILQEFESKL AML、NHL 362 ILTELGGFEV AML、PrC、BRCA、PC、CRC、HCC、HNSCC、黑色素瘤、NSCLC、OC、食管癌、GC、RCC、SCLC、膀胱癌、子宮癌 363 ITTEVVNELYV BRCA 365 LISPLLLPV SCLC、子宮癌、HNSCC 367 NLWSLVAKV 子宮癌 368 QLQPTDALLCV 子宮癌 370 SIFASPESV 子宮癌、膽囊癌、膽管癌、AML、HNSCC 374 VLSVITEEL SCLC、HCC、PrC、膀胱癌、子宮癌 375 VLWFKPVEL BRCA、OC、子宮癌、膽囊癌、膽管癌、HNSCC 377 VVDGTCVAV BRCA、子宮癌、HNSCC 378 YILGKFFAL 黑色素瘤、食管癌 379 YLAELVTPIL HCC、BRCA、子宮癌、AML、HNSCC 383 YLLREWVNL 黑色素瘤、子宮癌 384 YMIGSEVGNYL SCLC、子宮癌 385 YTIPLAIKL CRC Table 4B: The peptides of the present invention and their specific use in other proliferative diseases (especially other cancerous diseases). As in Table 4A, this table shows that for selected peptides of other tumor types, they are found to show excessive expression (including specific expressions) in more than 5% of tumor samples measured, or in more than 5% of tumor samples measured and geometrically averaged The ratio of tumor to normal tissue is greater than 3. Overperformance is defined as higher performance on tumor samples compared to the highest performing normal samples. The normal tissues that have been tested for overexpression are: adipose tissue, adrenal glands, arteries, bone marrow, brain, central nervous system, colon, duodenum, esophagus, eyes, gallbladder, heart, kidney, liver, lung, lymph nodes, mononuclear white blood cells, pancreas, nails Parathyroid glands, peripheral nerves, peritoneum, pituitary gland, pleura, rectum, salivary glands, skeletal muscle, skin, small intestine, spleen, stomach, thyroid, trachea, ureter, bladder, vein. Serial ID number sequence Other entities 5 ALIFKIASA Uterine cancer 7 ALLERTGYTL Uterine cancer, AML 8 ALAASALPALV AML, HNSCC 11 ALFTFSPLTV AML, HNSCC, NHL, NSCLC, OC, SCLC 13 VVDGMPPGV BRCA, uterine cancer 14 ALLRLLPGL BRCA, melanoma, HNSCC 15 ALPEVSVEA Esophageal cancer 25 ATYSGLESQSV AML 26 AVLLVLPLV NHL, NSCLC 27 AVLGLVWLL HNSCC 29 AVLQAVTAV Uterine cancer 30 ELLEGSEIYL CRC, BRCA, melanoma, uterine cancer 35 FILPSSLYL SCLC, uterine cancer 37 FIMEGGAMVL NHL 38 FIMEGGAMV HNSCC 40 FLDEDDMSL HNSCC 41 FLDPSLDPLL Uterine cancer 42 FLEEGGVVTV SCLC, AML 44 FLLSINDFL RCC, uterine cancer, AML, NHL 45 FLPELPADLEA NSCLC, uterine cancer, OC 48 FLSPQQPPLL HNSCC 49 FLTDLFAQL NHL, melanoma 51 FLVEAPHDWDL Uterine cancer 55 FMEPTLLML HNSCC 58 YIQQGIFSV NHL, NSCLC 59 FVFGDENGTVSL SCLC, HNSCC 67 GLDDVTVEV AML 68 GLDQQFAGLDL BRCA, OC, uterine cancer, gallbladder cancer, cholangiocarcinoma, HNSCC 76 GLLRIIPYL RCC 82 GLQSGVDIGV AML, esophageal cancer, PC 83 GQGEVLVYV NHL 84 GVMDVNTAL AML, HCC, HNSCC, NHL, NSCLC, GC, RCC 86 HLYPGAVTI AML 87 HQIEAVDGEEL PC 89 ILDFGTFQL BRCA 90 VIADLGLIRV Melanoma 91 ILDLNTYNV AML 93 ILFNTQINI Uterine cancer, HNSCC 94 ILFPLRFTL Gallbladder cancer, cholangiocarcinoma, AML, HNSCC 101 ILVVIEPLL Uterine cancer 107 ILYNLYDLL Melanoma 108 VIYTLIHYI BRCA, melanoma 111 KLWNVAAPLYL Melanoma 112 KTLDVDATYEI Uterine cancer 113 KVPAEEVLVAV NHL, NSCLC 114 LIPEGPPQV AML, HNSCC 115 LLFDKLYLL Uterine cancer 118 RLLILENILL HNSCC 119 VLPAEFFEV SCLC 124 LLLEETEKQAV HNSCC 125 LLLEIGEVGKLFV BRCA 126 LLPEGGITAI SCLC, HCC, melanoma, uterine cancer, HNSCC 127 LLPTAPTTV SCLC 128 LLSEEEYHL Bladder cancer, NHL, HNSCC 130 LLVLIPVYL Uterine cancer 136 NLDKLWTLV AML, CRC, melanoma, NSCLC, brain cancer 138 NLLDIDAPVTV Uterine cancer, AML 141 QILSEIVEA Uterine cancer 145 SALDTITTV NSCLC, RCC, GC, HCC, AML 146 SIIEGPIIKL HNSCC 147 SILETVATL Melanoma 152 SLIRILQTI SCLC 156 SLNIGDVQL AML 159 SLQEEKLIYV HNSCC 160 SLSFLVPSL Uterine cancer 161 SMDDGMINV SCLC, melanoma, uterine cancer, AML, HNSCC 162 SMKDDLENV PC, OC, HNSCC 164 SVHKGFAFV CRC, melanoma, HNSCC 172 TLYDMLASI SCLC, bladder cancer, uterine cancer 173 TVIENIHTI CRC, HCC, melanoma, NHL, NSCLC, OC, esophageal cancer, GC, RCC 174 VLAELPIIVV NSCLC, OC 176 VLAEQNIIPSA NSCLC, SCLC, GC, melanoma, NHL, HNSCC 179 VLLGLEMTL HNSCC 181 VLLSIPFVSV Melanoma, gallbladder cancer, cholangiocarcinoma 184 VLSHNLYTV NHL 187 VMDTHLVNI CRC, melanoma, uterine cancer 188 VMGDIPAAV Bladder cancer, HNSCC 189 VMLEMTPEL HNSCC 190 VVMGTVPRL Melanoma 191 YIFDGSDGGV Uterine cancer 192 YIQEYLTLL SCLC, bladder cancer, AML, HNSCC 197 YLQEVPILTL Uterine cancer 198 YLTFLPAEV BRCA, uterine cancer 199 YLVELSSLL Melanoma 200 YMFEEVPIVI BRCA, HNSCC 201 YQLELHGIEL NHL 202 YVDDVFLRV AML, HNSCC 203 ALLSSQLAL NHL, AML, NSCLC 204 GLLQINDKIAL BRCA, uterine cancer, AML 208 ILLKTEGINL Melanoma, HNSCC 211 LIDVKPLGV RCC, brain cancer, HCC, esophageal cancer, HNSCC 212 NIIEAINELLV Uterine cancer, AML, HNSCC 215 VMQDIVYKL HNSCC 216 WLAGDVPAA AML, HNSCC 217 ALDEPPYLTV AML 218 ALGEEWKGYVV AML 221 ALVSTIIMV Brain cancer 224 AMEALVVEV AML 226 AMNISVPQV AML, PrC, brain cancer C_CCC, HNSCC, NHL, NSCLC, OC, SCLC, uterine cancer 228 FLGGLSPGV SCLC, BRCA, uterine cancer, AML, HNSCC 231 FLYIRQLAI Uterine cancer 232 FMHQIIDQV Uterine cancer 234 GLDDAEYAL Uterine cancer, HNSCC 235 GLDDLLLFL BRCA, HNSCC 237 GLQENLDVVV AML 239 ILAGEMLSV SCLC, HCC 240 ILARDILEI Melanoma 242 ILLGIQELL BRCA 247 KLKDRLPSI HCC 257 RIINGIIISV AML 258 RLDYITAEI GC, uterine cancer 260 SLDDVEGMSV SCLC, HNSCC 261 SLVEAQGWLV AML 264 TILDYINVV HNSCC 266 TLLDQLDTQL CRC, BRCA, uterine cancer 268 TLLQVFHLL Uterine cancer 269 TLTDEQFLV BRCA 272 VLDELPPLI BRCA, uterine cancer, HNSCC 273 VLGEYSYLL NHL, HNSCC 274 VLLEYHIAYL Melanoma, HNSCC 275 VLLFIEHSV Melanoma 279 YMAPEVVEA HNSCC, NHL, NSCLC, SCLC, uterine cancer 280 YTLDSLYWSV SCLC, BRCA, melanoma, HNSCC 286 FLLEDLSQKL Uterine cancer 289 ILEEQPMDMLL Uterine cancer 293 ALDIMIPMV AML, CRC, brain cancer C_CCC, HNSCC, melanoma, NHL, NSCLC, OC, brain cancer, RCC, SCLC, bladder cancer, uterine cancer 294 ALLDQLHTLL AML 298 IILPVEVEV CRC, HNSCC 299 ILEENIPVL Uterine cancer, AML 303 ILSQPTPSL Uterine cancer 305 LLFPQIEGIKI Bladder cancer, HNSCC 306 VVAEELENV RCC 311 LLVDVEPKV Uterine cancer, HNSCC 312 LLYNSTDPTL Bladder Cancer 313 LMADLEGLHL Uterine cancer, AML 317 NLLAHIWAL NHL 321 RLLEFELAQL Brain cancer, PrC, uterine cancer, AML, HNSCC 323 SLDLFNCEV Uterine cancer, AML 324 SLYSALQQA SCLC, uterine cancer, AML 326 VLAFLVHEL Esophageal cancer, NSCLC, RCC 327 VLIKWFPEV Uterine cancer 333 VTLEFPQLIRV BRCA 334 YLLEEKIASL AML 335 YLYQEQYFI CRC, brain cancer C_CCC, NSCLC, uterine cancer 336 YMAVTTQEV Uterine cancer 339 GLWGTVVNI HNSCC 340 KLLEEICNL Melanoma, uterine cancer, AML, NHL 342 SLITPLQAV HNSCC 343 TLLEALDCI GC, uterine cancer, HNSCC 344 VLAFENPQV HNSCC 346 VLVQVSPSL HNSCC 348 ALAKPPVVSV NSCLC, bladder cancer, NHL, HNSCC 349 ALATHILSL HCC, melanoma 351 ALSEKLARL CRC, HNSCC, melanoma, NSCLC, OC, PC, brain cancer, RCC, uterine cancer 353 ATPMPTPSV BRCA, uterine cancer, gallbladder cancer, cholangiocarcinoma, AML, HNSCC 354 FIMDDPAGNSYL Brain cancer, HNSCC 355 FIWPMLIHI Melanoma, uterine cancer 357 FLIQEIKTL HCC 358 FLTDYLNDL AML 359 FMQDPMEVFV Melanoma, NHL, NSCLC, OC 360 HLIDTNKIQL NHL, NSCLC 361 ILQEFESKL AML, NHL 362 ILTELGGFEV AML, PrC, BRCA, PC, CRC, HCC, HNSCC, melanoma, NSCLC, OC, esophageal cancer, GC, RCC, SCLC, bladder cancer, uterine cancer 363 ITTEVVNELYV BRCA 365 LISPLLLPV SCLC, uterine cancer, HNSCC 367 NLWSLVAKV Uterine cancer 368 QLQPTDALLCV Uterine cancer 370 SIFASPESV Uterine cancer, gallbladder cancer, cholangiocarcinoma, AML, HNSCC 374 VLSVITEEL SCLC, HCC, PrC, bladder cancer, uterine cancer 375 VLWFKPVEL BRCA, OC, uterine cancer, gallbladder cancer, cholangiocarcinoma, HNSCC 377 VVDGTCVAV BRCA, uterine cancer, HNSCC 378 YILGKFFAL Melanoma, esophageal cancer 379 YLAELVTPIL HCC, BRCA, uterine cancer, AML, HNSCC 383 YLLREWVNL Melanoma, uterine cancer 384 YMIGSEVGNYL SCLC, uterine cancer 385 YTIPLAIKL CRC

NSCLC=非小細胞肺癌，SCLC=小細胞肺癌，RCC=腎癌，CRC =結腸或直腸癌，GC =胃癌， HCC = 肝癌，PC =胰腺癌，PrC=攝護腺癌，BRCA =乳腺癌， NHL=非霍奇金淋巴瘤，AML=急性骨髓性白血病，OC= 卵巢癌，HNSCC=頭頸部鱗狀細胞癌，CCC=膽管癌。NSCLC=non-small cell lung cancer, SCLC=small cell lung cancer, RCC=kidney cancer, CRC=colon or rectal cancer, GC=gastric cancer, HCC=liver cancer, PC=pancreatic cancer, PrC=prostate cancer, BRCA=breast cancer, NHL = non-Hodgkin's lymphoma, AML = acute myeloid leukemia, OC = ovarian cancer, HNSCC = head and neck squamous cell carcinoma, CCC = cholangiocarcinoma.

因此，本發明的另一個方面涉及根據SEQ ID No. 5、7、13、29、30、35、41、44、45、51、68、93、101、112、115、126、130、138、141、160、161、172、187、191、197、198、204、212、226、228、231、232、234、258、266、268、272、279、286、289、293、299、303、311、313、321、323、324、327、335、336、340、343、351、353、355、362、365、367、368、370、374、375、377、379、383 和 384 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療子宮癌。Therefore, another aspect of the present invention relates to according to SEQ ID No. 5, 7, 13, 29, 30, 35, 41, 44, 45, 51, 68, 93, 101, 112, 115, 126, 130, 138, 141, 160, 161, 172, 187, 191, 197, 198, 204, 212, 226, 228, 231, 232, 234, 258, 266, 268, 272, 279, 286, 289, 293, 299, 303, Any of 311, 313, 321, 323, 324, 327, 335, 336, 340, 343, 351, 353, 355, 362, 365, 367, 368, 370, 374, 375, 377, 379, 383 and 384 At least one peptide of the present invention (in a preferred embodiment combined) described in the item is used for the treatment of uterine cancer.

因此，本發明的另一個方面涉及根據SEQ ID No. 7、8、11、25、42、44、67、82、84、86、91、94、114、136、138、145、156、161、192、202、203、204、212、216、217、218、224、226、237、257、261、293、294、299、313、321、323、324、334、340、353、358、361、362、370 和 379 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療 AML。Therefore, another aspect of the present invention relates to according to SEQ ID No. 7, 8, 11, 25, 42, 44, 67, 82, 84, 86, 91, 94, 114, 136, 138, 145, 156, 161, 192, 202, 203, 204, 212, 216, 217, 218, 224, 226, 237, 257, 261, 293, 294, 299, 313, 321, 323, 324, 334, 340, 353, 358, 361, At least one peptide of the invention described in any one of 362, 370 and 379 (in combination in a preferred embodiment) is used for the treatment of AML.

因此，本發明的另一個方面涉及根據SEQ ID No. 8、11、14、27、38、40、48、55、59、68、84、93、94、114、118、124、126、128、146、159、161、162、164、176、179、188、189、192、200、202、208、211、212、215、216、226、228、234、235、260、264、272、273、274、279、280、293、298、305、311、321、339、342、343、344、346、348、351、353、354、362、365、370、375、377 和 379 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療 HNSCC。Therefore, another aspect of the present invention relates to according to SEQ ID No. 8, 11, 14, 27, 38, 40, 48, 55, 59, 68, 84, 93, 94, 114, 118, 124, 126, 128, 146, 159, 161, 162, 164, 176, 179, 188, 189, 192, 200, 202, 208, 211, 212, 215, 216, 226, 228, 234, 235, 260, 264, 272, 273, 274, 279, 280, 293, 298, 305, 311, 321, 339, 342, 343, 344, 346, 348, 351, 353, 354, 362, 365, 370, 375, 377 and 379 At least one peptide of the invention described (in combination in a preferred embodiment) is used to treat HNSCC.

因此，本發明的另一個方面涉及根據SEQ ID No. 11、26、37、44、49、58、83、84、113、128、173、176、184、201、203、226、273、279、293、317、340、348、359、360 和 361中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療 NHL。Therefore, another aspect of the present invention relates to according to SEQ ID No. 11, 26, 37, 44, 49, 58, 83, 84, 113, 128, 173, 176, 184, 201, 203, 226, 273, 279, At least one peptide of the invention described in any one of 293, 317, 340, 348, 359, 360 and 361 (in combination in a preferred embodiment) is used for the treatment of NHL.

因此，本發明的另一個方面涉及根據SEQ ID No. 11、58、84、26、113、136、45、145、173、174、176、203、226、279、293、326、335、348、351、359、360 和 362 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療 NSCLC。Therefore, another aspect of the present invention relates to according to SEQ ID No. 11, 58, 84, 26, 113, 136, 45, 145, 173, 174, 176, 203, 226, 279, 293, 326, 335, 348, At least one peptide of the present invention described in any one of 351, 359, 360 and 362 (in combination in a preferred embodiment) is used for the treatment of NSCLC.

因此，本發明的另一個方面涉及根據SEQ ID No. 11、45、68、162、173、174、226、293、351、359、362 和 375 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療 OC。Therefore, another aspect of the present invention relates to at least one peptide of the present invention according to any one of SEQ ID No. 11, 45, 68, 162, 173, 174, 226, 293, 351, 359, 362 and 375 (In combination in a preferred embodiment) for the treatment of OC.

因此，本發明的另一個方面涉及根據SEQ ID No. 11、35、42、59、119、126、127、152、161、172、176、192、226、228、239、260、280、293、324、362、365、374和384中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療SCLC。Therefore, another aspect of the present invention relates to according to SEQ ID No. 11, 35, 42, 59, 119, 126, 127, 152, 161, 172, 176, 192, 226, 228, 239, 260, 280, 293, At least one peptide of the present invention described in any one of 324, 362, 365, 374 and 384 (in combination in a preferred embodiment) is used for the treatment of SCLC.

因此，本發明的另一個方面涉及根據SEQ ID No. 13、14、30、68、89、108、125、198、200、204、228、235、242、266、269、272、280、333、353、362、363、375、377 和 379 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療 BRCA。Therefore, another aspect of the present invention relates to according to SEQ ID No. 13, 14, 30, 68, 89, 108, 125, 198, 200, 204, 228, 235, 242, 266, 269, 272, 280, 333, At least one peptide of the invention described in any one of 353, 362, 363, 375, 377 and 379 (in combination in a preferred embodiment) is used to treat BRCA.

因此，本發明的另一個方面涉及根據SEQ ID No. 14、30、49、90、107、108、111、126、136、147、161、164、173、176、181、187、190、199、208、240、274、275、280、293、340、349、351、355、359、362、378 和 383 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療黑色素瘤。Therefore, another aspect of the present invention relates to according to SEQ ID No. 14, 30, 49, 90, 107, 108, 111, 126, 136, 147, 161, 164, 173, 176, 181, 187, 190, 199, At least one peptide of the present invention according to any one of 208, 240, 274, 275, 280, 293, 340, 349, 351, 355, 359, 362, 378 and 383 (in combination in a preferred embodiment) For the treatment of melanoma.

因此，本發明的另一個方面涉及根據SEQ ID No. 15、82、173、211、326、362 和 378 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療食管癌。Therefore, another aspect of the present invention relates to the use of at least one peptide (in combination in a preferred embodiment) of the present invention according to any one of SEQ ID No. 15, 82, 173, 211, 326, 362 and 378 For the treatment of esophageal cancer.

因此，本發明的另一個方面涉及根據SEQ ID No. 30、136、164、173、187、266、293、298、335、351、362、363 和 385 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療 CRC。Therefore, another aspect of the present invention relates to at least one of the present invention according to any one of SEQ ID No. 30, 136, 164, 173, 187, 266, 293, 298, 335, 351, 362, 363 and 385 One peptide (in combination in a preferred embodiment) is used to treat CRC.

因此，本發明的另一個方面涉及根據SEQ ID No. 44、76、84、145、173、211、293、306、326、351 和 362 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療 RCC。Therefore, another aspect of the present invention relates to at least one peptide of the present invention according to any one of SEQ ID No. 44, 76, 84, 145, 173, 211, 293, 306, 326, 351 and 362 (in In a preferred embodiment, the combination) is used to treat RCC.

因此，本發明的另一個方面涉及根據SEQ ID No. 68、94、181、353、370 和 375 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療膽囊癌和/或膽管癌。Therefore, another aspect of the invention relates to at least one peptide of the invention (in combination in a preferred embodiment) according to any one of SEQ ID No. 68, 94, 181, 353, 370 and 375 for use in therapy Gallbladder cancer and/or cholangiocarcinoma.

因此，本發明的另一個方面涉及根據SEQ ID No. 82、87、162、351 和 362 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療 PC。Therefore, another aspect of the present invention relates to at least one peptide of the present invention (in combination in a preferred embodiment) according to any one of SEQ ID No. 82, 87, 162, 351 and 362 for the treatment of PC.

因此，本發明的另一個方面涉及根據SEQ ID No. 84、145、173、176、258、343 和 362 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療 GC。Therefore, another aspect of the present invention relates to the use of at least one peptide (in combination in a preferred embodiment) of the present invention according to any one of SEQ ID No. 84, 145, 173, 176, 258, 343 and 362 For the treatment of GC.

因此，本發明的另一個方面涉及根據SEQ ID No. 84、126、145、173、211、239、247、349、357、362、374 和 379 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療 HCC。Therefore, another aspect of the present invention relates to at least one peptide of the present invention according to any one of SEQ ID No. 84, 126, 145, 173, 211, 239, 247, 349, 357, 362, 374 and 379 (Combined in a preferred embodiment) for the treatment of HCC.

因此，本發明的另一個方面涉及根據SEQ ID No. 136、211、221、226、293、321、335、351 和 354 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療腦癌。Therefore, another aspect of the present invention relates to at least one peptide of the present invention according to any one of SEQ ID No. 136, 211, 221, 226, 293, 321, 335, 351 and 354 (in a preferred embodiment Zhonglian) is used to treat brain cancer.

因此，本發明的另一個方面涉及根據SEQ ID No. 128、172、188、192、293、305、312、321、348、362 和 374 中任一項所述的本發明的至少一種肽（在一種優選實施方案中聯合）用於治療膀胱癌。Therefore, another aspect of the present invention relates to at least one peptide of the present invention according to any one of SEQ ID No. 128, 172, 188, 192, 293, 305, 312, 321, 348, 362 and 374 (in In a preferred embodiment, the combination) is used in the treatment of bladder cancer.

因此，本發明的另一個方面涉及根據SEQ ID No. 226、321、362和374中任一項所述的本發明的至少一種肽（在一種優選實施方案的肽聯合）用於治療PrC。Therefore, another aspect of the present invention relates to at least one peptide of the present invention (in a preferred embodiment combination of peptides) according to any one of SEQ ID No. 226, 321, 362 and 374 for the treatment of PrC.

本發明還涉及本發明的肽，其具有與主要組織相容性複合體 (MHC) I或以拉長形式存在的例如長度變化的-MHC-II類分子結合的能力。The present invention also relates to the peptide of the present invention, which has the ability to bind to major histocompatibility complex (MHC) I or an elongated form of, for example, MHC-II molecules of varying length.

本發明進一步涉及本發明中的肽，其中所述肽（每種肽）系由或基本系由根據SEQ ID NO:1至SEQ ID NO:385的一個氨基酸序列組成。The present invention further relates to the peptides of the present invention, wherein the peptides (each peptide) consist or essentially consist of an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 385.

本發明進一步涉及本發明的肽，其中所述肽被修飾和/或包含非肽鍵。The present invention further relates to the peptide of the present invention, wherein the peptide is modified and/or contains non-peptide bonds.

本發明進一步涉及本發明的肽，其中所述肽為融合蛋白的一部分，特別是與 HLA-DR 抗原相關不變鏈 (Ii) 的 N-端氨基酸融合，或與抗體（例如，樹突狀細胞特定抗體）或抗體的序列融合。The present invention further relates to the peptide of the present invention, wherein the peptide is a part of a fusion protein, especially fused with the N-terminal amino acid of the HLA-DR antigen-related invariant chain (Ii), or with an antibody (for example, dendritic cell Specific antibody) or antibody sequence fusion.

本發明進一步涉及編碼本發明的鈦的核酸。本發明進一步涉及為DNA、cDNA、PNA、RNA或其組合的根據本發明的核酸。The invention further relates to nucleic acids encoding the titanium of the invention. The invention further relates to a nucleic acid according to the invention which is DNA, cDNA, PNA, RNA or a combination thereof.

本發明進一步涉及一種能表達和/或表達本發明核酸的表達載體。The present invention further relates to an expression vector capable of expressing and/or expressing the nucleic acid of the present invention.

本發明進一步涉及本發明的一種肽、本發明的一種核酸或本發明的一種治療疾病的藥用表達載體，特別是用於治療癌症。The present invention further relates to a peptide of the present invention, a nucleic acid of the present invention or a pharmaceutical expression vector of the present invention for treating diseases, especially for the treatment of cancer.

本發明進一步涉及本發明中肽或本發明中所述肽複合體（含有 MHC）的特定抗體以及製造這些抗體的方法。The present invention further relates to specific antibodies of the peptide of the present invention or the peptide complex (containing MHC) of the present invention and methods for producing these antibodies.

本發明進一步涉及本發明的 T細胞受體 (TCR)，特別是可溶性TCR (sTCRs) 和加工為自體或異體 T細胞的克隆 TCR，以及製造這些 TCR 的方法和載有所述 TCR 或所述 TCR 交叉反應的 NK 細胞的製造方法。The present invention further relates to the T cell receptors (TCRs) of the present invention, especially soluble TCRs (sTCRs) and cloned TCRs processed into autologous or allogeneic T cells, as well as methods for manufacturing these TCRs and containing the TCR or the TCR cross-reactive NK cell manufacturing method.

抗體和 TCR 是根據本發明的肽現有免疫治療用途的另外實施方案。Antibodies and TCRs are additional embodiments of existing immunotherapeutic uses of peptides according to the invention.

本發明進一步涉及含本發明核酸或前述表達載體的一種宿主細胞。本發明進一步涉及本發明的宿主細胞，其為抗原表現細胞，優選為樹突細胞。The present invention further relates to a host cell containing the nucleic acid of the present invention or the aforementioned expression vector. The present invention further relates to the host cell of the present invention, which is an antigen expressing cell, preferably a dendritic cell.

本發明進一步涉及配製本發明一種肽的一種方法，所述方法包括培養本發明的宿主細胞和從所述宿主細胞或其培養基中分離肽。The present invention further relates to a method of formulating a peptide of the present invention, the method comprising culturing the host cell of the present invention and isolating the peptide from the host cell or its culture medium.

本發明進一步涉及本發明中的所述方法，其中抗原透過與足夠量的含抗原提成細胞的抗原結合被載入表達於合適抗原表現細胞或人工抗原呈遞細胞表面的 I 或 II類MHC 分子。The present invention further relates to the method of the present invention, wherein the antigen is loaded into MHC class I or II molecules expressed on the surface of suitable antigen-expressing cells or artificial antigen-presenting cells by binding to a sufficient amount of antigen-containing antigen-producing cells.

本發明進一步涉及本發明的方法，其中抗原表現細胞由能表達含SEQ ID NO:1至 SEQ ID NO:385、優選為含 SEQ ID NO: 1至SEQ ID No: 202 所述肽的一個表達載體、或一個變體氨基酸序列組成。The present invention further relates to the method of the present invention, wherein the antigen-expressing cell is capable of expressing an expression vector containing the peptides of SEQ ID NO: 1 to SEQ ID NO: 385, preferably SEQ ID NO: 1 to SEQ ID No: 202 , Or a variant amino acid sequence composition.

本發明進一步涉及以本發明方法製造的活化T細胞，其中所述 T細胞有選擇性地識別一種細胞，該細胞表達含一種本發明氨基酸序列的多肽。The present invention further relates to an activated T cell produced by the method of the present invention, wherein the T cell selectively recognizes a cell that expresses a polypeptide containing an amino acid sequence of the present invention.

本發明進一步涉及一種殺傷患者靶細胞的方法，其中患者的靶細胞異常表達含本發明任何氨基酸序列的多肽，該方法包括給予患者按本發明方法製造的有效量 T細胞。The present invention further relates to a method for killing target cells of a patient, wherein the target cells of the patient abnormally express a polypeptide containing any amino acid sequence of the present invention, and the method comprises administering to the patient an effective amount of T cells produced by the method of the present invention.

本發明進一步涉及任何所述肽、本發明的核酸、本發明的表達載體、本發明的細胞、本發明的作為藥劑或製造藥劑的活化T淋巴細胞、T細胞受體或抗體或其他肽-和/或肽-MHC 結合分子的用途。所述藥劑優選為具有抗癌活性。The present invention further relates to any of the peptides, the nucleic acids of the present invention, the expression vectors of the present invention, the cells of the present invention, the activated T lymphocytes, T cell receptors or antibodies or other peptides of the present invention as medicaments or manufacturing medicaments-and / Or the use of peptide-MHC binding molecules. The agent preferably has anti-cancer activity.

優選情況為，所述藥劑為基於可溶性 TCR 或抗體的細胞治療藥物、疫苗或蛋白質。Preferably, the agent is a cell therapy drug, vaccine or protein based on soluble TCR or antibody.

本發明進一步涉及本發明中的用途，其中所述癌細胞為 CLL、急性骨髓性白血病、膽管癌、腦癌、乳腺癌、結直腸癌、食管癌、膽囊癌、胃癌、肝細胞癌、梅克爾細胞癌、黑色素瘤、非霍奇金淋巴瘤、非小細胞肺癌、卵巢癌、胰腺癌、攝護腺癌、腎細胞癌、小細胞肺癌、膀胱癌和子宮癌，優選為 CLL 細胞。The present invention further relates to the use in the present invention, wherein the cancer cells are CLL, acute myeloid leukemia, cholangiocarcinoma, brain cancer, breast cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, hepatocellular carcinoma, Merkel Cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and uterine cancer, preferably CLL cells.

本發明進一步涉及一種基於本發明肽的生物標誌物，在此成為「靶標」，其可用於診斷癌症，優選為 CLL。所述標誌物可以肽本身過度表現或相應基因過度表達。標誌物也可以用於預測治療成功的可能性，優選為免疫療法，最優選為靶向作用於該生物標誌物識別的相同靶標的免疫療法。例如，抗體或可溶性 TCR 可用於染色腫瘤切片以檢測是否存在相關肽與 MHC 複合。The present invention further relates to a biomarker based on the peptide of the present invention, referred to herein as a "target", which can be used to diagnose cancer, preferably CLL. The markers can be over-expressed in the peptide itself or over-expressed in the corresponding gene. Markers can also be used to predict the likelihood of successful treatment, preferably immunotherapy, and most preferably immunotherapy that targets the same target identified by the biomarker. For example, antibodies or soluble TCR can be used to stain tumor sections to detect the presence of related peptides complexed with MHC.

或者，抗體具有進一步的效應子功能，如免疫刺激域或毒素。本發明還涉及這些癌症治療中新靶點的用途。Alternatively, antibodies have further effector functions, such as immunostimulatory domains or toxins. The invention also relates to the use of these new targets in cancer treatment.

是否能刺激免疫反應取決於是否存在被宿主免疫系統視為異物的抗原。發現腫瘤相關抗原的存在增加了運用宿主免疫系統干預腫瘤生長的可能性。目前，針對癌症免疫治療，正在探索利用免疫系統的體液和細胞進行免疫的各種機制。Whether it can stimulate an immune response depends on the presence of an antigen that is regarded as a foreign body by the host immune system. The discovery of tumor-associated antigens increases the possibility of using the host immune system to interfere with tumor growth. At present, for cancer immunotherapy, various mechanisms of using the body fluids and cells of the immune system for immunity are being explored.

細胞免疫反應的特定元素能特異性地識別和破壞腫瘤細胞。從腫瘤浸潤細胞群或外周血中分離出的 T-細胞表明，這些細胞在癌症的天然免疫防禦中發揮了重要作用。特別是 CD8 陽性 T細胞在這種反應中發揮重要作用，TCD8+ 能識別通常8至10個源自蛋白或位於細胞質的缺損核糖體產物 (DRIP) 的氨基酸殘基的主要組織相容性複合體 (MHC) 所載的肽中所含的I類分子。人 MHC 分子也稱為人白細胞-抗原 (HLA)。Specific elements of cellular immune response can specifically recognize and destroy tumor cells. T-cells isolated from tumor-infiltrating cell populations or peripheral blood indicate that these cells play an important role in the natural immune defense of cancer. In particular, CD8-positive T cells play an important role in this reaction. TCD8+ can recognize 8 to 10 major histocompatibility complexes ( MHC) Class I molecules contained in peptides. Human MHC molecules are also called human leukocyte-antigens (HLA).

除非另有說明，否則本文使用的所有術語定義如下。Unless otherwise stated, all terms used herein are defined as follows.

術語「T細胞反應」是指由一種肽在體外或體內誘導的效應子功能的特異性擴散和活化。對於 MHC I類限制性細胞毒性 T細胞，效應子功能可能為溶解肽脈衝的、肽前體脈衝的或天然肽表現的靶細胞、分泌細胞因子，優選為肽誘導的干擾素-γ，TNF-α 或 IL-2，分泌效應分子，優選為肽誘導的顆粒酶或穿孔素，或脫顆粒。The term "T cell response" refers to the specific diffusion and activation of effector functions induced by a peptide in vitro or in vivo. For MHC class I restricted cytotoxic T cells, the effector function may be to dissolve peptide-pulsed, peptide precursor-pulsed or natural peptide-expressed target cells, secrete cytokines, preferably peptide-induced interferon-γ, TNF- α or IL-2, secretion effector molecule, preferably peptide-induced granzyme or perforin, or degranulation.

本文所用「肽」這一術語，系指一系列氨基酸殘基，通常透過相鄰氨基酸的α-氨基和羰基之間的肽鍵來連接。這些肽的長度優選為9個氨基酸，但至短可為8個氨基酸長度，至長可為10、11、12、13或14個氨基酸或更長，如果為MHC-II類肽時（本發明肽的拉長變體），至長可為15、16、17、18、19或20個氨基酸長度或更長。The term "peptide" as used herein refers to a series of amino acid residues, usually connected by peptide bonds between the α-amino group and the carbonyl group of adjacent amino acids. These peptides are preferably 9 amino acids in length, but can be as short as 8 amino acids in length, and as long as 10, 11, 12, 13, or 14 amino acids or longer. If they are MHC-II peptides (the present invention Elongated variants of peptides) can be 15, 16, 17, 18, 19 or 20 amino acids in length or longer.

因此，「肽」這一術語應包括一系列氨基酸殘基的鹽，通常透過相鄰氨基酸的 α-氨基和羰基之間的肽鍵來連接。優選的情況是，鹽為肽的藥用鹽，例如：氯化物或乙酸（三氟乙酸）鹽。必須注意的是，本發明肽的鹽與其體內狀態的肽基本上不同，因為該不是體內的鹽。Therefore, the term "peptide" shall include salts of a series of amino acid residues, usually connected by peptide bonds between the α-amino groups and carbonyl groups of adjacent amino acids. Preferably, the salt is a pharmaceutically acceptable salt of the peptide, such as chloride or acetic acid (trifluoroacetic acid) salt. It must be noted that the salt of the peptide of the present invention is basically different from the peptide in the in vivo state because it is not a salt in the body.

術語「肽」應也包括「寡肽」。本文使用的術語「寡肽」是指一系列氨基酸殘基，通常透過相鄰氨基酸的α-氨基和羰基之間的肽鍵來連接。寡肽的長度對於本發明來說並不十分關鍵，只要在寡肽中保持正確的表位即可。通常，寡肽長度約小於30個氨基酸殘基，約長於15個氨基酸。The term "peptide" shall also include "oligopeptides". The term "oligopeptide" as used herein refers to a series of amino acid residues, usually connected by peptide bonds between the α-amino group and the carbonyl group of adjacent amino acids. The length of the oligopeptide is not very critical to the present invention, as long as the correct epitope is maintained in the oligopeptide. Generally, oligopeptides are about less than 30 amino acid residues in length and about 15 amino acids in length.

「多肽」這一術語是指一系列氨基酸殘基，通常透過相鄰氨基酸的α-氨基和羰基之間的肽鍵來連接。多肽的長度對於本發明來說並不十分關鍵，只要保持正確的表位即可。與術語肽或寡肽相對，「多肽」這一術語是指包含多於約30個氨基酸殘基的分子。The term "polypeptide" refers to a series of amino acid residues, usually connected by peptide bonds between the α-amino group and the carbonyl group of adjacent amino acids. The length of the polypeptide is not very critical to the present invention, as long as the correct epitope is maintained. In contrast to the term peptide or oligopeptide, the term "polypeptide" refers to a molecule containing more than about 30 amino acid residues.

術語「全長多肽」是指多聚體蛋白的一種完全蛋白（氨基酸鏈）或完全亞基（氨基酸鏈）。The term "full-length polypeptide" refers to a complete protein (amino acid chain) or complete subunit (amino acid chain) of a multimeric protein.

一種肽、寡肽、蛋白質或編碼該分子的核苷酸如果能誘導免疫反應，則具有「免疫原性」（因此是本發明中的一種「免疫原」）。在本發明的情況下，免疫原性的更具體定義是誘導T細胞反應的能力。因此，「免疫原」是一種能夠誘導免疫反應的分子，並且在本發明的情況下，是一種能誘導T細胞反應的分子。在另一方面，所述免疫原可以是肽，肽與MHC的複合體、和/或用於提高特異性抗體或TCR抗性的蛋白。If a peptide, oligopeptide, protein, or nucleotide encoding the molecule can induce an immune response, it has "immunogenicity" (hence an "immunogen" in the present invention). In the context of the present invention, a more specific definition of immunogenicity is the ability to induce a T cell response. Therefore, an "immunogen" is a molecule that can induce an immune response, and in the case of the present invention, is a molecule that can induce a T cell response. In another aspect, the immunogen may be a peptide, a complex of a peptide and MHC, and/or a protein used to increase specific antibody or TCR resistance.

I 類 T細胞「表位」要求的是一種結合至MHCI類受體上的短肽，從而形成一種三元複合體（MHC I類α鏈、β-2-微球蛋白和肽），其可以透過T細胞負載匹配T細胞受體與具有適當親和力的MHC/肽複合物結合來識別。結合至MHC I類分子的肽的典型長度為8-14個氨基酸，最典型為9個氨基酸長度。The "epitope" of class I T cells requires a short peptide that binds to MHC class I receptors to form a ternary complex (MHC class I α chain, β-2-microglobulin and peptide), which can Recognition is achieved through the binding of T cell load-matched T cell receptors to MHC/peptide complexes with appropriate affinity. The typical length of peptides that bind to MHC class I molecules is 8-14 amino acids, most typically 9 amino acids in length.

在人類中，有三種編碼 MHC I類分子的不同基因位點（人 MHC分子也是指定的人白細胞抗原 (HLA)）：HLA-A、HLA-B 和 HLA-C。HLA-A*01、HLA-A*02 和 HLA-B*07 是可從這些基因位點表達的不同 MHC I類等位元基因的實例。In humans, there are three different genetic loci that encode MHC class I molecules (human MHC molecules are also designated human leukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02, and HLA-B*07 are examples of different MHC class I alleles that can be expressed from these gene loci.

表 5：HLA-A*02 和 HLA-A*24 和最常見 HLA-DR 血清類型的表達頻率 F。頻率根據 Mori 等人 (Mori et al., 1997) 使用的 Hardy-Weinberg 公式 F = 1 – (1-Gf)² 改編，從美國人群範圍內的單體型頻率中推導出。由於連鎖不平衡，某些 HLA-DR 等位基因內的 A*02 或 A*24 組合與其預期單一頻率相比，可能是濃縮的或頻率較低。有關詳細資訊，請參閱 Chanock 等人的文獻 (Chanock et al., 2004)。 等位基因 人群 根據等位元基因頻率算得的顯型 A*02 高加索人（北美） 49.1% A*02 非裔美國人（北美） 34.1% A*02 亞裔美國人（北美） 43.2% A*02 拉丁美洲（北美） 48.3% DR1 高加索人（北美） 19.4% DR2 高加索人（北美） 28.2% DR3 高加索人（北美） 20.6% DR4 高加索人（北美） 30.7% DR5 高加索人（北美） 23.3% DR6 高加索人（北美） 26.7% DR7 高加索人（北美） 24.8% DR8 高加索人（北美） 5.7% DR9 高加索人（北美） 2.1% DR1 非裔（北）美人 13.20% DR2 非裔（北）美人 29.80% DR3 非裔（北）美人 24.80% DR4 非裔（北）美人 11.10% DR5 非裔（北）美人 31.10% DR6 非裔（北）美人 33.70% DR7 非裔（北）美人 19.20% DR8 非裔（北）美人 12.10% DR9 非裔（北）美人 5.80% DR1 亞裔（北）美人 6.80% DR2 亞裔（北）美人 33.80% DR3 亞裔（北）美人 9.20% DR4 亞裔（北）美人 28.60% DR5 亞裔（北）美人 30.00% DR6 亞裔（北）美人 25.10% DR7 亞裔（北）美人 13.40% DR8 亞裔（北）美人 12.70% DR9 亞裔（北）美人 18.60% DR1 拉丁裔（北）美人 15.30% DR2 拉丁裔（北）美人 21.20% DR3 拉丁裔（北）美人 15.20% DR4 拉丁裔（北）美人 36.80% DR5 拉丁裔（北）美人 20.00% DR6 拉丁裔（北）美人 31.10% DR7 拉丁裔（北）美人 20.20% DR8 拉丁裔（北）美人 18.60% DR9 拉丁裔（北）美人 2.10% A*24 菲律賓 65% A*24 ***涅涅茨人 61% A*24:02 日本 59% A*24 馬來西亞 58% A*24:02 菲律賓 54% A*24 印度 47% A*24 韓國 40% A*24 斯里蘭卡人 37% A*24 中國 32% A*24:02 印度 29% A*24 澳大利亞西部人 22% A*24 美國 22% A*24 ***薩馬拉人 20% A*24 南美 20% A*24 歐洲 18% Table 5: Expression frequency F of HLA-A*02 and HLA-A*24 and the most common HLA-DR serotypes. The frequency is adapted from the Hardy-Weinberg formula F = 1 – (1-Gf)² used by Mori et al. (Mori et al., 1997) and is derived from haplotype frequencies in the American population. Due to linkage disequilibrium, A*02 or A*24 combinations within certain HLA-DR alleles may be concentrated or less frequent than their expected single frequency. For details, please refer to the paper by Chanock et al. (Chanock et al., 2004). Allele crowd Phenotype calculated based on allele frequency A*02 Caucasian (North America) 49.1% A*02 African American (North America) 34.1% A*02 Asian American (North America) 43.2% A*02 Latin America (North America) 48.3% DR1 Caucasian (North America) 19.4% DR2 Caucasian (North America) 28.2% DR3 Caucasian (North America) 20.6% DR4 Caucasian (North America) 30.7% DR5 Caucasian (North America) 23.3% DR6 Caucasian (North America) 26.7% DR7 Caucasian (North America) 24.8% DR8 Caucasian (North America) 5.7% DR9 Caucasian (North America) 2.1% DR1 African American (Northern) Beauty 13.20% DR2 African American (Northern) Beauty 29.80% DR3 African American (Northern) Beauty 24.80% DR4 African American (Northern) Beauty 11.10% DR5 African American (Northern) Beauty 31.10% DR6 African American (Northern) Beauty 33.70% DR7 African American (Northern) Beauty 19.20% DR8 African American (Northern) Beauty 12.10% DR9 African American (Northern) Beauty 5.80% DR1 Asian (Northern) Beauty 6.80% DR2 Asian (Northern) Beauty 33.80% DR3 Asian (Northern) Beauty 9.20% DR4 Asian (Northern) Beauty 28.60% DR5 Asian (Northern) Beauty 30.00% DR6 Asian (Northern) Beauty 25.10% DR7 Asian (Northern) Beauty 13.40% DR8 Asian (Northern) Beauty 12.70% DR9 Asian (Northern) Beauty 18.60% DR1 Latino (North) Beauty 15.30% DR2 Latino (North) Beauty 21.20% DR3 Latino (North) Beauty 15.20% DR4 Latino (North) Beauty 36.80% DR5 Latino (North) Beauty 20.00% DR6 Latino (North) Beauty 31.10% DR7 Latino (North) Beauty 20.20% DR8 Latino (North) Beauty 18.60% DR9 Latino (North) Beauty 2.10% A*24 Philippine 65% A*24 Nenets, Russia 61% A*24:02 Japan 59% A*24 Malaysia 58% A*24:02 Philippine 54% A*24 India 47% A*24 South Korea 40% A*24 Sri Lankan 37% A*24 China 32% A*24:02 India 29% A*24 Western Australia twenty two% A*24 U.S twenty two% A*24 Russian Samara 20% A*24 South America 20% A*24 Europe 18%

本發明的肽，優選當如本文描述納入本發明的疫苗時與A*02結合。疫苗還可能包括泛結合MHC II類肽。因此，本發明的疫苗可用於治療A*02陽性患者中的癌症，但不因為這些肽的廣泛結核性而必須選擇II類MHC同種異型。The peptides of the invention preferably bind to A*02 when incorporated into the vaccine of the invention as described herein. Vaccines may also include pan-binding MHC class II peptides. Therefore, the vaccine of the present invention can be used to treat cancer in A*02-positive patients, but MHC class II allotypes must not be selected because of the extensive tuberculosis of these peptides.

如果本發明的A*02肽與結合至另一等位基因例如A*24的肽組合，與單獨的MHC I類等位基因相比，可治療更高比例的患者群體。雖然在大多數人群中，低於50%的患者可由單獨的等位基因來解決問題，但是本發明中一種含HLA-A*24和HLA-A*02表位的疫苗可以治療任何相關人群中至少60%的患者。具體來說，各區域中，以下比例的患者這些等位基因中的至少一個有肯定效果：美國61%、西歐62%、中國75%、韓國77%、日本86%（根據www.allelefrequencies.net計算）。If the A*02 peptide of the present invention is combined with a peptide that binds to another allele, such as A*24, a higher proportion of the patient population can be treated than the MHC class I allele alone. Although in most populations, less than 50% of patients can solve the problem with a single allele, the vaccine of the present invention containing HLA-A*24 and HLA-A*02 epitopes can treat any related population At least 60% of patients. Specifically, in each region, at least one of these alleles in the following proportions of patients has a positive effect: 61% in the United States, 62% in Western Europe, 75% in China, 77% in South Korea, and 86% in Japan (according to www.allelefrequencies.net calculate).

在一項優選的實施方案中，術語「核苷酸序列」系指去氧核苷酸的雜聚物。In a preferred embodiment, the term "nucleotide sequence" refers to a heteropolymer of deoxynucleotides.

編碼特定肽、寡肽或多肽的核苷酸序列可為天然核苷酸序列，也可為合成核苷酸序列。一般來說，編碼肽、多肽以及本發明蛋白的DNA片段由cDNA片段和短寡核苷酸銜接物，或一系列寡核苷酸組成，以提供一種合成基因，該基因能夠在包含源自微生物或病毒操縱子的調節元素的重組轉錄單元中被表達。The nucleotide sequence encoding a specific peptide, oligopeptide or polypeptide may be a natural nucleotide sequence or a synthetic nucleotide sequence. Generally speaking, the DNA fragments encoding peptides, polypeptides and proteins of the present invention are composed of cDNA fragments and short oligonucleotide linkers, or a series of oligonucleotides, to provide a synthetic gene that can be used in Or the regulatory elements of the viral operon are expressed in the recombinant transcription unit.

如本文所用的術語「肽的核苷酸編碼」系指對肽進行核苷酸序列編碼，其中該肽包括與將由用於產生 TCR 的樹突細胞或另一細胞系統所表達該序列的生物系統相容的人工（人造）活化和停止密碼子。The term "nucleotide encoding of a peptide" as used herein refers to a nucleotide sequence encoding a peptide, wherein the peptide includes a biological system that will express the sequence by dendritic cells or another cell system used to produce TCR Compatible artificial (man-made) activation and stop codons.

本文提到的核酸序列既包括單鏈核酸也包括雙鏈核酸。因此，除非本文另有所指，否則，例如對於 DNA，具體的序列是該序列的單鏈 DNA、該序列與其互補序列的雙工（雙鏈 DNA）以及該序列的互補序列。The nucleic acid sequence mentioned herein includes both single-stranded nucleic acid and double-stranded nucleic acid. Therefore, unless otherwise indicated herein, for DNA, for example, the specific sequence is the single-stranded DNA of the sequence, the duplex of the sequence and its complementary sequence (double-stranded DNA), and the complementary sequence of the sequence.

「編碼區」這一術語是指在基因的天然基因組環境中天然或正常編碼該基因的表達產物的那部分基因，即，體內編碼該基因的天然表達產物的區域。The term "coding region" refers to the part of the gene that naturally or normally encodes the expression product of the gene in the natural genomic environment of the gene, that is, the region in the body that encodes the natural expression product of the gene.

編碼區可來自非突變（「正常」）基因、突變基因或異常基因，甚至還可以來自DNA序列，完全可在實驗室中使用本領域熟知的DNA合成方法合成。The coding region can be derived from non-mutated ("normal") genes, mutant genes, or abnormal genes, or even from DNA sequences, which can be synthesized in the laboratory using DNA synthesis methods well known in the art.

「表達產物」這一術語是指多肽或蛋白，它是基因和遺傳碼退化並因而編碼同樣的氨基酸所造成的任何核酸序列編碼同等物的翻譯產物。The term "expression product" refers to a polypeptide or protein, which is the translation product of any nucleic acid sequence encoding equivalent caused by the degradation of genes and genetic code and thus encoding the same amino acid.

「片斷」這一術語，當指的是一種編碼序列時，表示包含非完整編碼區的DNA的一部分，其表達產物與完整編碼區表達產物基本上具有相同的生物學功能或活性。The term "fragment", when referring to a coding sequence, refers to a part of DNA containing a non-complete coding region, the expression product of which has basically the same biological function or activity as the expression product of the complete coding region.

「DNA片段」這一術語是指一種DNA聚合物，以單獨的片段形式或一種較大DNA結構的組分形式存在，它們從至少分離過一次的DNA中以基本純淨的形式獲得，即不含污染性內源性材料，並且獲得的數量或濃度能夠使用標準生化方法，例如使用克隆載體，進行識別、操縱和回收該片段及其組分核苷酸序列。此類片段以開放閱讀框架（未被內部未翻譯序列打斷）或內含子（通常表現于真核基因中）的形式存在。未翻譯DNA序列可能存在於開放閱讀框架的下游，在那裏其不會干預編碼區的操縱或表達。The term "DNA fragment" refers to a DNA polymer that exists as a single fragment or as a component of a larger DNA structure. They are obtained in a substantially pure form from DNA that has been isolated at least once, that is, it does not Contaminate endogenous materials, and the obtained quantity or concentration can use standard biochemical methods, such as the use of cloning vectors, to identify, manipulate and recover the fragment and its component nucleotide sequence. Such fragments exist in the form of open reading frames (not interrupted by internal untranslated sequences) or introns (usually expressed in eukaryotic genes). Untranslated DNA sequences may exist downstream of the open reading frame, where they will not interfere with the manipulation or expression of the coding region.

「引物」這一術語表示一種短核酸序列，其可與一個DNA鏈配對，並在DNA聚合酶開始合成去氧核糖核酸鏈之處提供一個游離的3'-OH末端。The term "primer" refers to a short nucleic acid sequence that can pair with a DNA strand and provide a free 3'-OH end where the DNA polymerase begins to synthesize the deoxyribonucleic acid strand.

「啟動子」這一術語表示參與RNA聚合酶的結合從而啟動轉錄的DNA區域。The term "promoter" refers to a DNA region involved in the binding of RNA polymerase to initiate transcription.

術語「分離」表示一種物質從其原來的環境（例如，如果是天然發生的則是天然環境）中被移走。例如，活體動物中的天然核苷酸或多肽不是分離的，但是，從天然系統中一些或所有共存物質中分離出來的核苷酸或多肽是分離的。此類多核苷酸可能是載體的一部分和/或此類多核苷酸和多肽可能是一種組合物的一部分，並且由於該載體或組合物不是其天然環境的一部分，因此它仍然是分離的。The term "isolation" means that a substance is removed from its original environment (for example, the natural environment if it occurs naturally). For example, the natural nucleotides or polypeptides in living animals are not isolated, but the nucleotides or polypeptides isolated from some or all coexisting substances in the natural system are isolated. Such polynucleotides may be part of a vector and/or such polynucleotides and polypeptides may be part of a composition, and since the vector or composition is not part of its natural environment, it is still isolated.

本發明中披露的多核苷酸和重組或免疫原性多肽也可能以「純化」的形式存在。術語「純化」並非要求絕對的純度；它只是一個相對的定義，可以包括高度純化或部分純化的製劑，相關領域技術人員能理解這些術語。例如，各個從已用傳統方法純化為具有電泳同質性的cDNA庫中分離出的各種克隆物。明確考慮到將起始材料或天然物質純化至少一個數量級，優選為兩或三個數量級，更優選為四或五個數量級。此外，明確涵蓋所述多肽的純度優選為99.999%，或至少為99.99%或99.9%；甚而適宜為以重量計99%或更高。The polynucleotides and recombinant or immunogenic polypeptides disclosed in the present invention may also exist in a "purified" form. The term "purified" does not require absolute purity; it is only a relative definition, which can include highly purified or partially purified preparations, and those skilled in the relevant art can understand these terms. For example, various clones isolated from a cDNA library that has been purified by conventional methods to have electrophoretic homogeneity. It is explicitly considered that the starting material or natural substance is purified by at least one order of magnitude, preferably two or three orders of magnitude, and more preferably four or five orders of magnitude. In addition, it is clearly covered that the purity of the polypeptide is preferably 99.999%, or at least 99.99% or 99.9%; even suitably 99% by weight or higher.

根據本發明公開的核酸和多肽表達產物，以及包含此類核酸和/或多肽的表達載體可能以「濃縮的形式」存在。本文使用的術語「濃縮」是指材料的濃度至少是其自然濃度的大約2、5、10、100或1000倍，有優勢的是，按重量計為0.01%，優選為至少0.1%。也明確考慮到，按重量計約為0.5%、1%、5%、10%和20%的濃縮製劑。序列、構型、載體、克隆物以及包含本發明的其他材料可有優勢地以濃縮或分離的形式存在。「活性片段」這一術語是指產生免疫反應的片段（即具有免疫原性活性），通常是一種肽、多肽或核酸序列的片段，不論是單獨或可選地與合適的佐劑一起或在載體中給予一種動物，比如哺乳動物，例如兔子或小鼠，也包括人；這種免疫反應採用的形式是在接受動物（如：人）體內刺激T細胞反應。或者，「活性片段」也可用於誘導體外T細胞反應。The nucleic acid and polypeptide expression products disclosed according to the present invention, and the expression vector containing such nucleic acid and/or polypeptide may exist in a "concentrated form". As used herein, the term "concentrated" means that the concentration of a material is at least about 2, 5, 10, 100, or 1000 times its natural concentration. Advantageously, it is 0.01% by weight, preferably at least 0.1%. Concentrated formulations of approximately 0.5%, 1%, 5%, 10%, and 20% by weight are also explicitly considered. Sequences, configurations, vectors, clones, and other materials containing the present invention can advantageously be present in concentrated or isolated form. The term "active fragment" refers to a fragment that produces an immune response (ie, has immunogenic activity), usually a fragment of a peptide, polypeptide or nucleic acid sequence, whether alone or alternatively together with a suitable adjuvant or in The carrier is administered to an animal, such as mammals, such as rabbits or mice, as well as humans; this immune response takes the form of stimulating T cell responses in the receiving animal (such as human). Alternatively, "active fragments" can also be used to induce in vitro T cell responses.

本文使用的「部分(portion)」、「節段(segment)」、「片段(fragment)」這幾個術語，當與多肽相關地使用時是指殘基的連續序列，比如氨基酸殘基，其序列形成一個較大序列的子集。例如，如果一個多肽以任一種肽鏈內切肽酶（如胰蛋白酶或糜蛋白酶）進行處理，則該處理獲得的寡肽會代表起始多肽的部分、節段或片段。當與多核苷酸相關地使用時，這些術語系指用任何核酸內切酶處理所述多核苷酸產生的產物。The terms "portion", "segment", and "fragment" as used herein refer to a continuous sequence of residues, such as amino acid residues, when used in relation to polypeptides. The sequence forms a subset of a larger sequence. For example, if a polypeptide is treated with any endopeptidase (such as trypsin or chymotrypsin), the oligopeptide obtained by the treatment will represent a part, segment or fragment of the starting polypeptide. When used in connection with a polynucleotide, these terms refer to the product produced by treating the polynucleotide with any endonuclease.

根據本發明，術語「等同度百分比」或「等同百分比」，如果指的是序列，則表示在待對比序列（「被對比序列」）與所述序列或請求項的序列（「參考序列」）對準之後將被對比序列與所述序列或請求項的序列進行比較。然後根據下列公式計算等同度百分比： 等同度百分比= 100 [1 -(C/R)] 其中 C 是參考序列與被對比序列之間對準長度上參考序列與被對比序列之間的差異數量，其中 (i) 參考序列中每個堿基或氨基酸序列在被對比序列中沒有對應的對準堿基或氨基酸； (ii) 參考序列中每個空隙，以及 (iii) 參考序列中每個對準堿基或氨基酸與被比對比序列中對準堿基或氨基酸不同，即構成一個差異以及 (iiii) 必須在對準序列的第 1 位置開始對準； 並且 R 是參考序列與被對比序列對準長度上在參考序列中產生任何空隙也計算為一個堿基或氨基酸的參考序列中的堿基或氨基酸數目。According to the present invention, the term "equivalence percentage" or "equivalence percentage", if it refers to a sequence, means that the sequence to be compared (the "compared sequence") and the sequence of the sequence or the requested item (the "reference sequence") After alignment, the compared sequence is compared with the sequence or the sequence of the requested item. Then calculate the percentage of equivalence according to the following formula: Equivalence percentage = 100 [1 -(C/R)] Where C is the number of differences between the reference sequence and the compared sequence in the alignment length between the reference sequence and the compared sequence, where (i) Each base or amino acid sequence in the reference sequence does not have a corresponding alignment base or amino acid in the compared sequence; (ii) Each gap in the reference sequence, and (iii) Each alignment group or amino acid in the reference sequence is different from the alignment group or amino acid in the compared sequence, which constitutes a difference and (iiii) The alignment must start at the first position of the alignment sequence; And R is the length of the alignment between the reference sequence and the sequence to be compared, and any gaps in the reference sequence are also calculated as the number of yl groups or amino acids in the reference sequence.

如果「被對比序列」和「參考序列」之間存在的一個對準按上述計算的等同度百分比大致等於或大於指定的最低等同度百分比，則被對比序列與參考序列具有指定的最低等同度百分比，雖然可能存在按本文上述計算的等同度百分比低於指定等同度百分比的對準。If there is an alignment between the "compared sequence" and the "reference sequence", the percentage of identity calculated as above is approximately equal to or greater than the specified minimum identity percentage, then the compared sequence and the reference sequence have the specified minimum identity percentage , Although there may be alignments where the equivalent percentage calculated above is lower than the specified equivalent percentage.

因此，如上所述，本發明提出了一種肽，其包括選自SEQ ID NO:1至SEQ ID NO:385群組的一個序列、或與SEQ ID NO:1至SEQ ID NO:385具有88%同源性的其變體、或誘導與該肽發生T細胞交叉反應的一個變體。本發明所述的肽具有與主要組織相容性複合體 (MHC) I 或所述肽拉長版本的II類分子結合的能力。Therefore, as described above, the present invention provides a peptide comprising a sequence selected from the group of SEQ ID NO: 1 to SEQ ID NO: 385, or having 88% with SEQ ID NO: 1 to SEQ ID NO: 385. A variant of homology, or a variant that induces T cell cross-reaction with the peptide. The peptide of the present invention has the ability to bind to major histocompatibility complex (MHC) I or class II molecules of the elongated version of the peptide.

在本發明中，「同源性」一詞系指兩個氨基酸序列之間的同一度（參見上文的等同度百分比，如肽或多肽序列。前文所述的「同源」是透過將理想條件下調整的兩個序列與待比較序列進行比對後確定的。此類序列同源性可透過使用ClustalW等演算法創建一個排列而進行計算。也可用使用一般序列分析軟體，更具體地說，是Vector NTI、GENETYX或由公共資料庫提供的其他工具。In the present invention, the term "homology" refers to the degree of identity between two amino acid sequences (see above for the percentage of equivalence, such as peptide or polypeptide sequences. The aforementioned "homology" refers to the ideal The two sequences adjusted under the conditions are compared with the sequence to be compared and determined. Such sequence homology can be calculated by creating a permutation using algorithms such as ClustalW. Ordinary sequence analysis software can also be used, more specifically , Is Vector NTI, GENETYX or other tools provided by public database.

本領域技術人員能評估特定肽變體誘導的T細胞是否可與該肽本身發生交叉反應 (Appay et al., 2006; Colombetti et al., 2006; Fong et al., 2001; Zaremba et al., 1997)。Those skilled in the art can assess whether T cells induced by a specific peptide variant can cross-react with the peptide itself (Appay et al., 2006; Colombetti et al., 2006; Fong et al., 2001; Zaremba et al., 1997).

發明人用給定氨基酸序列的「變體」表示，一個或兩個氨基酸殘基等的側鏈透過被另一個天然氨基酸殘基的側鏈或其他側鏈取代而發生改變，這樣，這種肽仍然能夠以含有給定氨基酸序列（由SEQ ID NO:1至SEQ ID NO:385組成）的肽大致同樣的方式與 HLA 分子結合。例如，一種肽可能被修飾以便至少維持（如沒有提高）其能與 HLA-A*02 或 -DR 等合適 MHC 分子的結合槽相互作用和結合，以及至少維持（如沒有提高）其與活化T細胞的 TCR 結合的能力。The inventors used a "variant" of a given amino acid sequence to indicate that the side chain of one or two amino acid residues is changed by being replaced by the side chain of another natural amino acid residue or other side chains. In this way, this peptide The peptides containing the given amino acid sequence (consisting of SEQ ID NO: 1 to SEQ ID NO: 385) can still bind to HLA molecules in approximately the same way. For example, a peptide may be modified to at least maintain (if not increase) its ability to interact and bind to the binding groove of suitable MHC molecules such as HLA-A*02 or -DR, and at least maintain (if not increase) its ability to interact with activated T The ability of the cell's TCR to bind.

隨後，這些T細胞可與細胞和殺傷細胞發生交叉反應，這些細胞表達多肽（其中包含本發明中定義的同源肽的天然氨基酸序列）。正如科學文獻和資料庫 (Rammensee et al., 1999; Godkin et al., 1997) 中所述，HLA-A結合肽的某些位點通常為錨定殘基，可形成一種與HLA結合槽的結合模序相稱的核心序列，其定義由構成結合槽的多肽鏈的極性、電物理、疏水性和空間特性確定。因此，本領域技術人員能夠透過保持已知的錨殘基來修飾SEQ ID No: 1 至SEQ ID NO:385提出的氨基酸序列，並且能確定這些變體是否保持與 MHC I或II類分子結合的能力。本發明的變體保持與活化T細胞的TCR結合的能力，隨後，這些 T細胞可與表達一種包含本發明定義的同源肽的天然氨基酸序列的多肽的細胞發生交叉反應並殺死該等細胞。Subsequently, these T cells can cross-react with cells and killer cells, and these cells express polypeptides (which contain the natural amino acid sequence of the homologous peptide defined in the present invention). As described in scientific literature and database (Rammensee et al., 1999; Godkin et al., 1997), certain sites of HLA-A binding peptides are usually anchor residues, which can form a kind of HLA-binding groove. The definition of the core sequence commensurate with the binding motif is determined by the polarity, electrophysics, hydrophobicity and steric properties of the polypeptide chain that constitutes the binding groove. Therefore, those skilled in the art can modify the amino acid sequence set forth in SEQ ID No: 1 to SEQ ID NO: 385 by maintaining the known anchor residues, and can determine whether these variants maintain binding to MHC class I or II molecules. ability. The variants of the present invention maintain the ability to bind to the TCR of activated T cells, and subsequently, these T cells can cross-react with cells expressing a polypeptide comprising the natural amino acid sequence of the homologous peptide defined in the present invention and kill the cells .

如果無另有說明，那麼本文公開的原始（未修飾）肽可以透過在肽鏈內的不同（可能為選擇性）位點上取代一個或多個殘基而被修飾。優選情況是，這些取代位於氨基酸鏈的末端。此取代可能是保守性的，例如，其中一個氨基酸被具有類似結構和特點的另一個氨基酸所取代，比如其中一個疏水性氨基酸被另一個疏水性氨基酸取代。更保守的取代是具有相同或類似的大小和化學性質的氨基酸間的取代，例如，亮氨酸被異亮氨酸取代。在天然同源蛋白質家族序列變異的研究中，某些氨基酸的取代往往比其他氨基酸更具有耐受性，這些氨基酸往往表現出與原氨基酸的大小、電荷、極性和疏水性之間的相似性相關，這是確定「保守取代」的基礎。If not otherwise stated, the original (unmodified) peptides disclosed herein can be modified by substituting one or more residues at different (possibly selective) positions within the peptide chain. Preferably, these substitutions are at the end of the amino acid chain. This substitution may be conservative, for example, one of the amino acids is replaced by another amino acid with similar structure and characteristics, for example, one of the hydrophobic amino acids is replaced by another hydrophobic amino acid. More conservative substitutions are those between amino acids of the same or similar size and chemical properties, for example, replacement of leucine with isoleucine. In the study of sequence variation of natural homologous protein families, the substitution of certain amino acids is often more tolerant than other amino acids. These amino acids often show similarities in the size, charge, polarity and hydrophobicity of the original amino acids. This is the basis for determining "conservative substitution."

在本文中，保守取代定義為在以下五種基團之一的內部進行交換：基團1—小脂肪族、非極性或略具極性的殘基(Ala, Ser, Thr, Pro, Gly)；基團2—極性、帶負電荷的殘基及其醯胺(Asp, Asn, Glu, Gln)；基團3—極性、帶正電荷的殘基(His, Arg, Lys)；基團4—大脂肪族非極性殘基(Met, Leu, Ile, Val, Cys)以及基團5—大芳香殘基(Phe, Tyr, Trp)。In this article, conservative substitutions are defined as exchanges within one of the following five groups: group 1-small aliphatic, non-polar or slightly polar residues (Ala, Ser, Thr, Pro, Gly); Group 2—Polar, negatively charged residues and their amides (Asp, Asn, Glu, Gln); Group 3-Polar, positively charged residues (His, Arg, Lys); Group 4- Large aliphatic non-polar residues (Met, Leu, Ile, Val, Cys) and group 5-large aromatic residues (Phe, Tyr, Trp).

較不保守的取代可能涉及一個氨基酸被另一個具有類似特點但在大小上有所不同的氨基酸所取代，如：丙氨酸被異亮氨酸殘基取代。高度不保守的取代可能涉及一個酸性氨基酸被另一個具有極性或甚至具有鹼性性質的氨基酸所取代。然而，這種「激進」取代不能認為是無效的而不予考慮，因為化學作用是不完全可預測的，激進的取代可能會帶來其簡單化學原理中無法預見的偶然效果。Less conservative substitutions may involve the replacement of one amino acid by another amino acid with similar characteristics but different in size, such as the replacement of alanine with an isoleucine residue. Highly non-conservative substitutions may involve the substitution of an acidic amino acid by another amino acid that has polar or even basic properties. However, this kind of "radical" substitution cannot be considered ineffective and disregarded, because the chemical action is not completely predictable, and radical substitution may bring about accidental effects that cannot be foreseen in its simple chemical principles.

當然，這種取代可能涉及普通 L-氨基酸之外的其他結構。因此，D-氨基酸可能被本發明的抗原肽中常見的 L-氨基酸取代，也仍在本公開的範圍之內。此外，非標準氨基酸（即，除了常見的天然蛋白原氨基酸）也可以用於取代之目的，以生產根據本發明的免疫原和免疫原性多肽。Of course, this substitution may involve structures other than ordinary L-amino acids. Therefore, D-amino acids may be substituted by L-amino acids commonly found in the antigen peptides of the present invention, and it is still within the scope of the present disclosure. In addition, non-standard amino acids (ie, in addition to common natural proteogenic amino acids) can also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the present invention.

如果在一個以上位置上的取代發現導致肽的抗原活性基本上等於或大於以下定義值，則對這些取代的組合進行測試，以確定組合的取代是否產生對肽抗原性的疊加或協同效應。肽內被同時取代的位置最多不能超過 4 個。If substitutions at more than one position are found to cause the antigenic activity of the peptide to be substantially equal to or greater than the value defined below, then the combination of these substitutions is tested to determine whether the combined substitution produces an additive or synergistic effect on the antigenicity of the peptide. No more than 4 positions in the peptide can be substituted at the same time.

基本上由本文所指氨基酸序列組成的一種肽可能有一個或兩個非錨定氨基酸（見下面錨基序相關內容）被交換，而不存在這種情況，即相比於未修飾的肽，與人類主要組織相容性複合體(MHC)–I或II類分子的能力基本上被改變或受到不利影響。在另一實施方案中，在基本上由本文所述氨基酸序列組成的肽中，一個或兩個氨基酸可與其保守交換夥伴交換（見下文），而不存在這種情況，即相比於未修飾的肽，與人類主要組織相容性複合體(MHC)–I或II類分子的能力基本上被改變或受到不利影響。A peptide consisting essentially of the amino acid sequence referred to herein may have one or two non-anchor amino acids (see related content of anchor motifs below) exchanged, and there is no such situation, that is, compared to unmodified peptides, The ability to interact with the human major histocompatibility complex (MHC)-I or II molecules is basically altered or adversely affected. In another embodiment, in a peptide consisting essentially of the amino acid sequence described herein, one or two amino acids can be exchanged with its conservative exchange partner (see below), without this being the case, that is, as compared to unmodified The ability of the peptides to interact with the human major histocompatibility complex (MHC) – class I or class II molecules is basically altered or adversely affected.

這些基本不與T細胞受體互動的氨基酸殘基可透過取代其他幾乎不影響T細胞反應並不妨礙與相關MHC結合的氨基酸而得到修飾。因此，除了特定限制性條件外，本發明的肽可能為任何包括給定氨基酸序列或部分或其變體的肽（發明人所用的這個術語包括寡肽或多肽）。 表 6：根據SEQ ID NO: 2、5、8、62和53的肽的變體和基序 位置 1 2 3 4 5 6 7 8 9 10 11 SEQ ID NO. 2 A L H R P D V Y L     變體                 V                     I                     A       M                     M             V       M             I       M             A       A                     A             V       A             I       A             A       V                     V             V       V             I       V             A       T                     T             V       T             I       T             A       Q                     Q             V       Q             I       Q             A     位置 1 2 3 4 5 6 7 8 9 10 11 SEQ ID NO. 5 A L I F K I A S A     變體                 L                     I                     V       M                     M             L       M             I       M             V       A                     A             L       A             I       A             V       V                     V             L       V             I       V             V       T                     T             L       T             I       T             V       Q                     Q             L       Q             I       Q             V     位置 1 2 3 4 5 6 7 8 9 10 11 SEQ ID NO. 8 A L A A S A L P A L V 變體                     I                     L                     A   M                     M                 I   M                 L   M                 A   A                     A                 I   A                 L   A                 A   V                     V                 I   V                 L   V                 A   T                     T                 I   T                 L   T                 A   Q                     Q                 I   Q                 L   Q                 A 位置 1 2 3 4 5 6 7 8 9 SEQ ID NO. 62 G I I D G S P R L 變體   L             V   L             I   L                 L             A   M             V   M             I   M                 M             A   A             V   A             I   A                 A             A   V             V   V             I   V                 V             A   T             V   T             I   T                 T             A   Q             V   Q             I   Q                 Q             A 位置 1 2 3 4 5 6 7 8 9 SEQ ID NO.153 S L L A E L H V L 變體                   V                 I                 A   M             V   M             I   M                 M             A   A             V   A             I   A                 A             A   V             V   V             I   V                 V             A   T             V   T             I   T                 T             A   Q             V   Q             I   Q                 Q             A These amino acid residues that basically do not interact with T cell receptors can be modified by substituting other amino acids that hardly affect T cell response and do not hinder the binding of related MHCs. Therefore, except for certain restrictive conditions, the peptide of the present invention may be any peptide that includes a given amino acid sequence or part or variants thereof (the term used by the inventors includes oligopeptides or polypeptides). Table 6: Variants and motifs of peptides according to SEQ ID NO: 2, 5, 8, 62 and 53 Location 1 2 3 4 5 6 7 8 9 10 11 SEQ ID NO. 2 A L H R P D V Y L Variants V I A M M V M I M A A A V A I A A V V V V I V A T T V T I T A Q Q V Q I Q A Location 1 2 3 4 5 6 7 8 9 10 11 SEQ ID NO. 5 A L I F K I A S A Variants L I V M M L M I M V A A L A I A V V V L V I V V T T L T I T V Q Q L Q I Q V Location 1 2 3 4 5 6 7 8 9 10 11 SEQ ID NO. 8 A L A A S A L P A L V Variants I L A M M I M L M A A A I A L A A V V I V L V A T T I T L T A Q Q I Q L Q A Location 1 2 3 4 5 6 7 8 9 SEQ ID NO. 62 G I I D G S P R L Variants L V L I L L A M V M I M M A A V A I A A A V V V I V V A T V T I T T A Q V Q I Q Q A Location 1 2 3 4 5 6 7 8 9 SEQ ID NO.153 S L L A E L H V L Variants V I A M V M I M M A A V A I A A A V V V I V V A T V T I T T A Q V Q I Q Q A

較長（拉長）的肽也可能適合。MHC I類表位（通常長度為 8至11 個氨基酸）可能由肽從較長的肽或包含實際表位的蛋白中加工而產生。兩側有實際表位的殘基優選為在加工過程中幾乎不影響暴露實際表位所需蛋白裂解的殘基。Longer (elongated) peptides may also be suitable. MHC class I epitopes (usually 8 to 11 amino acids in length) may result from the processing of peptides from longer peptides or proteins containing actual epitopes. The residues flanked by the actual epitope are preferably residues that hardly affect the proteolytic cleavage required to expose the actual epitope during processing.

本發明的肽可被拉長多達四個氨基酸，即 1、2、3 或 4 個氨基酸，可按照 4:0 與 0:4之間的任何組合添加至任意一端。本發明的拉長組合可見表 7 。 表 7：本發明肽的拉長組合 C-端 N-端 4 0 3 0 或 1 2 0 或 1 或 2 1 0 或 1 或 2 或 3 0 0 或 1 或 2 或 3 或 4 N-端 C-端 4 0 3 0 或 1 2 0 或 1 或 2 1 0 或 1 或 2 或 3 0 0 或 1 或 2 或 3 或 4 The peptide of the present invention can be elongated up to four amino acids, namely 1, 2, 3 or 4 amino acids, and can be added to any end in any combination between 4:0 and 0:4. The elongated combination of the present invention can be seen in Table 7. Table 7: Elongation combinations of peptides of the invention C-terminal N-terminal 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4 N-terminal C-terminal 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

拉伸/延長的氨基酸可以是所述蛋白或任何其他氨基酸的原序列肽。拉長可用于增強所述肽的穩定性或溶解性。The stretched/elongated amino acid may be the original sequence peptide of the protein or any other amino acid. Elongation can be used to enhance the stability or solubility of the peptide.

因此，本發明所述的表位可能與天然腫瘤相關表位或腫瘤特異性表位相同，也可能包括來自參考肽的不超過四個殘基的不同肽，只要它們有基本相同的抗原活性即可。Therefore, the epitopes of the present invention may be the same as natural tumor-related epitopes or tumor-specific epitopes, or may include different peptides of no more than four residues from the reference peptide, as long as they have substantially the same antigenic activity. Can.

在一項替代實施方案中，肽的一邊或雙邊被拉長 4 個以上的氨基酸，優選最多 30 個氨基酸的總長度。這可形成 MHC-II類結合肽。結合至 MHC II類肽可透過本領域中已知的方法進行測試。In an alternative embodiment, one or both sides of the peptide are elongated by more than 4 amino acids, preferably a total length of up to 30 amino acids. This can form MHC class II binding peptides. Binding to MHC class II peptides can be tested by methods known in the art.

因此，本發明提出了MHC I類表位的肽和變體，其中所述肽或抗體的總長度為8至100個、優選為8至30個、最優選為8至14個氨基酸長度（即10、11、12、13、14個氨基酸，如果為拉長II類結合肽時，長度也可為15、16、17、18、19、20、21或22個氨基酸）。Therefore, the present invention proposes peptides and variants of MHC class I epitopes, wherein the total length of the peptide or antibody is 8 to 100, preferably 8 to 30, and most preferably 8 to 14 amino acids in length (ie 10, 11, 12, 13, 14 amino acids, if it is an elongated type II binding peptide, the length can also be 15, 16, 17, 18, 19, 20, 21 or 22 amino acids).

當然，本發明的肽或變體能與人主要組織相容性複合體 (MHC) I 或 II 類分子結合。肽或變體與 MHC 複合物的結合可用本領域內的已知方法進行測試。Of course, the peptides or variants of the present invention can bind to human major histocompatibility complex (MHC) class I or II molecules. The binding of the peptide or variant to the MHC complex can be tested by methods known in the art.

優選情況是，當本發明的肽特異性T細胞相比於取代肽受到檢測時，如果取代肽在相對於背景肽溶解度增加達到最大值的一半，則該肽濃度不超過約1 mM，優選為不超過約1 µM，更優選為不超過約1 nM，再優選為不超過約100 pM，最優選為不超過約10 pM。也優選為，取代肽被一個以上的T細胞識別，最少為2個，更優選為3個。Preferably, when the peptide-specific T cells of the present invention are tested compared to the substituted peptide, if the solubility of the substituted peptide relative to the background peptide increases to half of the maximum, the peptide concentration does not exceed about 1 mM, preferably Not more than about 1 µM, more preferably not more than about 1 nM, still more preferably not more than about 100 pM, and most preferably not more than about 10 pM. It is also preferred that the substituted peptide is recognized by more than one T cell, at least two, and more preferably three.

在本發明的一個特別優選實施方案中，肽系由或基本系由根據 SEQ ID NO: 1至SEQ ID NO: 385 所選的氨基酸序列組成。In a particularly preferred embodiment of the present invention, the peptide system consists or essentially consists of an amino acid sequence selected according to SEQ ID NO: 1 to SEQ ID NO: 385.

基本由「...組成」系指本發明的肽，除了根據SEQ ID NO: 1至SEQ ID NO: 385中的任一序列或其變體組成外，還含有位於其他N和/或C端延伸處的氨基酸，而它們不一定能形成作為MHC分子表位的肽。Basically consisting of "..." refers to the peptide of the present invention, in addition to being composed according to any one of SEQ ID NO: 1 to SEQ ID NO: 385 or a variant thereof, it also contains other N and/or C terminal Amino acids at the extension, and they may not necessarily form peptides that are epitopes of MHC molecules.

但這些延伸區域對有效將本發明中的肽引進細胞具有重要作用。在本發明的一實施例中，該肽為融合蛋白的一部分，含來自NCBI、GenBank登錄號X00497的HLA-DR抗原相關不變鏈（p33，以下稱為「Ii」）的80個N-端氨基酸等。在其他的融合中，本發明的肽可以被融合到本文所述的抗體、或其功能性部分，特別是融合入抗體的序列，以便所述抗體進行特異性靶向作用，或者，例如進入本文所述的樹突狀細胞特異性抗體。However, these extended regions play an important role in effectively introducing the peptides of the present invention into cells. In an embodiment of the present invention, the peptide is a part of the fusion protein, containing 80 N-terminals of the HLA-DR antigen-associated invariant chain (p33, hereinafter referred to as "Ii") from NCBI and GenBank accession number X00497 Amino acids, etc. In other fusions, the peptide of the present invention can be fused to the antibody described herein, or a functional part thereof, especially the sequence of the antibody, so that the antibody can perform a specific targeting effect, or, for example, into this article The dendritic cell specific antibody.

此外，該肽或變體可進一步修飾以提高穩定性和/或與 MHC 分子結合，從而引發更強的免疫反應。肽序列的該類優化方法是本領域內所熟知的，包括，例如，反式肽鍵和非肽鍵的引入。In addition, the peptide or variant can be further modified to improve stability and/or bind to MHC molecules, thereby eliciting a stronger immune response. Such optimization methods for peptide sequences are well known in the art and include, for example, the introduction of trans-peptide bonds and non-peptide bonds.

在反式肽鍵氨基酸中，肽(-CO-NH-)並未連接其殘基，但是其肽鍵是反向的。這種逆向反向模擬肽(retro-inverso peptidomimetics)可透過本領域已知的方法製備，例如：Meziere等人在(Meziere et al., 1997)中所述的方法，以引用的方式併入本文。這種方法涉及製備包含骨架（而並非側鏈）改變的模擬肽。Meziere等人(Meziere et al., 1997) 的研究顯示，這些類比肽有利於MHC的結合和輔助性T細胞的反應。以NH-CO鍵替代CO-NH肽鍵的逆向反向肽大大地提高了抗水解性能。Among trans peptide bond amino acids, the peptide (-CO-NH-) is not connected to its residues, but its peptide bond is reversed. Such retro-inverso peptidomimetics can be prepared by methods known in the art, such as the method described by Meziere et al. (Meziere et al., 1997), which is incorporated herein by reference. . This method involves the preparation of mimetic peptides that contain changes in the backbone (rather than the side chains). The study of Meziere et al. (Meziere et al., 1997) showed that these analog peptides are beneficial to MHC binding and helper T cell response. The reverse peptide with NH-CO bond instead of CO-NH peptide bond greatly improves the hydrolysis resistance.

非肽鍵為-CH2-NH、-CH2S-、-CH2CH2-、-CH=CH-、-COCH2-、-CH(OH)CH2-和 -CH2SO-等。美國 4897445 號專利提出了多肽鏈中非肽鍵 (-CH2-NH) 的非固相合成法，該方法涉及按標準程序合成的多肽以及透過氨基醛和一種含 NaCNBH3 的氨基酸相互作用而合成的非肽鍵。Non-peptide bonds are -CH2-NH, -CH2S-, -CH2CH2-, -CH=CH-, -COCH2-, -CH(OH)CH2- and -CH2SO-, etc. U.S. Patent No. 4897445 proposes a non-solid phase synthesis method for non-peptide bonds (-CH2-NH) in the polypeptide chain. Peptide bond.

含上述序列的肽可與其氨基和/或羧基末端的其他化學基團進行合成，從而提高肽的穩定性、生物利用度、和/或親和力等。例如，苄氧羰基、丹醯基等疏水基團或叔丁氧羰基團可加入肽的氨基末端。同樣，乙醯基或 9-芴甲氧羰基可能位於肽的氨基末端。此外，疏水基團、叔丁氧羰基團或氨基團都可能被加入肽的羧基末端。The peptides containing the above-mentioned sequences can be synthesized with other chemical groups at the amino and/or carboxyl terminal ends, thereby improving the stability, bioavailability, and/or affinity of the peptide. For example, hydrophobic groups such as benzyloxycarbonyl and dansyl or tert-butoxycarbonyl groups can be added to the amino terminal of the peptide. Similarly, the acetyl group or 9-fluorenylmethoxycarbonyl group may be located at the amino terminus of the peptide. In addition, hydrophobic groups, tert-butoxycarbonyl groups, or amino groups may all be added to the carboxyl end of the peptide.

另外，本發明中的所有肽都可能經合成而改變其空間構型。例如，可能使用這些肽的一個或多個氨基酸殘基的右旋體，通常不是其左旋體。更進一步地，本發明中肽的至少一個氨基酸殘基可被熟知的一個非天然氨基酸殘基取代。諸如此類的改變可能有助於增加本發明肽的穩定性、生物利用度和/或結合作用。In addition, all peptides in the present invention may be synthesized to change their spatial configuration. For example, it is possible to use a dextrorotatory form of one or more amino acid residues of these peptides, usually not their levorotatory form. Furthermore, at least one amino acid residue of the peptide of the present invention may be substituted by a well-known unnatural amino acid residue. Such changes may help increase the stability, bioavailability and/or binding of the peptides of the invention.

同樣，本發明中的肽或變體可在合成肽之前或之後透過特異氨基酸的反應而進行化學修飾。此類修飾的實施例為本領域所熟知，例如，在 R. Lundblad 所著的《Chemical Reagents for Protein Modification》 (3rd ed. CRC Press, 2004)  (Lundblad, 2004) 中有概述，以參考文獻的方式併入本文。雖然氨基酸的化學修飾方法無限制，但其包括（但不限於）透過以下方法修飾：醯基化、脒基化、賴氨酸吡哆基化、還原烷基化、以 2,4,6-三硝基苯磺酸 (TNBS) 三硝基苯基化氨基團、透過將半胱氨酸過甲酸氧化為磺基丙氨酸而對羧基團和巰基進行氨基修飾、形成易變衍生物、與其他巰基化合物形成混合二硫化合物、與馬來醯亞胺反應，與碘乙酸或碘乙醯胺羧甲基化、在鹼性 pH 值下與氰酸鹽甲氨醯化。在這方面，技術人員參考了《Current Protocols In Protein Science》 (Eds. Coligan et al. (John Wiley and Sons NY 1995-2000) )  (Coligan et al., 1995) 中第 15 章所述的在蛋白質化學修飾相關的廣泛方法。Similarly, the peptides or variants of the present invention can be chemically modified through specific amino acid reactions before or after the peptide is synthesized. Examples of such modifications are well known in the art. For example, they are summarized in "Chemical Reagents for Protein Modification" (3rd ed. CRC Press, 2004) (Lundblad, 2004) by R. Lundblad. The method is incorporated into this article. Although the chemical modification methods of amino acids are not limited, they include (but are not limited to) modification through the following methods: acylation, amidinolation, pyridoxylation of lysine, reductive alkylation, and 2,4,6- Trinitrobenzene sulfonic acid (TNBS) trinitrophenylated amino group, by oxidizing cysteine performic acid to cysteic acid to modify the carboxyl group and sulfhydryl group to form variable derivatives, and Other sulfhydryl compounds form mixed disulfide compounds, react with maleimine, carboxymethyl with iodoacetic acid or iodoacetamide, and methamine with cyanate at alkaline pH. In this regard, the technical staff refers to the "Current Protocols In Protein Science" (Eds. Coligan et al. (John Wiley and Sons NY 1995-2000)) (Coligan et al., 1995) in Chapter 15 A wide range of methods related to chemical modification.

簡言之，修飾蛋白質的精氨醯殘基等往往基於於鄰二羰基化合物（如苯甲醯甲醛、2,3 –丁二酮以及 1,2-烯巳二酮）的反應而形成加合物。另一個實施例是丙酮醛與精氨酸殘基的反應。半胱氨酸可在賴氨酸和組氨酸等親核位點不作隨同修飾的情況下就得到修飾。因此，有大量試劑可進行半胱氨酸的修飾。Sigma-Aldrich (http://www.sigma-aldrich.com) 等公司的網站含有具體試劑的資訊。In short, the arginine residues of modified proteins are often based on the reaction of o-dicarbonyl compounds (such as benzaldehyde, 2,3-butanedione, and 1,2-enandione) to form adducts. Things. Another example is the reaction of pyruvaldehyde with arginine residues. Cysteine can be modified without concomitant modification of nucleophilic sites such as lysine and histidine. Therefore, there are a large number of reagents for cysteine modification. The websites of companies such as Sigma-Aldrich (http://www.sigma-aldrich.com) contain information about specific reagents.

蛋白質中二硫鍵的選擇性還原也很普遍。二硫鍵可在生物制藥熱處理中形成和氧化。伍德沃德氏試劑 K 可用於修飾特定的谷氨酸殘基。N-（3-二甲氨基丙基）-N´-乙基-碳二亞胺可用于形成賴氨酸殘基和谷氨酸殘基的分子內交聯。例如：焦碳酸二乙酯是修飾蛋白質組氨酸殘基的試劑。組氨酸也可使用 4-羥基-2-壬烯醛進行修飾。賴氨酸殘基與其他α-氨基團的反應，例如，有利於肽結合到蛋白/肽的表面或交聯處。賴氨酸聚是多（乙烯）乙二醇的附著點，也是蛋白質糖基化的主要修飾位點。蛋白質的蛋氨酸殘基可透過碘乙醯胺、溴乙胺、氯胺 T 等被修飾。The selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during the heat treatment of biopharmaceuticals. Woodward's reagent K can be used to modify specific glutamate residues. N-(3-Dimethylaminopropyl)-N´-ethyl-carbodiimide can be used to form intramolecular crosslinks of lysine residues and glutamic acid residues. For example, diethyl pyrocarbonate is a reagent that modifies histidine residues in proteins. Histidine can also be modified with 4-hydroxy-2-nonenal. The reaction of lysine residues with other α-amino groups, for example, facilitates the binding of peptides to the surface or cross-links of proteins/peptides. Lysine poly is the attachment point of poly(ethylene) glycol and the main modification site for protein glycosylation. Methionine residues of proteins can be modified by iodoacetamide, bromoethylamine, chloramine T and so on.

四硝基甲烷和 N-乙醯基咪唑可用於酪氨酸殘基的修飾。經二酪氨酸形成的交聯可透過過氧化氫/銅離子完成。Tetranitromethane and N-acetylimidazole can be used for modification of tyrosine residues. The cross-linking formed by dityrosine can be accomplished through hydrogen peroxide/copper ions.

對色氨酸修飾的最近研究中使用了 N-溴代琥珀醯亞胺、2-羥基-5-硝基苄溴或 3-溴-3-甲基-2- (2 –硝苯巰基) -3H-吲哚 (BPNS-糞臭素)。Recent studies on the modification of tryptophan have used N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)- 3H-indole (BPNS-skatole).

當蛋白與戊二醛、聚乙二醇二丙烯酸酯和甲醛的交聯用於配製水凝膠時，治療性蛋白和含聚乙二醇的肽的成功修飾往往可延長迴圈半衰期。針對免疫治療的變態反應原化學修飾往往透過氰酸鉀的氨基甲醯化實現。When the cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde is used to formulate hydrogels, successful modification of therapeutic proteins and polyethylene glycol-containing peptides can often extend the loop half-life. Chemical modification of allergens for immunotherapy is often achieved through the aminomethylation of potassium cyanate.

一種肽或變體，其中肽被修飾或含非肽鍵，優選為本發明的實施例。A peptide or variant in which the peptide is modified or contains non-peptide bonds, preferably an embodiment of the present invention.

本發明的另一實施方案涉及一種非天然肽，其中所述肽系由或基本系由根據SEQ ID NO:1至SEQ ID NO:385的一個氨基酸序列組成，並經合成產生（即，合成）為一種藥用鹽。合成產生肽的方法是本領域公知的。本發明肽的鹽與其體內狀態的肽基本上不同，因為這些體內產生的肽不是鹽。該肽的非天然鹽形式介導肽的溶解度，特別是包含所述肽的藥物組合物的情況下，例如，本文所公開的肽疫苗。為了向需治療的受試者有效地提供肽，需要肽具有充分、至少基本的溶解度。優選地，鹽為肽的藥用鹽。本發明的這些鹽包括堿和堿土鹽類，諸如 Hofmeister 系列的鹽，包含陰離子 PO43-、SO42-、CH3COO-、Cl-、Br-、NO3-、ClO4-、I-、SCN-  和陽離子 NH4+、Rb+、K+、Na+、Cs+、Li+、Zn2+、Mg2+、Ca2+、Mn2+、Cu2+ 和 Ba2+。特別地，鹽選自 (NH4)3PO4、(NH4)2HPO4、(NH4)H2PO4、(NH4)2SO4、NH4CH3COO、NH4Cl、NH4Br、NH4NO3、NH4CIO4、NH4I、NH4SCN、Rb3PO4、Rb2HPO4、RbH2PO4、Rb2SO4、Rb4CH3COO、Rb4Cl、Rb4Br、Rb4NO3、Rb4CIO4、Rb4I、Rb4SCN、K3PO4、K2HPO4、KH2PO4、K2SO4、KCH3COO、KCl、KBr、KNO3、KClO4、KI、KSCN、Na3PO4、Na2HPO4、NaH2PO4、Na2SO4、NaCH3COO、NaCl、NaBr、NaNO3、NaCIO4、NaI、NaSCN、ZnCI2 Cs3PO4、Cs2HPO4、CsH2PO4、Cs2SO4、CsCH3COO、CsCl、CsBr、CsNO3、CsCIO4、CsI、CsSCN、Li3PO4、Li2HPO4、LiH2PO4、Li2SO4、LiCH3COO、LiCl、LiBr、LiNO3、LiClO4、LiI、LiSCN、Cu2SO4、Mg3(PO4)2、Mg2HPO4、Mg(H2PO4)2、Mg2SO4、Mg(CH3COO)2、MgCl2、MgBr2、Mg(NO3)2、Mg(ClO4)2、MgI2、Mg(SCN)2、MnCl2、Ca3(PO4),、Ca2HPO4、Ca(H2PO4)2、CaSO4、Ca(CH3COO)2、CaCl2、CaBr2、Ca(NO3)2、Ca(ClO4)2、CaI2、Ca(SCN)2、Ba3(PO4)2、Ba2HPO4、Ba(H2PO4)2、BaSO4、Ba(CH3COO)2、BaCl2、BaBr2、Ba(NO3)2、Ba(ClO4)2、BaI2 和 Ba(SCN)2。特別優選為 NH 乙酸、MgCl2、KH2PO4、Na2SO4、KCl、NaCl 和 CaCl2，例如：氯化物或乙酸鹽（三氟乙酸）鹽。Another embodiment of the present invention relates to a non-natural peptide, wherein the peptide is composed of or essentially consists of an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 385 and is produced synthetically (ie, synthetic) It is a medicinal salt. Methods of synthetically producing peptides are well known in the art. The salts of the peptides of the present invention are basically different from the peptides in the in vivo state because these peptides produced in the body are not salts. The non-natural salt form of the peptide mediates the solubility of the peptide, especially in the case of a pharmaceutical composition comprising the peptide, for example, the peptide vaccine disclosed herein. In order to effectively provide peptides to subjects in need of treatment, the peptides need to have sufficient, at least basic solubility. Preferably, the salt is a pharmaceutically acceptable salt of the peptide. These salts of the present invention include basal and basal soil salts, such as the Hofmeister series of salts, including anions PO43-, SO42-, CH3COO-, Cl-, Br-, NO3-, ClO4-, I-, SCN- and cationic NH4+ , Rb+, K+, Na+, Cs+, Li+, Zn2+, Mg2+, Ca2+, Mn2+, Cu2+ and Ba2+. In particular, the salt is selected from (NH4)3PO4, (NH4)2HPO4, (NH4)H2PO4, (NH4)2SO4, NH4CH3COO, NH4Cl, NH4Br, NH4NO3, NH4CIO4, NH4I, NH4SCN, Rb3PO4, Rb2HPO4, RbH2SO4, Rb2HPO4, RbH2SO4, Rb2HPO4, RbH2SO4, Rb4Cl, Rb4Br, Rb4NO3, Rb4CIO4, Rb4I, Rb4SCN, K3PO4, K2HPO4, KH2PO4, K2SO4, KCH3COO, KCl, KBr, KNO3, KClO4, KI, KSCN, Na3PO4, Na2HPO4, Na3COH2, Br NaCIO4, NaI, NaSCN, ZnCI2 Cs3PO4, Cs2HPO4, CsH2PO4, Cs2SO4, CsCH3COO, CsCl, CsBr, CsNO3, CsCIO4, CsI, CsSCN, Li3PO4, Li2HPO4, LiCOH, LiNOCl, Li2SO4, LiCN, LiCN , Cu2SO4, Mg3(PO4)2, Mg2HPO4, Mg(H2PO4)2, Mg2SO4, Mg(CH3COO)2, MgCl2, MgBr2, Mg(NO3)2, Mg(ClO4)2, MgI2, Mg(SCN)2, MnCl2 , Ca3(PO4),, Ca2HPO4, Ca(H2PO4)2, CaSO4, Ca(CH3COO)2, CaCl2, CaBr2, Ca(NO3)2, Ca(ClO4)2, CaI2, Ca(SCN)2, Ba3(PO4 )2, Ba2HPO4, Ba(H2PO4)2, BaSO4, Ba(CH3COO)2, BaCl2, BaBr2, Ba(NO3)2, Ba(ClO4)2, BaI2 and Ba(SCN)2. Particularly preferred are NH acetic acid, MgCl2, KH2PO4, Na2SO4, KCl, NaCl and CaCl2, such as chloride or acetate (trifluoroacetic acid) salt.

一般來說，肽和變體（至少含氨基酸殘基之間的肽聯接）可使用 Lukas 等人 (Lukas et al., 1981) 以及此處引用的參考文獻所披露的固相肽合成 Fmoc-聚醯胺模式進行合成。芴甲氧羰基 (Fmoc) 團對 N-氨基提供臨時保護。使用 N, N-二甲基甲醯胺中的 20% 二甲基呱啶中對這種堿高度敏感的保護基團進行重複***。由於它們的丁基醚 (在絲氨酸蘇氨酸和酪氨酸的情況下)、丁基酯 (在谷氨酸和天門冬氨酸的情況下)、叔丁氧羰基衍生物 (在賴氨酸和組氨酸的情況下)、三苯甲基衍生物 (在半胱氨酸的情況下) 及 4-甲氧基-2,3,6-三甲基苯磺醯基衍生物 (在精氨酸的情況下)，側鏈功能可能會受到保護。只要穀氨醯胺和天冬醯胺為 C-末端殘基，側鏈氨基功能保護所使用的是由 4,4'-二甲氧基二苯基團。固相支撐基於聚二甲基丙烯醯胺聚合物，其由三個單體二甲基丙烯醯胺（骨架單體）、雙丙烯醯乙烯二胺（交聯劑）和 N-丙烯醯肌胺酸甲酯（功能劑）構成。使用的肽-樹脂聯劑為酸敏感的 4 -羥甲基苯氧乙酸衍生物。所有的氨基酸衍生物均作為其預製對稱酸酐衍生物加入，但是天冬醯胺和穀氨醯胺除外，它們使用被逆轉的 N, N-二環己基碳二亞胺/1-羥基苯並***介導的耦合程序而加入。所有的耦合和脫保護反應用茚三酮、硝基苯磺酸或 isotin 測試程序監測。合成完成後，用濃度為 95% 含 50% 清道夫混合物的三氟醋酸，從伴隨去除側鏈保護基團的樹脂支承物中裂解肽。常用的清道夫混合物包括乙二硫醇、苯酚、苯甲醚和水，準確的選擇依據合成肽的氨基酸組成。此外，固相和液相方法結合使用對肽進行合成是可能的（例如，請參閱 (Bruckdorfer et al., 2004) 以及本文引用的參考文獻）In general, peptides and variants (at least containing peptide linkages between amino acid residues) can be synthesized using solid-phase peptides disclosed in Lukas et al. (Lukas et al., 1981) and the references cited herein. Synthesis is carried out in amide mode. The fluorenylmethyloxycarbonyl (Fmoc) group provides temporary protection to the N-amino group. The 20% dimethylpiperidine in N, N-dimethylformamide is used for repeated splitting of the protective group that is highly sensitive to this type. Due to their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), tert-butoxycarbonyl derivatives (in the case of lysine And histidine), trityl derivatives (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivatives (in the case of In the case of amino acids), the side chain function may be protected. As long as glutamine and asparagine are C-terminal residues, the functional protection of side chain amino groups is made of 4,4'-dimethoxydiphenyl groups. The solid support is based on polydimethylacrylamide polymer, which consists of three monomers: dimethylacrylamide (backbone monomer), bisacrylethylenediamine (crosslinking agent) and N-acrylamidoamine Composition of methyl acid methyl ester (functional agent). The peptide-resin coupling agent used is an acid-sensitive 4-hydroxymethylphenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives, except for asparagine and glutamine, which use reversed N, N-dicyclohexylcarbodiimide/1-hydroxybenzotrimide Azole-mediated coupling program was added. All coupling and deprotection reactions are monitored using ninhydrin, nitrobenzenesulfonic acid or isotin test procedures. After the synthesis is complete, use 95% trifluoroacetic acid with a 50% scavenger mixture to cleave the peptide from the resin support accompanied by the removal of the side chain protecting groups. Commonly used scavenger mixtures include ethanedithiol, phenol, anisole, and water. The exact selection depends on the amino acid composition of the synthetic peptide. In addition, it is possible to use a combination of solid-phase and liquid-phase methods to synthesize peptides (for example, see (Bruckdorfer et al., 2004) and the references cited herein)

三氟乙酸用真空中蒸發、隨後用承載粗肽的二乙基***滴定進行去除。用簡單萃取程序（水相凍幹後，該程序制得不含清道夫混合物的肽）清除任何存在的清道夫混合物。肽合成試劑一般可從Calbiochem- Novabiochem（英國諾丁漢）獲得。The trifluoroacetic acid was removed by evaporation in a vacuum, followed by titration with diethyl ether carrying the crude peptide. Use a simple extraction procedure (after freeze-drying the aqueous phase, the procedure produces peptides without the scavenger mixture) to remove any scavenger mixture present. Peptide synthesis reagents are generally available from Calbiochem-Novabiochem (Nottingham, UK).

純化可透過以下技術的任何一種或組合方法進行，如：再結晶法、體積排阻色譜法、離子交換色譜法、疏水作用色譜法以及（通常）反相高效液相色譜法（如使用乙腈/水梯度分離）。Purification can be carried out by any one or a combination of the following techniques, such as: recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (usually) reversed-phase high performance liquid chromatography (such as acetonitrile/ Water gradient separation).

可以使用薄層色譜法、電泳特別是毛細管電泳、固相萃取（CSPE）、反相高效液相色譜法、酸解後的氨基酸分析、快原子轟擊（FAB）質譜分析以及MALDI和ESI-Q-TOF質譜分析進行肽分析。Can use thin layer chromatography, electrophoresis, especially capillary electrophoresis, solid phase extraction (CSPE), reversed-phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, fast atom bombardment (FAB) mass spectrometry, MALDI and ESI-Q- TOF mass spectrometry for peptide analysis.

為了選擇過度表現的肽，計算了表現圖，其顯示樣本中位元表現量以及複製變化。該特點使相關腫瘤實體的樣本與正常組織樣本的基線值並列。可透過計算調節線性混合效應模型 (Pinheiro et al., 2015)的p值將以上每個特點併入過度表現分數中，從而透過假發現率 (Benjamini and Hochberg, 1995) 調整多項檢驗（參見實施例 1、圖 1）。In order to select over-expressed peptides, a performance map was calculated, which shows the amount of bit expression and replication changes in the sample. This feature aligns the baseline values of samples of related tumor entities with normal tissue samples. The p-value of the linear mixed-effects model (Pinheiro et al., 2015) can be calculated and adjusted to incorporate each of the above characteristics into the overperformance score, so that multiple tests can be adjusted through the false discovery rate (Benjamini and Hochberg, 1995) (see Example 1. Figure 1).

對於透過質譜法對 HLA 配體的識別和相對定量，對來自衝擊冷凍組織樣本的 HLA 分子進行純化並對  HLA 相關肽進行分離。分離的肽分開，並透過線上納米-電噴霧-電離 (nanoESI) 液相色譜- 譜 (LC-MS) 實驗進行鑒定。由此產生的肽序列的驗證方法是，將 CLL 樣本（N = 17 個 A*02 陽性樣本）中記錄的自然腫瘤相關肽 (TUMAP) 的片段模式與相同序列相應合成參考肽的片段模式進行比較。由於這些肽被直接鑒定為原發性腫瘤 HLA 分子的配體，因此這些結果為來自 16 名 CLL 患者的原發癌症組織上確定肽的自然加工和表現提供了直接證據。For the identification and relative quantification of HLA ligands by mass spectrometry, HLA molecules from shock-frozen tissue samples are purified and HLA-related peptides are separated. The separated peptides are separated and identified by online nano-electrospray-ionization (nanoESI) liquid chromatography-spectroscopy (LC-MS) experiments. The resulting peptide sequence is verified by comparing the fragment pattern of natural tumor-associated peptide (TUMAP) recorded in the CLL sample (N = 17 A*02 positive samples) with the fragment pattern of the corresponding synthetic reference peptide of the same sequence . Since these peptides were directly identified as primary tumor HLA molecule ligands, these results provided direct evidence for the natural processing and performance of the peptides identified on primary cancer tissues from 16 CLL patients.

發現管道XPRESIDENT® v2.1（例如，參見 US 2013-0096016，並在此透過引用將其整體併入本文）考慮到識別和選擇相關過量表現的候選肽疫苗，這基於與幾種不同的非癌組織和器官相比癌症或其他受感染組織的HLA限制肽水準直接相對定量結果。這透過以下方法實現：使用專有資料分析管道處理的LC-MS採集資料、結合序列識別演算法、譜聚類、計算離子、保留時間調整、充電狀態卷積以及正態化而開發無標記差異化定量方法。The discovery pipeline XPRESIDENT® v2.1 (for example, see US 2013-0096016, which is hereby incorporated by reference in its entirety) takes into account the identification and selection of candidate peptide vaccines related to overexpression, which is based on a number of different non-cancer vaccines. Tissues and organs compare the HLA-restricted peptide levels of cancer or other infected tissues directly to quantitative results. This is achieved through the following methods: LC-MS collected data processed by a proprietary data analysis pipeline, combined with sequence recognition algorithms, spectral clustering, ion calculation, retention time adjustment, charge state convolution, and normalization to develop unmarked differences Quantification method.

為每種肽和樣本確立了表現水準，包括誤差估計值。腫瘤組織大量表現的肽以及腫瘤與非腫瘤組織和器官中過量表現的肽已經得到確定。A performance level was established for each peptide and sample, including error estimates. A large number of peptides expressed in tumor tissues and excessively expressed peptides in tumor and non-tumor tissues and organs have been identified.

對來自CLL組織樣本的HLA肽複合物進行純化，並且對HLA相關肽使用LC-MS進行分離和分析（見實施例）。本申請中包含的所有TUMAP使用原發性CLL樣本的方法進行鑒定，確認其在原發性CLL上的表現。Purify HLA peptide complexes from CLL tissue samples, and use LC-MS to separate and analyze HLA-related peptides (see Examples). All TUMAPs included in this application are identified using the method of primary CLL samples to confirm their performance on primary CLL.

在多個CLL和正常組織上確定的TUMAP用無標記LC-MS資料的離子計數方法進行量化。該方法假定肽的LC-MS信號區域與樣本中其豐度相關。各種LC-MS實驗中肽的所有量化信號在集中趨勢基礎上進行正常化，根據每個樣品進行平均，併合併入柱狀圖（被稱為表現圖）。表現圖整合了不同分析方法，如：蛋白資料庫檢索、譜聚類、充電狀態卷積（除電）和保留時間校準和正態化。The TUMAP determined on multiple CLLs and normal tissues was quantified by the ion counting method of unlabeled LC-MS data. This method assumes that the LC-MS signal area of the peptide is related to its abundance in the sample. All quantitative signals of peptides in various LC-MS experiments are normalized on the basis of central tendency, averaged according to each sample, and merged into a histogram (referred to as a performance graph). The performance map integrates different analysis methods, such as: protein database search, spectral clustering, state-of-charge convolution (in addition to charge), retention time calibration and normalization.

此外，發現平臺技術 XPRESIDENT® v2.x（例如參見 PCT/EP2011/056056 和 PCT/EP2015/079873）可對癌症或其他感染組織上的 MHC-肽，優選為 HLA 限制性肽進行直接的絕對定量。簡言之，總細胞計數根據被分析的組織樣本的總 DNA 含量來計算。組織樣本中 TUMAP 的總肽量用 nanoLC-MS/MS 測定為天然 TUMAP 的比率以及TUMAP 同位素標記版本的已知量，稱為內部標準。TUMAP 分離效率確定方法：把肽：所有選定 TUMAP 的 MHC 在 TUMAP 分離程序儘早的時間點加入組織裂解液，並在肽分離成後完成後通過 nanoLC-MS/MS 檢測。總細胞計數和總肽量根據每份組織樣本三次測量值來計算。所述肽特異性隔離效率計算為三次測量 10 次加標實驗的平均值（見實施例 6 和表 12）。In addition, it was discovered that the platform technology XPRESIDENT® v2.x (see for example PCT/EP2011/056056 and PCT/EP2015/079873) can directly quantify MHC-peptides, preferably HLA-restricted peptides, on cancer or other infected tissues. In short, the total cell count is calculated based on the total DNA content of the tissue sample being analyzed. The total peptide amount of TUMAP in the tissue sample is determined by nanoLC-MS/MS as the ratio of natural TUMAP and the known amount of the isotope-labeled version of TUMAP, which is called the internal standard. TUMAP separation efficiency determination method: add peptides: all MHCs of selected TUMAP to the tissue lysate at the earliest time point of the TUMAP separation program, and detect by nanoLC-MS/MS after the separation of the peptides is completed. The total cell count and total peptide amount are calculated based on three measurements for each tissue sample. The peptide-specific isolation efficiency is calculated as the average value of three measurements and 10 addition experiments (see Example 6 and Table 12).

除了過量表現肽之外，也測試了潛在基因的 mRNA 表達。mRNA 資料通過 RNA 測序分析正常組織和癌組織獲得（見實施例 2、圖3）。正常組織資料的額外來源是從 3000 個正常組織樣本中公開獲得的 RNA 表達資料的資料庫 (Lonsdale, 2013)。獲得自蛋白的肽在癌組織中顯示高表達編碼mRNA，但是在重要正常組織中非常低或不存在，這些肽作為優選肽納入本發明。In addition to overexpressing peptides, the mRNA expression of potential genes was also tested. The mRNA data was obtained by RNA sequencing analysis of normal tissues and cancer tissues (see Example 2, Figure 3). An additional source of normal tissue data is a database of RNA expression data publicly obtained from 3000 normal tissue samples (Lonsdale, 2013). Peptides obtained from proteins show high expression of encoding mRNA in cancer tissues, but are very low or absent in important normal tissues, and these peptides are included in the present invention as preferred peptides.

本發明提出了有利於治療癌腫/腫瘤，優選為治療過量表現或只表現本發明肽的CLL。這些肽由質譜分析法直接顯示出，而由HLA分子自然表現于原發性CLL樣本中。The present invention proposes to be beneficial for the treatment of cancer/tumor, preferably for the treatment of over-expression or CLL that only expresses the peptide of the present invention. These peptides are directly revealed by mass spectrometry, while HLA molecules are naturally expressed in primary CLL samples.

與正常組織相比，癌症中高度過量表達肽來源的許多源基因/蛋白質（也指定為「全長蛋白」或「潛在蛋白」）- 本發明相關的「正常組織」是健康脂肪組織、腎上腺、血細胞、血管、骨髓、腦、軟骨、食道、眼、膽囊、心臟、腎、大腸、肝、肺、淋巴結、神經、胰腺、甲狀旁腺、腹膜、垂體、胸膜、唾液腺、骨骼肌、皮膚、小腸、脾、胃、胸腺、甲狀腺、氣管、輸尿管、膀胱細胞或其他正常組織細胞，這表明腫瘤與這些源基因的高度關聯性（見實施例 2）。此外，這些肽本身也在腫瘤組織中過度表現（本發明相關的「腫瘤組織」是指來自 CLL 患者的樣本），但不在正常組織中過度表現（見實施例 1）。Compared with normal tissues, many source genes/proteins derived from peptides (also designated as "full-length proteins" or "potential proteins") are highly overexpressed in cancer-the "normal tissues" related to the present invention are healthy adipose tissue, adrenal glands, and blood cells , Blood vessels, bone marrow, brain, cartilage, esophagus, eyes, gallbladder, heart, kidney, large intestine, liver, lungs, lymph nodes, nerves, pancreas, parathyroid glands, peritoneum, pituitary gland, pleura, salivary glands, skeletal muscle, skin, small intestine , Spleen, stomach, thymus, thyroid, trachea, ureter, bladder cells or other normal tissue cells, which indicates that tumors are highly related to these source genes (see Example 2). In addition, these peptides themselves are also over-represented in tumor tissues (the “tumor tissue” in the present invention refers to samples from CLL patients), but not over-represented in normal tissues (see Example 1).

HLA 結合肽能夠被免疫系統識別，特別是 T 淋巴細胞。T細胞可破壞表現被識別 HLA/肽複合體的細胞（如：表現衍生肽的 CLL 細胞）。HLA-binding peptides can be recognized by the immune system, especially T lymphocytes. T cells can destroy cells that express recognized HLA/peptide complexes (such as CLL cells that express derived peptides).

本發明的所有肽已被證明具有刺激T細胞反應的能力，並過量表現，因而可用于製備本發明的抗體和/或TCR，例如可溶性TCR（參見實施例3和實施例4）。此外，肽與相應的MHC組合時，也可用于製備本發明的抗體和/或TCR，特別是sTCR。各個方法均為技術人員所熟知，並在各個文獻中可找到。因此，本發明的肽可用于在患者中產生免疫反應，從而能夠毀滅腫瘤細胞。患者的免疫反應能夠透過直接給予患者所述肽或前體物質（如，加長肽、蛋白或編碼這些肽的核酸），較理想是與加強免疫原性的製劑相結合，而進行誘導。源自該治療性疫苗的免疫反應預期能夠高度特異性地對抗腫瘤細胞，因為本發明的目標肽在正常組織上表現的複製數目較少，防止患者發生對抗正常細胞的不良自體免疫反應的風險。All peptides of the present invention have been proven to have the ability to stimulate T cell responses and are over-expressed, so they can be used to prepare antibodies and/or TCRs of the present invention, such as soluble TCR (see Example 3 and Example 4). In addition, when the peptide is combined with the corresponding MHC, it can also be used to prepare the antibody and/or TCR of the present invention, especially sTCR. Each method is well-known to the skilled person and can be found in various documents. Therefore, the peptide of the present invention can be used to generate an immune response in a patient, thereby being able to destroy tumor cells. The patient's immune response can be induced by directly administering the peptides or precursor substances (eg, lengthened peptides, proteins, or nucleic acids encoding these peptides) to the patient, and preferably combined with preparations that enhance immunogenicity. The immune response derived from the therapeutic vaccine is expected to be highly specific against tumor cells, because the target peptide of the present invention exhibits a small number of replications in normal tissues, preventing the risk of adverse autoimmune reactions against normal cells in patients .

本說明書還涉及包含一個 α 鏈和一個 β鏈 (「α/β TCR」) 的 T細胞受體 (TCR)。還提供了由 MHC 分子表現時可與 TCR 和抗體結合的本發明的肽。本說明書還涉及核酸、載體和用於表達 TCR 的宿主細胞和本說明書的肽；以及使用它們的方法。This specification also relates to a T cell receptor (TCR) that contains an alpha chain and a beta chain ("α/β TCR"). The peptide of the present invention that can bind to TCR and antibody when expressed by MHC molecules is also provided. This specification also relates to nucleic acids, vectors and host cells for expressing TCR and peptides of this specification; and methods of using them.

術語「T細胞受體」（縮寫TCR）是指一種異二聚體分子，其包含一個α多肽鏈（α鏈）和一個β多肽鏈（β鏈），其中所述異二聚體受體能夠結合由HLA分子表現的肽抗原。該術語還包括所謂的γ/δTCR。The term "T cell receptor" (abbreviated as TCR) refers to a heterodimeric molecule that contains an α polypeptide chain (α chain) and a β polypeptide chain (β chain), wherein the heterodimeric receptor is capable of Binds peptide antigens expressed by HLA molecules. The term also includes the so-called gamma/delta TCR.

在一個實施方案中，本說明書提供了如本文中所描述的產生 TCR 的方法，該方法包括在適於促進 TCR 表達的條件下培養能夠表達 TCR 的宿主細胞。In one embodiment, this specification provides a method for producing TCR as described herein, the method comprising culturing a host cell capable of expressing TCR under conditions suitable for promoting TCR expression.

另一個方面，本說明書涉及一種根據本說明書的方法，其中所述抗原透過與足夠量的含抗原提成細胞的抗原結合被載入表達於合適抗原表現細胞或人工抗原呈遞細胞表面的I或II類MHC分子，或該抗原透過四聚化被載入I或II類MHC四聚體/I或II類MHC複合單體。In another aspect, the present specification relates to a method according to the present specification, wherein the antigen is loaded into a class I or II type expressed on the surface of a suitable antigen-expressing cell or artificial antigen-presenting cell by binding to a sufficient amount of antigen-containing antigen-producing cells The MHC molecule, or the antigen, is loaded into the MHC class I or II tetramer/MHC class II complex monomer through tetramerization.

α/β TCR的α和β鏈和γ/δ TCR的γ和δ鏈通常被視為各自有兩個「結構域」，即可變和恒定結構域。可變結構域由可變區(V)和連接區(J)的組合。可變結構域還可能包括一個前導區(L)。β和δ鏈還可能包括一個多樣區(D)。α和β恒定結構域還可能包括錨定α和β鏈至細胞膜的C末端跨膜(TM)結構域。The alpha and beta chains of the alpha/beta TCR and the gamma and delta chains of the gamma/delta TCR are generally considered to have two "domains" each, namely variable and constant domains. The variable domain consists of a combination of a variable region (V) and a junction region (J). The variable domain may also include a leader region (L). The β and δ chains may also include a diverse region (D). The alpha and beta constant domains may also include a C-terminal transmembrane (TM) domain that anchors the alpha and beta chains to the cell membrane.

相對於 γ/δ 的 TCR，如本文所用的術語 「TCRγ可變域」是指無前導區 (L) 的  TCR γ V (TRGV) 區與 TCR γ (TRGJ) 區的組合，術語 TCR γ恒定結構域是指細胞外TRGC區域，或 C-末端截短 TRGC 序列。同樣地，「TCRδ可變域」是指無前導區 (L) 的  TCR δ V (TRDV) 區與TCRδD/J (TRDD/TRDJ) 區的組合，術語 「TCRδ恒定結構域」是指細胞外TRDC區域，或 C-末端截短 TRDC 序列。Relative to the TCR of γ/δ, the term "TCRγ variable domain" as used herein refers to the combination of the TCR γ V (TRGV) region and the TCR γ (TRGJ) region without the leader (L). The term TCR γ constant structure Domain refers to the extracellular TRGC region, or C-terminal truncated TRGC sequence. Similarly, “TCRδ variable domain” refers to the combination of TCR δ V (TRDV) region without leader region (L) and TCRδD/J (TRDD/TRDJ) region. The term “TCRδ constant domain” refers to the extracellular TRDC Region, or C-terminal truncated TRDC sequence.

本說明書的TCR優選結合至肽HLA分子複合體，其具有約 100 µM或更小、約 50 µM或更小、約 25 µM或更小或約 10 µM或更小的結合親和力 (KD)。更為優選的情況是具有約 1µM或更小、約100 nM或更小、約50nM 或更小或約 25nM或更小結合親和力的高親和力TCR。本發明TCR優選結合親和力範圍的非限制性示例包括約 1 nM 至約 10 nM；約 10 nM 至約 20 nM；約 20 nM 至約 30 nM；約 30 nM 至約 40 nM；約 40 nM 至約 50 nM；約 50 nM 至約 60 nM；約 60 nM 至約 70 nM；約 70 nM 至約 80 nM；約 80 nM 至約 90 nM；以及約 90 nM 至約 100 nM。The TCR of the present specification preferably binds to a peptide HLA molecular complex having a binding affinity (KD) of about 100 µM or less, about 50 µM or less, about 25 µM or less, or about 10 µM or less. A more preferred case is a high-affinity TCR having a binding affinity of about 1 µM or less, about 100 nM or less, about 50 nM or less, or about 25 nM or less. Non-limiting examples of preferred binding affinity ranges of the TCR of the present invention include about 1 nM to about 10 nM; about 10 nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM; about 40 nM to about 40 nM; 50 nM; about 50 nM to about 60 nM; about 60 nM to about 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; and about 90 nM to about 100 nM.

與本說明書 TCR 相關，本文使用的「特異性結合」及其語法變體用於表示對 100μM 或更小的肽-HLA 分子複合體有結合親和力 (KD) 的 TCR。In relation to TCR in this specification, "specific binding" and its grammatical variants used herein are used to mean TCR with binding affinity (KD) for peptide-HLA molecular complexes of 100μM or less.

本說明書的 α/β 異二聚體 TCR可能具有其恒定結構域之間的引入二硫鍵。這種類型的優選 TCR 包括那些具有一個 TRAC 恒定域序列和  TRBC1 或 TRBC2 恒定域序列的 TCR，除非 TRAC 的蘇氨酸 48 和 TRBC1 或 TRBC2 的絲氨酸 57被半胱氨酸殘基取代，所述半胱氨酸形成 TRAC 恒定域序列和 TCR 的 TRBC1 或 TRBC2 恒定區序列之間的二硫鍵。The α/β heterodimer TCR of this specification may have introduced disulfide bonds between its constant domains. Preferred TCRs of this type include those with a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, unless TRAC's threonine 48 and TRBC1 or TRBC2's serine 57 are replaced by cysteine residues. Cystine forms a disulfide bond between the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of TCR.

不論具有或不具有上述的引入鏈間鍵，本說明書的α/β雜二聚體TCR可能具有一個TRAC恒定域序列和一個TRBC1或TRBC2恒定結構域序列，並且TRAC恒定結構域序列和TCR的TRBC1或TRBC2恒定結構域序列可能透過TRAC外顯子2的Cys4和TRBC1或TRBC2外顯子2的Cys4之間的天然二硫鍵相連。Regardless of having or not having the above-mentioned introduced inter-chain bond, the α/β heterodimer TCR of this specification may have a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, and the TRAC constant domain sequence and TCR TRBC1 Or the TRBC2 constant domain sequence may be connected by a natural disulfide bond between Cys4 in TRAC exon 2 and Cys4 in TRBC1 or TRBC2 exon 2.

本說明書的 TCR 可能包括選自由放射性核素、螢光團和生物素組成組中的可檢測標記。本說明書的 TCR可能共軛至治療活性劑，如放射性核素、化學治療劑或毒素。The TCR in this specification may include a detectable label selected from the group consisting of radionuclides, fluorophores, and biotin. The TCR of this specification may be conjugated to a therapeutically active agent, such as a radionuclide, a chemotherapeutic agent, or a toxin.

在一個實施方案中，具有在 α 鏈中至少一個突變和/或具有在 β鏈中至少一個突變的 TCR 與未突變 TCR 相比，已經修改了糖基化。In one embodiment, a TCR with at least one mutation in the alpha chain and/or with at least one mutation in the beta chain has modified glycosylation compared to an unmutated TCR.

在一個實施方案中，在 TCR α 鏈和/或 TCR β鏈中包括至少一個突變的 TCR 對肽 HLA 分子複合體有結合親和力和/或結合半衰期，其是包含未突變 TCR α 鏈和/或未突變 TCR β鏈的 TCR 的結合親和力的至少兩倍。腫瘤特異性 TCR 親和力增強及其開發依賴於存在最佳 TCR 親和力的窗口。這樣窗口的存在是根據觀察結果：HLA-A2 限制性病原體特異性 TCR 與 HLA-A2 限制性腫瘤相關自身抗原特異性 TCR 相比， KD 值通常大約低 10 倍。現已知，儘管腫瘤抗原可能具有免疫原性，但是因為腫瘤來自個體自身的細胞，因此僅突變蛋白質或翻譯加工改變的蛋白將被免疫系統視為外來物質。上調或過度表達（所謂的自體抗原）的抗原不一定誘導針對腫瘤的功能免疫應答：表達對這些抗原具有高度反應性的TCR的T細胞會在一種稱為中樞耐受的程序中在胸腺內被不利選擇，也就是說只有對自身抗原具有低親和力TCR的細胞才仍然存在。因此，本說明書的 TCR或變體對肽的親和力可透過本領域熟知的方法來增強。In one embodiment, the TCR that includes at least one mutation in the TCR α chain and/or TCR β chain has binding affinity and/or binding half-life for the peptide HLA molecule complex, which is comprised of an unmutated TCR α chain and/or non-mutated TCR α chain. The binding affinity of the TCR of the mutant TCR β chain is at least twice that of. The enhancement of tumor-specific TCR affinity and its development depend on the existence of the optimal TCR affinity window. The existence of such a window is based on the observation that the KD value of HLA-A2-restricted pathogen-specific TCR and HLA-A2-restricted tumor-associated autoantigen-specific TCR is usually about 10 times lower. It is now known that although tumor antigens may be immunogenic, because tumors come from the individual’s own cells, only mutant proteins or proteins with altered translation processing will be treated as foreign substances by the immune system. Up-regulated or over-expressed (so-called self-antigens) antigens do not necessarily induce a functional immune response against tumors: T cells expressing TCRs that are highly responsive to these antigens will be in the thymus in a procedure called central tolerance Unfavorable selection, that is, only cells with low affinity TCR for self-antigens still exist. Therefore, the affinity of the TCR or variants of the present specification to peptides can be enhanced by methods well known in the art.

本說明書還涉及一種識別和分離本發明 TCR 的一種方法，所述方法包括：用A2/肽單體從 HLA-A*02  陰性健康供體孵育PBMC，用四聚體-藻紅蛋白(PE)孵育PBMC並透過螢光啟動細胞分選 (FACS)–Calibur方法分析分離高親和力 T細胞。This specification also relates to a method for identifying and isolating the TCR of the present invention. The method includes: incubating PBMC from HLA-A*02 negative healthy donors with A2/peptide monomer, and using tetramer-phycoerythrin (PE) Incubate PBMC and analyze and isolate high-affinity T cells by fluorescence-activated cell sorting (FACS)-Calibur method.

本說明書還涉及一種識別和分離本發明 TCR 的一種方法，所述方法包括：獲得含整個人體 TCRαβ 基因位點 (1.1 and 0.7 Mb) 的轉基因小鼠（其 T細胞表達多樣化人類 TCR，用於補償小鼠 TCR 缺乏），用一種肽對小鼠進行免疫處理，用四聚體 - 藻紅蛋白 (PE) 孵育從轉基因小鼠中獲得的PBMC，並透過螢光啟動細胞分選 (FACS) – Calibur方法分析分離高親和力 T細胞。This specification also relates to a method for identifying and isolating the TCR of the present invention, the method comprising: obtaining a transgenic mouse containing the entire human TCRαβ gene locus (1.1 and 0.7 Mb) (its T cells express diversified human TCR, for To compensate for the lack of TCR in mice), the mice are immunized with a peptide, PBMC obtained from transgenic mice are incubated with tetramer-phycoerythrin (PE), and cell sorting (FACS) is activated by fluorescence- The Calibur method analyzes and isolates high-affinity T cells.

一方面，為了獲得表達本說明書TCR的T細胞，編碼本說明書TCR-α和/或TCR-β鏈的核酸被克隆入表達載體，諸如γ反轉錄病毒或慢病毒。重組病毒產生，然後測試功能，如抗原專一性和功能性親合力。然後，最終產品的等分試樣被用於轉導靶T細胞群體（一般純化自患者的PBMC），在輸入患者前展開。On the one hand, in order to obtain T cells expressing the TCR of the specification, the nucleic acid encoding the TCR-α and/or TCR-β chain of the specification is cloned into an expression vector, such as a γ retrovirus or a lentivirus. The recombinant virus is produced and then tested for functions such as antigen specificity and functional affinity. Then, an aliquot of the final product is used to transduce the target T cell population (usually purified from the patient's PBMC), which is unfolded before being imported into the patient.

另一方面，為了獲得表達本說明書TCR的T細胞，TCR RNA透過本領域中已知的技術（例如，體外轉錄系統）合成。然後，體外合成的TCR RNA透過電穿孔來重新表達腫瘤特異性 TCR-α和/或TCR-β鏈被引入獲得自健康供體的初級CD8+ T細胞。On the other hand, in order to obtain T cells expressing the TCR of the present specification, TCR RNA is synthesized by a technique known in the art (for example, an in vitro transcription system). Then, the TCR RNA synthesized in vitro is electroporated to re-express the tumor-specific TCR-α and/or TCR-β chain and is introduced into primary CD8+ T cells obtained from healthy donors.

為了增加表達，編碼本說明書 TCR 的核酸在操作上可連接到強啟動子，例如逆轉錄病毒長末端重複序列 (LTR)、巨細胞病毒 (CMV)、鼠幹細胞病毒 (MSCV) U3、磷酸甘油酸激酶 (PGK)、β 肌動蛋白、泛素蛋白和猿猴病毒 40 (SV40)/CD43複合啟動子、延伸因子 (EF) -1a和脾臟病灶形成病毒 (SFFV) 啟動子。在一優選實施方案中，啟動子與被表達的核酸異源。除了強啟動子外，本說明書的 TCR 表達盒可能含有附加的元素，可提高轉基因表達，包括中樞多聚嘌呤區 (CPPT)， 其促進了慢病毒構建體的核易位 (Follenzi et al., 2000)， 和土撥鼠肝炎病毒轉錄後調控元素 (WPRE)， 其透過提高 RNA 穩定性增加轉基因表達水準 (Zufferey et al., 1999)。In order to increase expression, the nucleic acid encoding the TCR of this specification can be operatively linked to a strong promoter, such as retrovirus long terminal repeat (LTR), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate Kinase (PGK), β-actin, ubiquitin protein and simian virus 40 (SV40)/CD43 composite promoter, elongation factor (EF) -1a and spleen foci forming virus (SFFV) promoter. In a preferred embodiment, the promoter is heterologous to the nucleic acid being expressed. In addition to strong promoters, the TCR expression cassette of this specification may contain additional elements that can increase transgene expression, including the central polypurine region (CPPT), which promotes nuclear translocation of lentiviral constructs (Follenzi et al., 2000), and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which increases transgene expression levels by improving RNA stability (Zufferey et al., 1999).

本發明TCR的α和β鏈可由位於分開的載體核酸進行編碼，或者可透過位於同一載體的多核苷酸編碼。The alpha and beta chains of the TCR of the present invention can be encoded by separate vector nucleic acids, or can be encoded by polynucleotides located on the same vector.

實現高水準的TCR表面表達需要引入TCR的TCR-α和TCR-β鏈高水準轉錄。為了實現它，本說明書的TCR-α和TCR-β鏈可在單一的載體中被克隆入雙順反子構建體，其已被證明能夠克服這一障礙。使用TCR-α和TCR-β鏈在之間的病毒核糖體間進入位元點(IRES)導致兩鏈的協同表達，因為TCR-α和TCR-β鏈均由在翻譯過程中分成兩個蛋白質的單一轉錄物產生，從而確保了產生TCR-α和TCR-β鏈的相等摩爾比(Schmitt et al. 2009)。Achieving high-level TCR surface expression requires the introduction of high-level transcription of TCR-α and TCR-β chains. To achieve this, the TCR-α and TCR-β chains of this specification can be cloned into a bicistronic construct in a single vector, which has been shown to overcome this obstacle. The use of TCR-α and TCR-β chains in the viral inter-ribosome entry site (IRES) between the two chains leads to the coordinated expression of the two chains, because both TCR-α and TCR-β chains are divided into two proteins during translation A single transcript is produced, thereby ensuring the production of equal molar ratios of TCR-α and TCR-β chains (Schmitt et al. 2009).

編碼本說明書TCR的核酸可以是被優化以從宿主細胞增加表達的密碼子。遺傳密碼冗餘讓一些氨基酸被一個以上的密碼子編碼，但某些密碼子沒有其他密碼子「優化」，因為匹配tRNA以及其他因子的相對可用性 (Gustafsson et al., 2004)。修改TCR-α和TCR-β基因序列使得每個氨基酸被用於哺乳動物基因表達的最佳密碼子編碼，以及消除mRNA不穩定性基序或隱蔽剪接位元點，已顯示可顯著提高TCR-α和TCR-β基因表達 (Scholten et al., 2006)。The nucleic acid encoding the TCR of the present specification may be a codon optimized to increase expression from the host cell. The redundancy of the genetic code allows some amino acids to be encoded by more than one codon, but some codons are not "optimized" by other codons because of the relative availability of matching tRNAs and other factors (Gustafsson et al., 2004). Modifying the TCR-α and TCR-β gene sequences so that each amino acid is used to encode the best codons for mammalian gene expression, as well as eliminating mRNA instability motifs or cryptic splice sites, has been shown to significantly increase TCR- α and TCR-β gene expression (Scholten et al., 2006).

此外，引入的和內源性TCR鏈之間的錯配可能會導致獲得特異性，其構成自身免疫的顯著風險。例如，混合TCR二聚體的形成可能會減少可用以形成正確配對TCR複合體的CD3分子數目，因此，可以顯著降低表達所引入TCR的細胞的功能性親合力 (Kuball et al., 2007)。In addition, mismatches between the introduced and endogenous TCR chains may lead to gaining specificity, which constitutes a significant risk of autoimmunity. For example, the formation of mixed TCR dimers may reduce the number of CD3 molecules that can be used to form the correct paired TCR complex, and therefore, can significantly reduce the functional affinity of cells expressing the introduced TCR (Kuball et al., 2007).

為了減少錯配，本說明書引入的TCR鏈的C-末端結構域可以進行修改以促進鏈間親和力，同時降低引入鏈與內源TCR配對的能力。這些策略可能包括用鼠配對物取代人類TCR-α和TCR-βC端結構域（鼠化C端結構域）；透過引入第二個半胱氨酸殘基到引入TCR的TCR-α和TCR-β鏈產生C末端結構域的第二個鏈間二硫鍵（半胱氨酸修飾）；交換TCR-α和TCR-β鏈C端結構域的相互作用殘基（「杵臼結構」）；直接融合TCR-α和TCR-β鏈可變結構域至CD3ζ（CD3ζ融合）(Schmitt et al. 2009)。In order to reduce mismatches, the C-terminal domain of the TCR chain introduced in this specification can be modified to promote inter-chain affinity while reducing the ability of the introduced chain to pair with the endogenous TCR. These strategies may include replacing the C-terminal domains of human TCR-α and TCR-β with mouse counterparts (murine C-terminal domains); by introducing a second cysteine residue into the TCR-α and TCR-β The β chain generates the second interchain disulfide bond (cysteine modification) of the C-terminal domain; exchanges the interacting residues of the C-terminal domain of the TCR-α and TCR-β chains (“knob and socket structure”); direct Fusion of TCR-α and TCR-β chain variable domains to CD3ζ (CD3ζ fusion) (Schmitt et al. 2009).

在一實施方案中，宿主細胞被改變結構以表達本說明書的TCR。在一優選實施方案中，宿主細胞為人T細胞或 T細胞祖細胞。在一些實施方案中，T細胞或T細胞祖細胞從癌症患者中獲得。在另一些實施方案中，T細胞或T細胞祖細胞從健康供體中獲得。本說明書的宿主細胞相對於待治療的患者可以為同種異體或自體的。在一實施方案中，宿主是被轉化以表達α/β TCR的γ/δ T細胞。In one embodiment, the host cell is modified in structure to express the TCR of the present specification. In a preferred embodiment, the host cell is a human T cell or T cell progenitor cell. In some embodiments, T cells or T cell progenitor cells are obtained from cancer patients. In other embodiments, T cells or T cell progenitor cells are obtained from healthy donors. The host cell of the present specification can be allogeneic or autologous relative to the patient to be treated. In one embodiment, the host is a gamma/delta T cell transformed to express alpha/beta TCR.

「藥物組合物」是指適合在醫療機構用於人體的組合物。優選地，藥物組合物為無菌狀態，並根據 GMP 指南生產。"Pharmaceutical composition" refers to a composition suitable for use on the human body in a medical institution. Preferably, the pharmaceutical composition is sterile and produced according to GMP guidelines.

藥物組合物包括游離形式或以一種藥用鹽形式存在的肽（也參見上文）。此處使用的「藥用鹽」系指所公開的肽的一種衍生物，其中該肽由制酸或藥劑的堿鹽進行改性。例如，用與適合的酸反應的游離堿（通常其中的中性藥物有一個中性–NH2 基團）製備酸式鹽。適合製備酸鹽的酸包括有機酸，如：乙酸、丙酸、羥基酸、丙酮酸、草酸、蘋果酸、丙二酸、丁二酸、馬來酸、富馬酸、酒石酸、檸檬酸、苯甲酸酸、肉桂酸、扁桃酸、甲磺酸、甲磺酸、苯磺酸、水楊酸等等、以及無機酸，如：鹽酸、氫溴酸、硫酸、硝酸和磷酸等。相反，可在一種肽上表現的酸性基團的堿鹽製劑使用藥用堿基進行製備，如氫氧化鈉、氫氧化鉀、氫氧化銨、氫氧化鈣、三甲胺等等。The pharmaceutical composition includes the peptide in free form or in the form of a pharmaceutically acceptable salt (see also above). "Pharmaceutical salt" as used herein refers to a derivative of the disclosed peptide, wherein the peptide is modified by an antacid or a pharmaceutical salt. For example, the acid salt is prepared with free radicals (usually the neutral drug in which the neutral drug has a neutral -NH2 group) reacted with a suitable acid. Acids suitable for the preparation of acid salts include organic acids such as acetic acid, propionic acid, hydroxy acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzene Formic acid, cinnamic acid, mandelic acid, methanesulfonic acid, methanesulfonic acid, benzenesulfonic acid, salicylic acid, etc., as well as inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid. On the contrary, the preparation of a salt of an acidic group that can be expressed on a peptide is prepared using a pharmaceutically acceptable group, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like.

在特別優選的實施方案中，藥物組合物包括乙酸（醋酸鹽），三氟乙酸鹽或鹽酸（氯化物）形式的肽。In a particularly preferred embodiment, the pharmaceutical composition includes the peptide in the form of acetic acid (acetate), trifluoroacetate or hydrochloric acid (chloride).

本發明中所述的藥劑優選為一種免疫治療藥劑，例如，一種疫苗。該疫苗可直接給到患者的受影響器官，也可i.d.、i.m.、s.c.、i.p. 和 i.v. 注射方式全身給藥，或體外應用到來自患者或其細胞株的細胞（隨後再將這些細胞注入到患者中），或體外用於從來自患者的免疫細胞的一個細胞亞群（然後再將細胞重新給予患者）。如果核酸體外注入細胞，可能有益於細胞轉染，以共同表達免疫刺激細胞因子（如白細胞介素-2）。肽可完全單獨給藥，也可與免疫刺激佐劑相結合（見下文）、或與免疫刺激細胞因子聯合使用、或以適當的輸送系統給藥（例如脂質體）。該肽也可共軛形成一種合適的載體（如鑰孔蟲戚血藍蛋白 (KLH) 或甘露）到合適的載體 （參閱WO 95/18145及(Longenecker et al., 1993)）。肽也可能被標記，可能是融合蛋白，或可能是雜交分子。在本發明中給出序列的肽預計能刺激 CD4 或 CD8 T細胞。然而，在有 CD4 T-輔助細胞的幫助時，CD8  T細胞刺激更加有效。因此，對於刺激 CD8 T細胞的 MHC-I類表位，一種雜合分子的融合夥伴或片段提供了刺激 CD4 陽性 T細胞的適當表位。CD4- 和 CD8 刺激表位為本領域所熟知、並包括本發明中確定的表位。The agent described in the present invention is preferably an immunotherapeutic agent, for example, a vaccine. The vaccine can be directly administered to the affected organs of the patient, or systemically administered by id, im, sc, ip, and iv injections, or applied to cells from the patient or its cell line in vitro (these cells are then injected into the patient) Middle), or a subpopulation of cells used in vitro from immune cells from a patient (the cells are then re-administered to the patient). If nucleic acid is injected into cells in vitro, it may be beneficial for cell transfection to co-express immunostimulatory cytokines (such as interleukin-2). Peptides can be administered alone, combined with immunostimulatory adjuvants (see below), or combined with immunostimulatory cytokines, or administered with an appropriate delivery system (for example, liposomes). The peptide can also be conjugated to form a suitable carrier (such as keyhole limpet hemocyanin (KLH) or manna) to a suitable carrier (see WO 95/18145 and (Longenecker et al., 1993)). The peptide may also be labeled, it may be a fusion protein, or it may be a hybrid molecule. The peptides of the sequence given in the present invention are expected to stimulate CD4 or CD8 T cells. However, with the help of CD4 T-helper cells, CD8 T cell stimulation is more effective. Therefore, for MHC class I epitopes that stimulate CD8 T cells, a fusion partner or fragment of a hybrid molecule provides an appropriate epitope to stimulate CD4-positive T cells. CD4- and CD8 stimulating epitopes are well known in the art and include the epitopes identified in the present invention.

一方面，疫苗包括至少含有SEQ ID NO:1至SEQ ID NO:385中提出的一種肽以及至少另外一種肽，優選為 2至50 個、更優選為 2至25 個、再優選為 2至20 個、最優選為 2、3、4、5、6、7、8、9、10、11、12 、13、14、15、16、17 或 18 個肽。肽可能從一個或多個特定 TAA 中衍生，並且可能與 MHC I類分子結合。In one aspect, the vaccine includes at least one peptide set forth in SEQ ID NO: 1 to SEQ ID NO: 385 and at least another peptide, preferably 2 to 50, more preferably 2 to 25, even more preferably 2 to 20. One, most preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 peptides. Peptides may be derived from one or more specific TAAs and may bind to MHC class I molecules.

另一方面，本發明提出了一種編碼本發明中肽或肽變體的核酸（如多聚核苷酸）。多聚核苷酸可能為，例如，DNA、cDNA、PNA、RNA或其組合物，它們可為單鏈和/或雙鏈、或多聚核苷酸的原生或穩定形式（如：具有硫代磷酸骨架的多聚核苷酸），並且只要它編碼肽，就可能包含也可能不包含內含子。當然，多聚核苷酸只能編碼加入天然肽鍵並含有天然氨基酸殘基的肽。另一個方面，本發明提出了一種可根據本發明表達多肽的表達載體。On the other hand, the present invention provides a nucleic acid (such as a polynucleotide) encoding the peptide or peptide variant of the present invention. Polynucleotides may be, for example, DNA, cDNA, PNA, RNA, or a combination thereof, and they may be single-stranded and/or double-stranded, or the native or stable form of polynucleotides (e.g., with thio Phosphate backbone polynucleotide), and as long as it encodes a peptide, it may or may not contain introns. Of course, polynucleotides can only encode peptides that incorporate natural peptide bonds and contain natural amino acid residues. In another aspect, the present invention provides an expression vector that can express polypeptides according to the present invention.

對於連接多核苷酸，已經開發出多種方法，尤其是針對DNA，可透過向載體補充可連接性末端等方法進行連接。例如，可向DNA片段加入補充性均聚物軌道，之後DNA片段被***到載體DNA。然後，透過補充性均聚物尾巴的氫鍵結合，將載體和DNA片段結合，從而形成重組DNA分子。For linking polynucleotides, various methods have been developed, especially for DNA, which can be linked by adding linkable ends to the vector. For example, a complementary homopolymer track can be added to the DNA fragment, after which the DNA fragment is inserted into the vector DNA. Then, through the hydrogen bonding of the complementary homopolymer tail, the vector and the DNA fragment are combined to form a recombinant DNA molecule.

含有一個或多個酶切位點的合成接頭為 DNA 片段與載體連接提供了另一種方法。含各種限制性核酸內切酶的合成接頭可透過多種管道購得，其中包括從國際生物技術公司（International Biotechnologies Inc, New Haven, CN, 美國）購得。Synthetic linkers containing one or more restriction sites provide an alternative method for linking DNA fragments to vectors. Synthetic linkers containing various restriction endonucleases can be purchased through a variety of channels, including from International Biotechnologies Inc. (International Biotechnologies Inc, New Haven, CN, USA).

編碼本發明多肽的 DNA 理想修飾方法是使用 Saiki 等人 (Saiki et al., 1988) 所採用的聚合酶鏈反應方法。此方法可用於將 DNA 引入合適的載體（例如，透過設計合適的酶切位點），也可用於本領域已知的其他有用方法修飾 DNA。如果使用病毒載體，痘病毒載體或腺病毒載體為優選。The ideal modification method for DNA encoding the polypeptide of the present invention is to use the polymerase chain reaction method adopted by Saiki et al. (Saiki et al., 1988). This method can be used to introduce DNA into a suitable vector (for example, by designing a suitable restriction site), and it can also be used in other useful methods known in the art to modify DNA. If a viral vector is used, a poxvirus vector or an adenovirus vector is preferred.

之後，DNA （或在逆轉錄病毒載體情況下，RNA）可能表達於合適的宿主，從而製成含本發明肽或變體的多肽。因此，可根據已知技術使用編碼本發明肽或變體的 DNA，用本文所述方法適當修飾後，構建表達載體，然後表達載體用於轉化合適宿主細胞，從而表達和產生本發明中的多肽。此類技術包括那些公開於，例如，美國專利 4,440,859、4,530,901、4,582,800、4,677,063、4,678,751、4,704,362、4,710,463、4,757,006、4,766,075 和 4,810,648。Thereafter, DNA (or RNA in the case of retroviral vectors) may be expressed in a suitable host, thereby making a polypeptide containing the peptide or variant of the present invention. Therefore, the DNA encoding the peptide or variant of the present invention can be used according to known techniques, after being appropriately modified by the methods described herein, an expression vector can be constructed, and then the expression vector can be used to transform a suitable host cell to express and produce the polypeptide of the present invention. . Such techniques include those disclosed in, for example, U.S. Patent Nos. 4,440,859, 4,530,901, 4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648.

編碼含本發明化合物多肽的 DNA （或在逆轉錄病毒載體情況下，RNA）可能被加入到其他多種 DNA 序列，從而引入到合適的宿主中。同伴 DNA 將取決於宿主的性質、DNA 引入宿主的方式、以及是否需要保持為游離體還是要相互結合。The DNA (or RNA in the case of retroviral vectors) encoding the polypeptide containing the compound of the present invention may be added to various other DNA sequences to be introduced into a suitable host. Companion DNA will depend on the nature of the host, the way the DNA is introduced into the host, and whether it needs to be kept as an episome or combined with each other.

一般來說，DNA 可以適當的方向和正確的表達閱讀框架附著到一種表達載體（如質粒）中。如有必要，該 DNA 可能與所需宿主所識別的相應轉錄和翻譯調節控制核苷酸序列連接，儘管表達載體中一般存在此類控制功能。然後，該載體透過標準方法被引入宿主。一般來說，並不是所有的宿主都會被載體轉化。因此，有必要選擇轉化過的宿主細胞。選擇方法包括用任何必要的控制元素向表達載體***一個 DNA 序列，該序列對轉化細胞中的可選擇性屬性（如抗生素耐藥性）進行編碼。Generally speaking, DNA can be attached to an expression vector (such as a plasmid) in the proper orientation and the correct reading frame for expression. If necessary, the DNA may be linked to the corresponding transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such control functions generally exist in expression vectors. Then, the vector is introduced into the host by standard methods. Generally speaking, not all hosts will be transformed by the vector. Therefore, it is necessary to select transformed host cells. The selection method involves inserting a DNA sequence into the expression vector with any necessary control elements, which encodes a selectable attribute (such as antibiotic resistance) in the transformed cell.

另外，有這種選擇屬性的基因可在另外一個載體上，該載體用來協同轉化所需的宿主細胞。In addition, genes with such selective properties can be on another vector, which is used to co-transform the desired host cells.

然後，本發明中的重組 DNA 所轉化的宿主細胞在本文中所述本領域技術人員熟悉的合適條件下培養足夠長的時間，從而表達之後可回收的肽。Then, the host cell transformed by the recombinant DNA of the present invention is cultured for a sufficient period of time under suitable conditions familiar to those skilled in the art as described herein, so as to express the peptide that can be recovered afterwards.

有許多已知的表達系統，包括細菌（如大腸桿菌和枯草芽孢桿菌）、酵母（如酵母菌）、絲狀真菌（如曲黴菌）、植物細胞、動物細胞及昆蟲細胞。該系統可優選為哺乳動物細胞，如來自 ATCC 細胞生物學庫 (Cell Biology Collection) 中的 CHO 細胞。There are many known expression systems, including bacteria (such as E. coli and Bacillus subtilis), yeast (such as yeast), filamentous fungi (such as Aspergillus), plant cells, animal cells, and insect cells. The system may preferably be mammalian cells, such as CHO cells from the ATCC Cell Biology Collection.

典型的哺乳動物細胞組成型表達載體質粒包括 CMV 或含一個合適的多聚 A 尾巴的 SV40 啟動子以及抗性標誌物（如新黴素）。一個實例為從 Pharmacia 公司（Piscataway，新澤西，美國）獲得的 pSVL。一種可誘導型哺乳動物表達載體的例子是 pMSG，也可以從 Pharmacia 公司獲得。有用的酵母質粒載體是 pRS403-406 和 pRS413-416，一般可從 Stratagene Cloning Systems 公司（La Jolla, CA 92037，美國）獲得。質粒 pRS403、pRS404、pRS405 和 pRS406 是酵母整合型質粒 (YIp)，並***了酵母可選擇性標記物 HIS3、TRP1、LEU2 和 URA3。pRS413-416 質粒為酵母著絲粒質粒 (Ycp)。基於 CMV 啟動子的載體（如，來自於 Sigma-Aldrich 公司）提供了暫態或穩定的表達、胞漿表達或分泌，以及 FLAG、3xFLAG、c-myc 或 MATN 不同組合物中的 N-端或 C-端標記。這些融合蛋白可用於檢測、純化及分析重組蛋白。雙標融合為檢測提供了靈活性。Typical mammalian cell constitutive expression vector plasmids include CMV or SV40 promoter with a suitable poly A tail and resistance markers (such as neomycin). An example is pSVL obtained from Pharmacia (Piscataway, New Jersey, USA). An example of an inducible mammalian expression vector is pMSG, which is also available from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416, which are generally available from Stratagene Cloning Systems (La Jolla, CA 92037, USA). Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integrative plasmids (YIp), and have inserted yeast selectable markers HIS3, TRP1, LEU2 and URA3. The pRS413-416 plasmid is a yeast centromere plasmid (Ycp). Vectors based on the CMV promoter (for example, from Sigma-Aldrich) provide transient or stable expression, cytoplasmic expression or secretion, as well as the N-terminal or N-terminal in different combinations of FLAG, 3xFLAG, c-myc or MATN. C-terminal mark. These fusion proteins can be used for detection, purification and analysis of recombinant proteins. The dual-standard fusion provides flexibility for detection.

強勁的人巨細胞病毒 (CMV) 啟動子調控區使得 COS 細胞中的組成蛋白表達水準高達 1 mg/L。對於較弱的細胞株，蛋白水準一般低於 0.1 mg/L。SV40 複製原點的出現將導致 DNA 在 SV40 複製容納性 COS 細胞中高水準複製。例如，CMV 載體可包含細菌細胞中的 pMB1（pBR322 的衍生物）複製原點、細菌中進行氨苄青黴素抗性選育的 鈣-內醯胺酶基因、hGH polyA 和 f1 的原點。含前胰島素原引導 (PPT) 序列的載體可使用抗 FLAG 抗體、樹脂和板引導 FLAG 融合蛋白分泌到進行純化的培養基中。其他與各種宿主細胞一起應用的載體和表達系統是本領域熟知眾所周知的。The powerful human cytomegalovirus (CMV) promoter regulatory region makes the expression level of constituent proteins in COS cells up to 1 mg/L. For weaker cell lines, the protein level is generally less than 0.1 mg/L. The emergence of SV40 replication origin will lead to high-level replication of DNA in SV40 replication-accommodating COS cells. For example, a CMV vector can contain the origin of replication of pMB1 (a derivative of pBR322) in bacterial cells, the origin of the calcium-endominidase gene for ampicillin resistance breeding in bacteria, and the origin of hGH polyA and f1. The vector containing the preproinsulin guide (PPT) sequence can use anti-FLAG antibodies, resins, and plates to guide the FLAG fusion protein secreted into the purified medium. Other vectors and expression systems used with various host cells are well known in the art.

在另一個實施方案中，對本發明的兩個或更多的肽或肽變體進行編碼，因此，以一個連續順序（類似於「一串珠子」的構建體）表達。在達到目標，所述肽或肽變體可能透過連接子氨基酸的延伸處（例如LLLLLL）連接或融合一起，也可能他們之間沒有任何附加的肽而被連接。這些構建體也可用於癌症治療，可誘導涉及 MHC I 和 MHC II類分子的免疫應答。In another embodiment, two or more peptides or peptide variants of the invention are encoded and therefore expressed in a continuous sequence (similar to a "string of beads" construct). To achieve the goal, the peptides or peptide variants may be connected or fused together through the extension of the linker amino acid (such as LLLLLL), or they may be connected without any additional peptides between them. These constructs can also be used in cancer therapy, and can induce immune responses involving MHC I and MHC class II molecules.

本發明還涉及一種宿主細胞，其以本發明的多核苷酸載體構建轉化而來。宿主細胞可為原核細胞，也可為真核細胞。在有些情況下，細菌細胞為優選原核宿主細胞，典型為大腸桿菌株，例如，大腸桿菌菌株 DH5（從 Bethesda Research Laboratories 公司（Bethesda, MD, 美國）獲得）和 RR1（從美國菌種保藏中心（ATCC, Rockville, MD, 美國），ATCC 編號31343 獲得）。首選的真核宿主細胞包括酵母、昆蟲和哺乳動物細胞，優選為脊椎動物細胞，如：小鼠、大鼠、猴子或人成纖維細胞和結腸癌細胞株中的細胞。酵母宿主細胞包括 YPH499、YPH500 和 YPH501，一般可從 Stratagene Cloning Systems 公司（La Jolla, CA 92037, 美國）獲得。首選哺乳動物宿主細胞包括中國倉鼠卵巢 (CHO) 細胞為 ATCC 中的 CCL61 細胞、NIH 瑞士小鼠胚胎細胞 NIH/3T3 為 ATCC 中的 CRL 1658 細胞、猴腎源性 COS-1 細胞為 ATCC 中的 CRL 1650 細胞以及人胚胎腎細胞的 293 號細胞。首選昆蟲細胞為 Sf9 細胞，可用杆狀病毒表達載體轉染。有關針對表達選擇合適宿主細胞的概要，可從教科書 (Paulina Balbás and Argelia Lorence 《Methods in Molecular Biology Recombinant Gene Expression, Reviews and Protocols》Part One, Second Edition, ISBN 978-1-58829-262-9) 和技術人員知道的其他文獻中查到。The present invention also relates to a host cell, which is transformed with the polynucleotide vector construction of the present invention. The host cell can be a prokaryotic cell or a eukaryotic cell. In some cases, bacterial cells are preferred prokaryotic host cells, typically E. coli strains, for example, E. coli strain DH5 (obtained from Bethesda Research Laboratories (Bethesda, MD, USA)) and RR1 (from the American Culture Collection ( ATCC, Rockville, MD, USA), ATCC No. 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells, such as cells in mouse, rat, monkey or human fibroblasts and colon cancer cell lines. Yeast host cells include YPH499, YPH500 and YPH501, which are generally available from Stratagene Cloning Systems (La Jolla, CA 92037, USA). The preferred mammalian host cells include Chinese hamster ovary (CHO) cells as CCL61 cells in ATCC, NIH Swiss mouse embryonic cells NIH/3T3 as CRL 1658 cells in ATCC, and monkey kidney-derived COS-1 cells as CRL in ATCC 1650 cells and 293 cells of human embryonic kidney cells. The first insect cell is Sf9 cell, which can be transfected with baculovirus expression vector. For an overview of selecting a suitable host cell for expression, you can read from the textbook (Paulina Balbás and Argelia Lorence "Methods in Molecular Biology Recombinant Gene Expression, Reviews and Protocols" Part One, Second Edition, ISBN 978-1-58829-262-9) and Find it in other literature known to the technician.

含本發明 DNA 結構的適當宿主細胞的轉化可使用大家熟知的方法完成，通常取決於使用載體的類型。關於原核宿主細胞的轉化，請參見，例如，Cohen 等人的文獻 (Cohen et al., 1972) 和  (Green and Sambrook, 2012)。酵母細胞的轉化在 Sherman 等人的文章 (Sherman et al., 1986) 中進行了描述。Beggs (Beggs, 1978) 中所述的方法也很有用。對於脊椎動物細胞，轉染這些細胞的試劑等，例如，磷酸鈣和 DEAE-葡聚糖或脂質體配方，可從 Stratagene Cloning Systems 公司或 Life Technologies 公司（Gaithersburg, MD 20877，美國）獲得。電穿孔也可用於轉化和/或轉染細胞，是本領域用於轉化酵母細胞、細菌細胞、昆蟲細胞和脊椎動物細胞大家熟知的方法。The transformation of a suitable host cell containing the DNA structure of the present invention can be accomplished using well-known methods, and usually depends on the type of vector used. For the transformation of prokaryotic host cells, see, for example, Cohen et al. (Cohen et al., 1972) and (Green and Sambrook, 2012). The transformation of yeast cells is described in Sherman et al. (Sherman et al., 1986). The method described in Beggs (Beggs, 1978) is also useful. For vertebrate cells, reagents for transfecting these cells, for example, calcium phosphate and DEAE-dextran or liposome formulations can be obtained from Stratagene Cloning Systems or Life Technologies (Gaithersburg, MD 20877, USA). Electroporation can also be used to transform and/or transfect cells, and is a well-known method in the art for transforming yeast cells, bacterial cells, insect cells, and vertebrate cells.

被成功轉化的細胞（即含本發明 DNA 結構的細胞）可用大家熟知的方法（如 PCR）進行識別。另外，上清液存在的蛋白可使用抗體進行檢測。Successfully transformed cells (ie, cells containing the DNA structure of the present invention) can be identified by well-known methods (such as PCR). In addition, the protein present in the supernatant can be detected using antibodies.

應瞭解，本發明中的某些宿主細胞用於製備本發明中的肽，例如細菌細胞、酵母細胞和昆蟲細胞。但是，其他宿主細胞可能對某些治療方法有用。例如，抗原表現細胞（如樹突狀細胞）可用于表達本發明中的肽，使他們可以加載入相應的 MHC 分子中。因此，本發明提出了含本發明中核酸或表達載體的一種宿主細胞。It should be understood that certain host cells in the present invention are used to prepare the peptides in the present invention, such as bacterial cells, yeast cells, and insect cells. However, other host cells may be useful for certain treatment methods. For example, antigen-expressing cells (such as dendritic cells) can be used to express the peptides of the present invention so that they can be loaded into the corresponding MHC molecules. Therefore, the present invention proposes a host cell containing the nucleic acid or expression vector of the present invention.

在一個優選實施方案中，宿主細胞為抗原表現細胞，尤其是樹突狀細胞或抗原表現細胞。2010 年 4 月 29 日，美國食品和藥物管理局 (FDA) 批准載有含攝護腺酸性磷酸酶 (PAP) 的重組融合蛋白可用於治療無症狀或症狀輕微的轉移性 HRPC  (Rini et al., 2006; Small et al., 2006)。In a preferred embodiment, the host cell is an antigen-expressing cell, especially a dendritic cell or an antigen-expressing cell. On April 29, 2010, the U.S. Food and Drug Administration (FDA) approved a recombinant fusion protein containing prostate acid phosphatase (PAP) for the treatment of asymptomatic or mildly symptomatic metastatic HRPC (Rini et al. , 2006; Small et al., 2006).

另一方面，本發明提出了一種配製一種肽及其變體的方法，該方法包括培養宿主細胞和從宿主細胞或其培養基中分離肽。In another aspect, the present invention provides a method for formulating a peptide and a variant thereof, which method includes culturing a host cell and isolating the peptide from the host cell or its culture medium.

在另一個實施方案中，本發明中的肽、核酸或表達載體用於藥物中。例如，肽或其變體可製備為靜脈(i.v.)注射劑、皮下(s.c.)注射劑、皮內(i.d.)注射劑、腹膜內(i.p.)注射劑、肌肉(i.m.)注射劑。肽注射的優選方法包括s.c.、i.d.、i.p.、i.m.和i.v.注射。DNA注射的優選方法為i.d.、i.m.、s.c.、i.p.和i.v.注射。例如，給予50 µg至1.5 mg，優選為125 µg至500 µg的肽或DNA，這取決於具體的肽或DNA。上述劑量範圍在以前的試驗中成功使用 (Walter et al., 2012)。In another embodiment, the peptide, nucleic acid or expression vector of the present invention is used in medicine. For example, peptides or variants thereof can be prepared as intravenous (i.v.) injections, subcutaneous (s.c.) injections, intradermal (i.d.) injections, intraperitoneal (i.p.) injections, intramuscular (i.m.) injections. Preferred methods of peptide injection include s.c., i.d., i.p., i.m. and i.v. injections. The preferred methods of DNA injection are i.d., i.m., s.c., i.p. and i.v. injection. For example, 50 µg to 1.5 mg, preferably 125 µg to 500 µg of peptide or DNA are administered, depending on the specific peptide or DNA. The above dose range has been successfully used in previous trials (Walter et al., 2012).

用於主動免疫接種的多聚核苷酸可為基本純化形式，也可包被於載體或輸送系統。核酸可能為 DNA、cDNA、PNA、RNA，也可能為其組合物。這種核酸的設計和引入方法為本領域所熟知。例如，文獻中有其概述  (Teufel et al., 2005)。多核苷酸疫苗很容易製備，但這些載體誘導免疫反應的作用模式尚未完全瞭解。合適的載體和輸送系統包括病毒 DNA 和/或 RNA，如基於腺病毒、牛痘病毒、逆轉錄病毒、皰疹病毒、腺相關病毒或含一種以上病毒元素的混合病毒的系統。非病毒輸送系統包括陽離子脂質體和陽離子聚合物，是 DNA 輸送所屬領域內熟知的系統。也可使用物理輸送系統，如透過「基因槍」。肽或核酸編碼的肽可以是一種融合蛋白，例如，含刺激 T細胞進行上述 CDR 的表位。The polynucleotide used for active immunization may be in a substantially purified form, or it may be coated in a carrier or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA, or a combination thereof. The design and introduction methods of such nucleic acids are well known in the art. For example, there is an overview in the literature (Teufel et al., 2005). Polynucleotide vaccines are easy to prepare, but the mode of action of these vectors to induce immune responses is not yet fully understood. Suitable vectors and delivery systems include viral DNA and/or RNA, such as systems based on adenovirus, vaccinia virus, retrovirus, herpes virus, adeno-associated virus, or mixed virus containing more than one viral element. Non-viral delivery systems include cationic liposomes and cationic polymers, and are well-known systems in the field of DNA delivery. Physical delivery systems can also be used, such as through a "gene gun". The peptide or the peptide encoded by the nucleic acid may be a fusion protein, for example, containing an epitope that stimulates T cells to perform the above-mentioned CDR.

本發明的藥劑也可能包括一種或多種佐劑。佐劑是那些非特異性地增強或加強免疫反應的物質（例如，透過 CD8-陽性 T細胞和輔助 T(TH) 細胞介導的對一種抗原的免疫應答，因此被視為對本發明的藥劑有用。適合的佐劑包括（但不僅限於）1018ISS、鋁鹽、AMPLIVAX®、AS15、BCG、CP-870,893、CpG7909、CyaA、dSLIM、鞭毛蛋白或鞭毛蛋白衍生的 TLR5 配體、FLT3 配體、GM-CSF、IC30、IC31、咪喹莫特 (ALDARA®)、resiquimod、ImuFact IMP321、白細胞介素 IL-2、IL-13、IL-21、干擾素 α 或 β，或其聚乙二醇衍生物、IS Patch、ISS、ISCOMATRIX、ISCOMs、JuvImmune®、LipoVac、MALP2、MF59、單磷醯脂A、Montanide IMS 1312、Montanide ISA 206、Montanide ISA 50V、Montanide ISA-51、水包油和油包水乳狀液、OK-432、OM-174、OM-197-MP-EC、ONTAK、OspA、PepTel® 載體系統、基於聚丙交酯複合乙交酯 [PLG] 和右旋糖苷微粒、重組人乳鐵傳遞蛋白 SRL172、病毒顆粒和其他病毒樣顆粒、YF-17D、VEGF trap、R848、β-葡聚糖、Pam3Cys、源自皂角苷、分支桿菌提取物和細菌細胞壁合成模擬物的 Aquila 公司的 QS21 刺激子，以及其他專有佐劑，如：Ribi's Detox、Quil 或 Superfos。優選佐劑如：弗氏佐劑或 GM-CSF。前人對一些樹突狀細胞特異性免疫佐劑（如 MF59）及其製備方法進行了描述(Allison and Krummel, 1995)。也可能使用細胞因子。一些細胞因子直接影響樹突狀細胞向淋巴組織遷移（如，TNF-），加速樹突狀細胞成熟為 T 淋巴細胞的有效抗原表現細胞（如，GM-CSF、IL-1 和 IL-4）（美國 5849589 號專利，特別以其完整引用形式併入本文），並充當免疫佐劑（如 IL-12、IL-15、IL-23、IL-7、IFN-α、IFN-β） (Gabrilovich et al., 1996)。The medicament of the present invention may also include one or more adjuvants. Adjuvants are substances that non-specifically enhance or strengthen the immune response (for example, an immune response to an antigen mediated by CD8-positive T cells and helper T (TH) cells, and therefore are considered useful for the medicament of the present invention Suitable adjuvants include (but are not limited to) 1018ISS, aluminum salt, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligand, FLT3 ligand, GM- CSF, IC30, IC31, imiquimod (ALDARA®), resiquimod, ImuFact IMP321, interleukin IL-2, IL-13, IL-21, interferon alpha or beta, or its polyethylene glycol derivatives, IS Patch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59, Monophosphoryl A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, oil-in-water and water-in-oil emulsion Liquid, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® carrier system, based on polylactide complex glycolide [PLG] and dextran microparticles, recombinant human lactoferrin SRL172, virus particles and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulator derived from saponin, mycobacterial extract and bacterial cell wall synthesis mimic , And other proprietary adjuvants, such as Ribi's Detox, Quil or Superfos. Preferred adjuvants such as Freund’s adjuvant or GM-CSF. Predecessors have specific immune adjuvants for some dendritic cells (such as MF59) and The preparation method is described (Allison and Krummel, 1995). Cytokines may also be used. Some cytokines directly affect the migration of dendritic cells to lymphoid tissues (eg, TNF-), accelerating the maturation of dendritic cells into T lymphocytes Effective antigen-expressing cells (eg, GM-CSF, IL-1, and IL-4) (US Patent No. 5849589, specifically incorporated herein by reference in its entirety), and act as immune adjuvants (eg, IL-12, IL-15) , IL-23, IL-7, IFN-α, IFN-β) (Gabrilovich et al., 1996).

據報告，CpG 免疫刺激寡核苷酸可提高佐劑在疫苗中的作用。如果沒有理論的約束， CpG 寡核苷酸可透過 Toll 樣受體 (TLR) （主要為 TLR9）活化先天（非適應性）免疫系統從而起作用。CpG 引發的 TLR9 活化作用提高了對各種抗原的抗原特異性體液和細胞反應，這些抗原包括肽或蛋白抗原、活病毒或被殺死的病毒、樹突狀細胞疫苗、自體細胞疫苗以及預防性和治療性疫苗中的多糖結合物。更重要的是，它會增強樹突狀細胞的成熟和分化，導致 TH1 細胞的活化增強以及細胞毒性 T 淋巴細胞 (CTL) 生成加強，甚至 CD4 T細胞説明的缺失。甚至有疫苗佐劑的存在也能維持 TLR9 活化作用誘發的 TH1 偏移，這些佐劑如：正常促進 TH2 偏移的明礬或弗氏不完全佐劑 (IFA)。CpG 寡核苷酸與以下其他佐劑或配方一起製備或聯合給藥時，表現出更強的佐劑活性，如微粒、納米粒子、脂肪乳或類似製劑，當抗原相對較弱時，這些對誘發強反應尤為必要。他們還能加速免疫反應，使抗原劑量減少約兩個數量級，在有些實驗中，對不含CpG 的全劑量疫苗也能產生類似的抗體反應  (Krieg, 2006)。美國 6406705 B1 號專利對 CpG 寡核苷酸、非核酸佐劑和抗原結合使用促使抗原特異性免疫反應進行了描述。一種 CpG TLR9 拮抗劑為 Mologen 公司（德國柏林）的 dSLIM（雙幹環免疫調節劑），這是本發明藥物組合物的優選成分。也可使用其他如 TLR 結合分子，如：RNA 結合 TLR7、TLR8 和/或 TLR9。It has been reported that CpG immunostimulatory oligonucleotides can improve the effect of adjuvants in vaccines. If not bound by theory, CpG oligonucleotides can activate the innate (non-adaptive) immune system through Toll-like receptors (TLR) (mainly TLR9) to act. The activation of TLR9 triggered by CpG improves the antigen-specific humoral and cellular responses to various antigens, including peptide or protein antigens, live viruses or killed viruses, dendritic cell vaccines, autologous cell vaccines, and preventive And polysaccharide conjugates in therapeutic vaccines. More importantly, it will enhance the maturation and differentiation of dendritic cells, resulting in increased activation of TH1 cells and increased production of cytotoxic T lymphocytes (CTL), and even the absence of CD4 T cell instructions. Even the presence of vaccine adjuvants can maintain TH1 excursions induced by TLR9 activation, such as alum that normally promotes TH2 excursions or Freund's incomplete adjuvant (IFA). When CpG oligonucleotides are prepared or administered in combination with the following other adjuvants or formulations, they exhibit stronger adjuvant activity, such as microparticles, nanoparticles, fat emulsions or similar preparations. When the antigen is relatively weak, these pairs It is especially necessary to induce a strong response. They can also accelerate the immune response and reduce the antigen dose by about two orders of magnitude. In some experiments, a full-dose vaccine without CpG can also produce a similar antibody response (Krieg, 2006). US Patent No. 6406705 B1 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants and antigens to promote antigen-specific immune responses. A CpG TLR9 antagonist is dSLIM (Double Dry Loop Immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other binding molecules such as TLR can also be used, such as RNA binding to TLR7, TLR8 and/or TLR9.

其他有用的佐劑例子包括（但不限於）化學修飾性 CpG （如 CpR、Idera）、dsRNA 模擬物，如，Poly(I:C) 及其衍生物（如：AmpliGen、Hiltonol、多聚-(ICLC)、多聚 (IC-R)、多聚 (I:C12U)）、非 CpG 細菌性 DNA 或 RNA 以及免疫活性小分子和抗體，如：環磷醯胺、舒尼替單抗、貝伐單抗®、西樂葆、NCX-4016、西地那非、他達拉非、伐地那非、索拉非尼、替莫唑胺、temsirolimus、XL-999、CP-547632、帕唑帕尼、VEGF Trap、ZD2171、AZD2171、抗-CTLA4、免疫系統的其他抗體靶向性主要結構（如：抗-CD40、抗-TGFβ、抗-TNFα受體) 和 SC58175，這些藥物都可能有治療作用和/或充當佐劑。技術人員無需過度進行不當實驗就很容易確定本發明中有用的佐劑和添加劑的數量和濃度。Examples of other useful adjuvants include (but are not limited to) chemically modified CpG (such as CpR, Idera), dsRNA mimics, such as Poly(I:C) and its derivatives (such as AmpliGen, Hiltonol, poly-( ICLC), poly(IC-R), poly(I:C12U)), non-CpG bacterial DNA or RNA and immunologically active small molecules and antibodies, such as: cyclophosphamide, sunitizumab, bevac Mab®, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap , ZD2171, AZD2171, anti-CTLA4, other main structures targeted by immune system antibodies (such as: anti-CD40, anti-TGFβ, anti-TNFα receptor) and SC58175, these drugs may have therapeutic effects and/or act as Adjuvant. The skilled person can easily determine the number and concentration of adjuvants and additives useful in the present invention without excessively performing improper experiments.

首選佐劑是抗-CD40、咪喹莫特、瑞喹莫德、GM-CSF、環磷醯胺、舒尼替尼、貝伐單抗、干擾素α、CpG 寡核苷酸及衍生物、多聚(I:C)及衍生物、RNA、西地那非和PLG或病毒顆粒的微粒製劑。The preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon alpha, CpG oligonucleotides and derivatives, Poly(I:C) and derivatives, RNA, sildenafil and PLG or microparticle preparations of virus particles.

本發明藥物組合物的一個優選實施方案中，佐劑從含集落刺激因子製劑中選擇，如粒細胞巨噬細胞集落刺激因子（GM-CSF，沙格司亭）、環磷醯胺、咪喹莫特、resiquimod 和干擾素-α。In a preferred embodiment of the pharmaceutical composition of the present invention, the adjuvant is selected from preparations containing colony stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, and imiquine Mott, resiquimod and interferon-α.

本發明藥物組合物的一個優選實施方案中，佐劑從含集落刺激因子製劑中選擇，如粒細胞巨噬細胞集落刺激因子（GM-CSF，沙格司亭）、環磷醯胺、咪喹莫特和 resimiquimod。在本發明藥物組合物的一個優選實施方案中，佐劑為環磷醯胺、咪喹莫特或 resiquimod。更優選的佐劑是 Montanide IMS 1312、Montanide ISA 206、Montanide ISA 50V、Montanide ISA-51、聚-ICLC (Hiltonol®) 和抗CD40 mAB或其組合物。In a preferred embodiment of the pharmaceutical composition of the present invention, the adjuvant is selected from preparations containing colony stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, and imiquine Mott and resimiquimod. In a preferred embodiment of the pharmaceutical composition of the present invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod. More preferred adjuvants are Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB or combinations thereof.

此組合藥物為非腸道注射使用，如皮下、皮內、肌肉注射，也可口服。為此，肽和其他選擇性分子在藥用載體中分解或懸浮，優選為水載體。此外，組合物可包含輔料，如：緩衝劑、結合劑、衝擊劑、稀釋劑、香料、潤滑劑等。這些肽也可與免疫刺激物質合用，如：細胞因子。可用於此類組合物的更多輔料可在從 A. Kibbe 所著的 Handbook of Pharmaceutical Excipients (Kibbe, 2000) 等書中獲知。此組合藥物可用於阻止、預防和/或治療腺瘤或癌性疾病。例如，EP2112253 中有示例製劑。This combination medicine is used for parenteral injection, such as subcutaneous, intradermal, intramuscular injection, or oral administration. To this end, peptides and other selective molecules are decomposed or suspended in a pharmaceutical carrier, preferably an aqueous carrier. In addition, the composition may contain auxiliary materials, such as buffers, binders, impact agents, diluents, perfumes, lubricants, and the like. These peptides can also be used in combination with immunostimulatory substances, such as cytokines. More excipients that can be used in such compositions can be found in books such as Handbook of Pharmaceutical Excipients (Kibbe, 2000) by A. Kibbe. This combination medicine can be used to prevent, prevent and/or treat adenomas or cancerous diseases. For example, there are example formulations in EP2112253.

重要的是要認識到，透過本發明的疫苗引發的免疫應答在不同的細胞階段和開發的不同階段攻擊癌症。而且不同的癌症相關信號通路被攻擊。這相對於其他疫苗的優勢，這些疫苗只針對一個或幾個靶標，這可能會導致腫瘤很容易適應於攻擊（腫瘤逃逸）。此外，並非所有的個體腫瘤都表達相同模式的抗原。因此，幾個腫瘤相關肽的組合確保了每個腫瘤都承擔至少一些靶標。該組合物以這樣的方式設計，預期每個腫瘤可表達幾種抗原並覆蓋腫瘤生長和維持所需要的幾種獨立的途徑。因此，疫苗可易於「現成的」用於較大患者群體。這意味著，預選擇接受疫苗治療的患者可限制為HLA分型，無需抗原表達的任何額外的生物標誌物評估，但仍然確保多個靶標同時被誘導的免疫應答攻擊，這對於療效很重要(Banchereau et al., 2001; Walter et al., 2012)。It is important to realize that the immune response triggered by the vaccine of the present invention attacks cancer at different cellular stages and at different stages of development. And different cancer-related signaling pathways were attacked. This has advantages over other vaccines, which only target one or a few targets, which may cause tumors to easily adapt to attack (tumor escape). In addition, not all individual tumors express the same pattern of antigens. Therefore, the combination of several tumor-associated peptides ensures that each tumor bears at least some targets. The composition is designed in such a way that each tumor is expected to express several antigens and cover several independent pathways required for tumor growth and maintenance. Therefore, vaccines can be easily "off-the-shelf" for larger patient populations. This means that pre-selected patients for vaccine treatment can be restricted to HLA typing without any additional biomarker evaluation of antigen expression, but still ensure that multiple targets are attacked by the induced immune response at the same time, which is important for efficacy ( Banchereau et al., 2001; Walter et al., 2012).

本文所用的「支架」一詞是指與（如抗原）決定因子特異性結合的分子。在一項實施方案中，支架是能夠引導其所連接的實體（例如，（第二）抗原結合部分） 至目標靶點，例如，至特定類型的腫瘤細胞或承載抗原決定簇的腫瘤基質（如根據目前申請中肽和MHC的複合體）。在另一項實施例中，支架能夠透過其靶抗原（例如T細胞受體複合體抗原）活化信號通路。支架包括但不限於抗體及其片段，抗體的抗原結合區，其包含抗體重鏈可變區和抗體輕鏈可變區，結合的蛋白包括至少一個錨蛋白重複序列基元和單域抗原結合(SDAB)分子、適體、（可溶）TCR和（經修飾的）細胞，例如同種異體或自體T細胞。為了評估某個分子是否是結合至靶點的支架，可進行結合測定。The term "scaffold" as used herein refers to a molecule that specifically binds to (eg, antigen) determinants. In one embodiment, the scaffold is capable of guiding the entity to which it is attached (e.g., (second) antigen binding moiety) to a target site, e.g., to a specific type of tumor cell or tumor matrix that carries antigenic determinants (e.g., According to the complex of peptide and MHC in the current application). In another embodiment, the scaffold can activate the signaling pathway through its target antigen (eg, T cell receptor complex antigen). The scaffold includes but is not limited to antibodies and fragments thereof, the antigen binding region of the antibody, which includes the variable region of the antibody heavy chain and the variable region of the antibody light chain, and the bound protein includes at least one ankyrin repeat sequence motif and single domain antigen binding ( SDAB) molecules, aptamers, (soluble) TCRs and (modified) cells, such as allogeneic or autologous T cells. To assess whether a molecule is a scaffold that binds to the target, a binding assay can be performed.

「特定」結合系指，與其他天然肽-MHC 複合體相比，該支架與感興趣的肽-MHC複合體更好地結合，結合程度為，擁有能夠殺死承載特定靶點細胞的活性分子的支架不能夠殺死無特定靶點但表現一個或多個其他肽-MHC複合體的另一細胞。如果交叉反應性肽-MHC 的肽並不是天然的，即，並非來自人 HLA-多肽組，則結合至其他肽-MHC 複合體是無關緊要的。評估靶細胞殺傷的測試在本領域中是公知的。它們應該含有未改變的肽-MHC 表現的靶細胞（原發細胞或細胞系）或載有肽的細胞進行，以便達到天然肽-MHC 的水準。"Specific" binding means that, compared with other natural peptide-MHC complexes, the scaffold binds to the peptide-MHC complex of interest better. The degree of binding is that it possesses active molecules that can kill cells carrying specific targets. The scaffold cannot kill another cell that does not have a specific target but exhibits one or more other peptide-MHC complexes. If the cross-reactive peptide-MHC peptide is not natural, that is, it is not from the human HLA-peptide group, binding to other peptide-MHC complexes is not important. Tests to assess the killing of target cells are well known in the art. They should contain unaltered peptide-MHC target cells (primary cells or cell lines) or peptide-loaded cells in order to achieve the level of natural peptide-MHC.

各支架可包括一個標記，其透過確定是否存在或不存在標籤所提供的信號可檢測到結合支架。例如，該支架可用螢光染料或任何其他適用的細胞標記分子進行標記。此類標記分子是本領域中公知的。例如，透過螢光染料進行的螢光標記可透過螢光或鐳射掃描顯微術或流式細胞術提供結合適體的視覺化。Each stent may include a label, which can detect the bound stent by determining the presence or absence of the signal provided by the label. For example, the scaffold can be labeled with fluorescent dyes or any other suitable cell labeling molecules. Such labeling molecules are well known in the art. For example, fluorescent labeling through fluorescent dyes can provide visualization of the fit body through fluorescent or laser scanning microscopy or flow cytometry.

各支架可與第二個活性分子（例如 IL-21、抗 CD3、抗 CD28）共軛。Each scaffold can be conjugated to a second active molecule (eg IL-21, anti-CD3, anti-CD28).

關於多肽支架的進一步資訊，可參閱，例如，在  WO 2014/071978A1 背景技術部分，並作為參考文獻引用。For further information on polypeptide scaffolds, please refer to, for example, in the background art section of WO 2014/071978A1, which is cited as a reference.

本發明還涉及適體。適體（例如，參見WO 2014/191359及其中引用的文獻）是短的單鏈核酸分子，其可以折疊為所定義的三維結構並識別特定的靶標結構。它們似乎是開發靶向治療的合適替代方法。適體已顯示可選擇性與具有高親和力和特異性的複合體靶標相結合。The invention also relates to aptamers. Aptamers (for example, see WO 2014/191359 and the documents cited therein) are short single-stranded nucleic acid molecules that can fold into a defined three-dimensional structure and recognize a specific target structure. They appear to be a suitable alternative to the development of targeted therapies. Aptamers have been shown to selectively bind to complex targets with high affinity and specificity.

識別細胞表面分子的適體在過去十年內已經確定，並為開發診斷和治療方法提供了手段。由於適體已顯示幾乎無毒性和免疫原性，因此，它們是生物醫學應用中有前景的候選物質。事實上適體，例如攝護腺特異性膜抗原識別適體，已被成功地用於靶向治療並在體內模型的異種移植物中顯示出功能。此外，認識到特定腫瘤細胞系的適體也已確定。Aptamers that recognize cell surface molecules have been identified in the past ten years and have provided a means for the development of diagnostic and therapeutic methods. Since aptamers have shown almost no toxicity and immunogenicity, they are promising candidates for biomedical applications. In fact, aptamers, such as prostate-specific membrane antigen recognition aptamers, have been successfully used in targeted therapy and have shown function in xenografts in in vivo models. In addition, aptamers that recognize specific tumor cell lines have also been identified.

可選擇 DNA 適體來揭示各種癌細胞的廣譜識別屬性，特別是那些來自於實體瘤的細胞，而非致瘤和主要健康細胞不被識別。如果所識別的適體不僅識別腫瘤特異性子類型，而且與一系列腫瘤相互作用，這使適體適用于作為所謂的廣譜診斷和治療手段。DNA aptamers can be selected to reveal the broad-spectrum recognition properties of various cancer cells, especially those cells derived from solid tumors, and non-tumorigenic and mainly healthy cells are not recognized. If the identified aptamer not only recognizes tumor-specific subtypes, but also interacts with a series of tumors, this makes the aptamer suitable as a so-called broad-spectrum diagnosis and treatment method.

此外，用流式細胞儀對細胞結合行為的研究顯示，適體在納摩爾範圍內顯示出很好的親和力。In addition, studies on cell binding behavior using flow cytometry have shown that aptamers show good affinity in the nanomolar range.

適體用於診斷和治療目的。此外，也可能顯示，一些適體被腫瘤細胞吸取，因而可作為抗癌劑靶向遞送的分子賦形劑，例如 siRNA 進入腫瘤細胞。Aptamers are used for diagnostic and therapeutic purposes. In addition, it may also be shown that some aptamers are absorbed by tumor cells, and thus can be used as molecular excipients for targeted delivery of anticancer agents, such as siRNA into tumor cells.

可選擇適體針對複合體的靶標，如細胞和組織以及包含、優選包括根據任何SEQ ID NO 1至SEQ ID NO 385的一個序列、根據當前發明的肽複合體與MHC分子，使用細胞SELEX（透過指數富集的配體系統進化）技術。The aptamer can be selected to target the target of the complex, such as cells and tissues and including, preferably including a sequence according to any of SEQ ID NO 1 to SEQ ID NO 385, peptide complexes and MHC molecules according to the current invention, using cell SELEX (permeable Ligand systematic evolution of exponential enrichment) technology.

本發明中的肽可用于生成和開發出針對MHC/肽複合物的特定抗體。這些抗體可用於治療，將毒素或放射性物質靶向病變組織。這些抗體的另一用途是為了成像之目的（如PET）將放射性核素靶向病變組織。這可有助於檢測小轉移灶或確定病變組織的大小和準確位置。The peptides of the present invention can be used to generate and develop specific antibodies against MHC/peptide complexes. These antibodies can be used in therapy to target toxins or radioactive substances to diseased tissues. Another use of these antibodies is to target radionuclides to diseased tissues for imaging purposes (such as PET). This can help detect small metastases or determine the size and exact location of diseased tissue.

因此，本發明的另一方面是提出產生特異性結合至與HLA限制性抗原絡合的I或II類人主要組織相容性複合體(MHC)的一種重組抗體的方法，該方法包括：用可溶形式的與HLA限制性抗原絡合的(MHC) I或II類分子對包含表達所述主要組織相容性說複合體(MHC) I或II類的基因工程非人哺乳動物進行免疫；將mRNA分子與產生所述非人哺乳動物細胞的抗體分離；產生一個噬菌體顯示庫，顯示由所述mRNA分子編碼的蛋白分子；以及將至少一個噬菌體與所述噬菌體顯示庫分離，所述的至少一個噬菌體顯示所述抗體特異性地結合至與HLA限制性抗原絡合的所述人主要組織相容性說複合體(MHC) I或II類。Therefore, another aspect of the present invention is to propose a method for producing a recombinant antibody that specifically binds to a human major histocompatibility complex (MHC) of type I or II complexed with an HLA-restricted antigen, the method comprising: A soluble form of molecules complexed with HLA-restricted antigens (MHC) class I or class II immunizes non-human mammals containing genetically engineered mammals expressing the major histocompatibility complex (MHC) class I or class II; Separating mRNA molecules from the antibodies that produce the non-human mammalian cells; generating a phage display library to display the protein molecules encoded by the mRNA molecules; and separating at least one phage from the phage display library, the at least A phage showed that the antibody specifically binds to the human major histocompatibility complex (MHC) class I or II complexed with an HLA-restricted antigen.

本發明的另一方面提出一種抗體，其特異性結合至與一種 HLA 限制性抗原絡合的 I 或 II 類人主要組織相容性說複合體 (MHC)，其中該抗體優選為多克隆抗體、單克隆抗體、雙特異性抗體和/或嵌合抗體。Another aspect of the present invention provides an antibody that specifically binds to a human major histocompatibility complex (MHC) of class I or II complexed with an HLA-restricted antigen, wherein the antibody is preferably a polyclonal antibody, Monoclonal antibodies, bispecific antibodies and/or chimeric antibodies.

產生這種抗體和單鏈 I 類主要組織相容性複合物的相應方法，以及產生這些抗體的其他工具在 WO 03/068201、WO 2004/084798、WO 01/72768、WO 03/070752 以及出版物 (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg et al., 2003) 中進行了披露，為了本發明之目的，所有參考文獻透過引用被完整地併入本文。The corresponding methods for producing such antibodies and single-chain type I major histocompatibility complexes, as well as other tools for producing these antibodies are described in WO 03/068201, WO 2004/084798, WO 01/72768, WO 03/070752 and publications (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg et al., 2003). For the purpose of the present invention, all references are fully incorporated herein by reference.

優選地，該抗體與複合體的結合親和力低於 20 納摩爾，優選為低於 10 納摩爾，這在本發明情況下也被視為具有「特異性」。Preferably, the binding affinity of the antibody to the complex is less than 20 nanomolar, preferably less than 10 nanomolar, which is also regarded as "specific" in the context of the present invention.

本發明涉及一種肽，包含選自SEQ ID NO:1至SEQ ID NO:385組成的組的一個序列或該序列的與SEQ ID NO:1至SEQ ID NO:385具有88%同源性（優選為相同）的一種變體，或誘導與所述變異肽發生 T細胞交叉反應的一種變體，其中，所述肽不是基本的全長多肽。The present invention relates to a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 385 or the sequence has 88% homology with SEQ ID NO: 1 to SEQ ID NO: 385 (preferably Is a variant of the same), or a variant that induces T cell cross-reaction with the variant peptide, wherein the peptide is not a basic full-length polypeptide.

本發明進一步涉及一種肽，包含選自SEQ ID NO:1至SEQ ID NO:385組成的組的一個序列、或與SEQ ID NO:1至SEQ ID NO:385具有至少88%同源性（優選為相同）的一種變體，其中所述肽或變體的總長度為8至100個、優選為8至30個、最優選為8至14個氨基酸。The present invention further relates to a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 385, or having at least 88% homology with SEQ ID NO: 1 to SEQ ID NO: 385 (preferably Is a variant of the same), wherein the total length of the peptide or variant is 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids.

本發明進一步涉及本發明的肽，其具有與主要組織相容性複合體 (MHC) I 或 II 類分子結合的能力。The present invention further relates to the peptides of the present invention, which have the ability to bind to major histocompatibility complex (MHC) class I or II molecules.

本發明進一步涉及本發明中的肽，其中肽系由或基本系由根據 SEQ ID NO:1至SEQ ID NO:385的一個氨基酸序列組成。The present invention further relates to the peptide of the present invention, wherein the peptide system consists of or essentially consists of an amino acid sequence according to SEQ ID NO:1 to SEQ ID NO:385.

本發明進一步涉及本發明的肽，其中該肽（在化學上）被修飾和/或包含非肽鍵。The invention further relates to a peptide of the invention, wherein the peptide is (chemically) modified and/or contains non-peptide bonds.

本發明進一步涉及本發明的肽，其中該肽為融合蛋白的一部分，特別包括 HLA-DR 抗原相關不變鏈 (Ii ) 的 N-端氨基酸，或其中該肽與一種抗體（例如，樹突狀細胞特定抗體）融合。The present invention further relates to the peptide of the present invention, wherein the peptide is a part of a fusion protein, particularly including the N-terminal amino acid of the HLA-DR antigen-related invariant chain (Ii), or wherein the peptide is associated with an antibody (eg, dendritic Cell specific antibody) fusion.

本發明進一步涉及一種核酸，其編碼本發明所述肽，前提是該肽並非完整（完全）的人蛋白。The present invention further relates to a nucleic acid encoding the peptide of the present invention, provided that the peptide is not a complete (complete) human protein.

本發明進一步涉及一種本發明的核酸，為 DNA、cDNA、PNA、RNA，也可能為其組合物。The present invention further relates to a nucleic acid of the present invention, which is DNA, cDNA, PNA, RNA, or a combination thereof.

本發明進一步涉及一種能表達本發明核酸的表達載體。The present invention further relates to an expression vector capable of expressing the nucleic acid of the present invention.

本發明進一步涉及本發明的一種肽、本發明的一種核酸或本發明的一種藥用表達載體，特別是用於治療 CLL。The present invention further relates to a peptide of the present invention, a nucleic acid of the present invention or a pharmaceutical expression vector of the present invention, especially for the treatment of CLL.

本發明進一步涉及含本發明核酸或本發明表達載體的一種宿主細胞。The invention further relates to a host cell containing the nucleic acid of the invention or the expression vector of the invention.

本發明進一步涉及本發明的宿主細胞，其為抗原表現細胞，優選為樹突細胞。The present invention further relates to the host cell of the present invention, which is an antigen expressing cell, preferably a dendritic cell.

本發明進一步涉及配製本發明一種肽的一種方法，所述方法包括培養本發明的宿主細胞和從所述宿主細胞或其培養基中分離肽。The present invention further relates to a method of formulating a peptide of the present invention, the method comprising culturing the host cell of the present invention and isolating the peptide from the host cell or its culture medium.

本發明進一步涉及本發明中的方法，其中抗原透過與足夠量的含抗原提成細胞的抗原結合被載入表達於合適抗原表現細胞表面的 I 或 II類MHC 分子。The present invention further relates to the method of the present invention, wherein the antigen is loaded into MHC class I or II molecules expressed on the surface of suitable antigen-expressing cells by binding to a sufficient amount of antigen containing antigen-derived cells.

本發明進一步涉及本發明的方法，其中該抗原表現細胞包括一個表達載體，該載體有能力表達或正在表達含SEQ ID NO:1至SEQ ID NO:385的肽或所述變體氨基酸序列。The present invention further relates to the method of the present invention, wherein the antigen-expressing cell includes an expression vector capable of expressing or being expressing the peptide or the amino acid sequence of the variant containing SEQ ID NO: 1 to SEQ ID NO: 385.

本發明進一步涉及以本發明方法製造的活化T細胞，其中所述 T細胞有選擇性地識別一種細胞，該細胞異常表達含一種本發明氨基酸序列的多肽。The present invention further relates to activated T cells produced by the method of the present invention, wherein the T cells selectively recognize a cell that abnormally expresses a polypeptide containing an amino acid sequence of the present invention.

本發明進一步涉及一種殺傷患者靶細胞的方法，其中患者的靶細胞異常表達含本發明任何氨基酸序列的多肽，該方法包括給予患者本發明的有效量 T細胞。The present invention further relates to a method for killing target cells of a patient, wherein the target cells of the patient abnormally express a polypeptide containing any amino acid sequence of the present invention, and the method comprises administering an effective amount of T cells of the present invention to the patient.

本發明進一步涉及任何所述肽、本發明的一種核酸、本發明的一種表達載體、本發明的一種細胞、本發明一種作為藥劑或製造藥劑的活化細胞毒性 T 淋巴細胞的用途。本發明進一步涉及一種本發明的用途，其中藥劑可有效抗癌。The present invention further relates to the use of any of the peptides, a nucleic acid of the present invention, an expression vector of the present invention, a cell of the present invention, and an activated cytotoxic T lymphocyte of the present invention as a medicament or manufacturing a medicament. The present invention further relates to a use of the present invention, wherein the medicament can effectively fight cancer.

本發明進一步涉及一種本發明的用途，其中該藥劑為一種疫苗。本發明進一步涉及一種本發明的用途，其中藥劑可有效抗癌。The present invention further relates to a use of the present invention, wherein the medicament is a vaccine. The present invention further relates to a use of the present invention, wherein the medicament can effectively fight cancer.

本發明進一步涉及本發明中的用途，其中所述癌細胞為 CLL 細胞或其他實體或血液腫瘤細胞，如急性骨髓性白血病、膽管癌、腦癌、乳腺癌、結直腸癌、食管癌、膽囊癌、胃癌、肝細胞癌、梅克爾細胞癌、黑色素瘤、非霍奇金淋巴瘤、非小細胞肺癌、卵巢癌、胰腺癌、攝護腺癌、腎細胞癌、小細胞肺癌、膀胱癌和子宮癌。The present invention further relates to the use of the present invention, wherein the cancer cells are CLL cells or other solid or blood tumor cells, such as acute myeloid leukemia, cholangiocarcinoma, brain cancer, breast cancer, colorectal cancer, esophageal cancer, gallbladder cancer , Stomach cancer, hepatocellular carcinoma, Merkel cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and uterus cancer.

本發明進一步涉及一種基於本發明肽的特定標誌物蛋白和生物標誌物，在此成為「靶標」，其可用於診斷和/或判斷 CLL 的預後。本發明還涉及這些供癌症治療使用的新靶點。The present invention further relates to a specific marker protein and biomarker based on the peptide of the present invention, which are referred to herein as "targets", which can be used to diagnose and/or judge the prognosis of CLL. The invention also relates to these new targets for cancer treatment.

本文中術語「抗體」為廣義上的定義，既包括多克隆也包括單克隆抗體。除了完整或「全部」的免疫球蛋白分子，「抗體」這一術語還包括這些免疫球蛋白分子和人源化免疫球蛋白分子的片段（如，CDR、Fv、Fab 和 Fc 片段）或聚合物，只要它們表現出本發明的任何期望屬性（例如，CLL 標誌物（多）肽的特異性結合、將毒素傳遞給癌症標誌物基因表達水準增加時的 CLL 細胞和/或抑制 CLL 標誌物多肽的活性）。The term "antibody" as used herein is defined in a broad sense, including both polyclonal and monoclonal antibodies. In addition to complete or "entire" immunoglobulin molecules, the term "antibody" also includes these immunoglobulin molecules and fragments of humanized immunoglobulin molecules (eg, CDR, Fv, Fab and Fc fragments) or polymers , As long as they exhibit any desired properties of the present invention (for example, specific binding of CLL marker (poly) peptides, delivery of toxins to CLL cells when the expression level of cancer marker genes is increased, and/or inhibition of CLL marker polypeptides) active).

只要有可能，本發明的抗體可從商業來源購買。本發明的抗體也可能使用已知的方法制得。技術人員會瞭解全長 CLL 標誌物多肽或其片段可用于製備本發明的抗體。用於產生本發明抗體的多肽可部分或全部地由天然源經純化而得，也可利用重組 DNA 技術生產。Whenever possible, the antibodies of the invention can be purchased from commercial sources. The antibodies of the present invention may also be prepared using known methods. The skilled person will understand that full-length CLL marker polypeptides or fragments thereof can be used to prepare the antibodies of the present invention. The polypeptides used to produce the antibodies of the present invention can be partially or completely purified from natural sources, or can be produced by recombinant DNA technology.

例如，本發明的編碼肽的 cDNA，例如，該肽為根據SEQ ID NO:1至SEQ ID NO:385多肽的肽，或其中一個變體或片段，可在原核細胞中（如：細菌）或真核細胞（如：酵母、昆蟲或哺乳動物細胞）中表達，之後，可純化重組蛋白，並用於產生一種特異性結合用於產生本發明抗體的 CLL 標誌物多肽的單克隆或多克隆抗體製劑。For example, the cDNA encoding the peptide of the present invention, for example, the peptide is a peptide according to SEQ ID NO: 1 to SEQ ID NO: 385, or a variant or fragment thereof, can be in prokaryotic cells (such as bacteria) or After being expressed in eukaryotic cells (such as yeast, insect or mammalian cells), the recombinant protein can be purified and used to produce a monoclonal or polyclonal antibody preparation that specifically binds to the CLL marker polypeptide used to produce the antibody of the present invention .

本領域的技術人員會認識到，兩種或兩種以上不同集合的單克隆抗體或多克隆抗體能最大限度地增加獲得一種含預期用途所需的特異性和親和力（例如，ELISA 法、免疫組織化學、體內成像、免疫毒素療法）的抗體的可能性。根據抗體的用途，用已知的方法對其期望活性進行測試（例如，ELISA 法、免疫組織化學、免疫治療等；要獲取產生和測試抗體的進一步指導，請參閱，例如，Greenfield, 2014 (Greenfield, 2014)）。例如，該抗體可用 ELISA 法或免疫印跡法、免疫組織化學染色福馬林固定的癌組織或冰凍的組織切片進行檢測。在初次體外表徵後，用於治療或體內診斷用途的抗體根據已知的臨床測試方法進行檢測。Those skilled in the art will recognize that two or more different collections of monoclonal antibodies or polyclonal antibodies can maximize the specificity and affinity required for the intended use (for example, ELISA method, immune tissue Chemistry, in vivo imaging, immunotoxin therapy). Depending on the purpose of the antibody, use known methods to test its expected activity (for example, ELISA, immunohistochemistry, immunotherapy, etc.; for further guidance on the production and testing of antibodies, please refer to, for example, Greenfield, 2014 (Greenfield) , 2014)). For example, the antibody can be detected by ELISA or Western blotting, immunohistochemical staining of formalin-fixed cancer tissues or frozen tissue sections. After the initial in vitro characterization, antibodies for therapeutic or in vivo diagnostic purposes are tested according to known clinical testing methods.

此處使用的術語「單克隆抗體」系指從大量同質抗體中獲得的一種抗體，即，由相同的抗體組成的抗體群，但可能少量表現的自然突變除外。此處所述的單克隆抗體具體包括「嵌合」抗體，其中一部分重鏈和/或輕鏈與從特定物種中獲得的抗體或屬於特定抗體類型和分類型抗體的相應序列相同（同質），同時，剩餘鏈與從其他物種中獲得的抗體或屬於特定抗體類型和子類型抗體的相應序列以及這些抗體的片段相同（同質），只要他們表現出預期的拮抗活性（美國 4816567 號專利，其在此以其整體併入）。The term "monoclonal antibody" as used herein refers to an antibody obtained from a large number of homogeneous antibodies, that is, an antibody group composed of the same antibodies, except for natural mutations that may be expressed in a small amount. The monoclonal antibodies described herein specifically include "chimeric" antibodies, in which a part of the heavy chain and/or light chain is the same (homogeneous) as the antibody obtained from a specific species or the corresponding sequence of the antibody belonging to a specific antibody type and subtype, At the same time, the remaining chains are the same (homogeneous) as the corresponding sequences of antibodies obtained from other species or belonging to specific antibody types and subtypes and fragments of these antibodies, as long as they exhibit the expected antagonistic activity (US Patent No. 4,816,567, which is here Incorporate in its entirety).

本發明的單克隆抗體可能使用雜交瘤方法制得。在雜交瘤方法中，老鼠或其他適當的宿主動物，通常用免疫製劑以引發產生或能產生將特異性結合至免疫製劑的抗體。或者，淋巴細胞可在體外進行免疫。The monoclonal antibody of the present invention may be produced using the hybridoma method. In the hybridoma method, mice or other suitable host animals are usually used with immune agents to induce or produce antibodies that specifically bind to the immune agents. Alternatively, lymphocytes can be immunized in vitro.

單克隆抗體也可由 DNA 重組方法制得，如：美國 4816567 號專利所述。編碼本發明單克隆抗體的 DNA 可很容易地使用傳統程序進行分離和測序（例如：透過使用能與編碼鼠抗體重鏈和輕鏈的基因特異性結合的寡核苷酸探針）。Monoclonal antibodies can also be prepared by DNA recombination methods, such as described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibody of the present invention can be easily separated and sequenced using traditional procedures (for example, by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the murine antibody).

體外方法也適用於製備單價抗體。抗體消化以產生抗體的片段，尤其是 Fab 片段，可以透過使用本領域已知的常規技術完成。例如，可以透過使用木瓜蛋白酶完成消化。木瓜蛋白酶消化的實施例在 WO 94/29348和美國 4342566 號專利中有描述。抗體的木瓜蛋白酶消化通常產生兩種相同的抗原結合性片段，稱為 Fab 片段（每個片段都有一個抗原結合點）和殘餘 Fc 片段。胃蛋白酶處理產生一個 F(ab')2 片段和一個 pFc' 片段。In vitro methods are also suitable for preparing monovalent antibodies. Antibody digestion to produce antibody fragments, especially Fab fragments, can be accomplished by using conventional techniques known in the art. For example, digestion can be accomplished by using papain. Examples of papain digestion are described in WO 94/29348 and U.S. Patent No. 4,342,566. Papain digestion of antibodies usually produces two identical antigen-binding fragments, called Fab fragments (each fragment has an antigen binding site) and residual Fc fragments. Pepsin treatment produces an F(ab')2 fragment and a pFc' fragment.

抗體片段，不論其是否附著於其他序列，均可包括特定區域或特定氨基酸殘基的***、刪除、替換、或其他選擇性修飾，但前提是，片段的活性與非修飾的抗體或抗體片段相比沒有顯著的改變或損害。這些修飾可提供一些額外的屬性，如：刪除/添加可與二硫鍵結合的氨基酸，以增加其生物壽命、改變其分泌特性等。在任何情況下，抗體片段必須擁有生物活性的特性，如：結合活性、調節結合域的結合力等。抗體的功能性或活性區域可透過蛋白特定區域的基因突變、隨後表達和測試所表達的多肽進行確定。這些方法為本行業技術人員所熟知，可包括編碼抗體片段的核酸的特定位點基因突變。Antibody fragments, regardless of whether they are attached to other sequences, can include insertions, deletions, substitutions, or other selective modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is comparable to that of the non-modified antibody or antibody fragment. Than no significant changes or damage. These modifications can provide some additional properties, such as deleting/adding amino acids that can be combined with disulfide bonds to increase their biological life span and change their secretion properties. In any case, the antibody fragment must possess the characteristics of biological activity, such as: binding activity, regulating the binding force of the binding domain, etc. The functionality or active region of an antibody can be determined by gene mutation in a specific region of the protein, subsequent expression and testing of the expressed polypeptide. These methods are well known to those skilled in the art, and may include gene mutations at specific sites in nucleic acids encoding antibody fragments.

本發明的抗體可進一步包括人源化抗體或人抗體。非人（如：鼠）抗體的人源化形式為嵌合抗體免疫球蛋白、免疫球蛋白鏈或其片段（如：Fv、Fab、Fab' 或抗體的其他抗原結合序列），其中包含從非人免疫球蛋白中獲得的最小序列。人源化抗體包括人免疫球蛋白（受體抗體），其中來自受體互補決定區 (CDR) 的殘基被來自非人物種（供體抗體）（如具有與其特異性、親和力和能力的小鼠、大鼠或兔子）CDR 的殘基取代。在某些情況下，人類免疫球蛋白的 Fv 框架 (FR) 殘基被相應的非人殘基取代。人源化抗體可能還包括既非受體抗體、也非輸入 CDR 或框架序列中發現的殘基。一般來說，人源化抗體將包括幾乎所有的至少一個、通常為二個可變域，其中，全部或幾乎全部的 CDR 區域均對應於非人免疫球蛋白的區域並且全部或幾乎全部的 FR區域均為人免疫球蛋白相同序列的區域。理想情況是，人源化抗體還將包括至少免疫球蛋白恒定區 (Fc) 的一部分，通常是人免疫球蛋白的恒定區的一部分。The antibodies of the present invention may further include humanized antibodies or human antibodies. Humanized forms of non-human (such as murine) antibodies are chimeric antibody immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab' or other antigen-binding sequences of antibodies), which contain non-human The smallest sequence obtained in human immunoglobulin. Humanized antibodies include human immunoglobulins (acceptor antibodies), in which residues from the receptor complementarity determining region (CDR) are derived from non-human species (donor antibodies) (such as small antibodies with specificity, affinity, and ability). Mouse, rat or rabbit) CDR residues. In some cases, the Fv framework (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. Humanized antibodies may also include residues found in neither the recipient antibody nor the imported CDR or framework sequences. Generally speaking, a humanized antibody will include almost all of at least one and usually two variable domains, wherein all or almost all of the CDR regions correspond to the regions of non-human immunoglobulin and all or almost all of the FRs The regions are all regions of the same sequence of human immunoglobulin. Ideally, the humanized antibody will also include at least a part of the constant region of an immunoglobulin (Fc), usually a part of the constant region of a human immunoglobulin.

人源化非人抗體的方法為本行業所熟知。一般來說，人源化抗體具有一個或多個從非人源頭引入的氨基酸殘基。這些非人氨基酸殘基往往被稱為「輸入」殘基，通常從「輸入」可變域中獲得。人源化基本上可以透過將齧齒動物 CDR 或 CDR 序列取代為相應的人抗體序列而完成。因此，這種「人源化」抗體為嵌合抗體（美國 4816567 號專利），其中大大少於完整的人可變域被來自於非人物種的相應序列取代。在實踐中，人源化抗體通常為人抗體，其中有些 CDR 殘基以及可能的一些 FR 殘基被來自齧齒動物抗體中的類似位點的殘基取代。The method of humanizing non-human antibodies is well known in the industry. Generally, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are usually obtained from the "import" variable domain. Humanization can basically be accomplished by substituting rodent CDR or CDR sequences with corresponding human antibody sequences. Therefore, this "humanized" antibody is a chimeric antibody (US Patent No. 4,816,567), in which much less than the complete human variable domain is replaced with a corresponding sequence from a non-human species. In practice, humanized antibodies are usually human antibodies, in which some CDR residues and possibly some FR residues are replaced by residues from similar sites in rodent antibodies.

可使用免疫後在內源性免疫球蛋白產生缺失時能產生完整人抗體的轉基因動物（如：小鼠）。例如，它被描述為，嵌合和種系突變小鼠中的抗體重鏈連接區域基因的純合性缺失導致內源性抗體生成的完全抑制。在此種系變種小鼠中人種系免疫球蛋白基因陣列的轉移在抗原挑戰後將導致人抗體的生成。人抗體也可在噬菌體展示庫中產生。Transgenic animals (such as mice) that can produce intact human antibodies can be used after immunization. For example, it has been described that the homozygous deletion of the antibody heavy chain junction region gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. The transfer of human germline immunoglobulin gene arrays in this strain of mutant mice will lead to the production of human antibodies after antigen challenge. Human antibodies can also be produced in phage display libraries.

本發明的抗體優選為透過藥用載體的形式給予受試者。通常，在製劑中使用適量的藥用鹽，以使製劑等滲。藥用載體的例子包括生理鹽水、林格氏液和葡萄糖溶液。溶液的 pH 值優選為約 5 至8，更優選為約 7 至7.5。此外，載體還包括緩釋製劑，如：含有抗體的固體疏水性聚合物半透性基質，其中基質為有形物品形式，如：薄膜、脂質體或微粒。本行業的技術人員熟知，某些載體可能為更優選，取決於例如，抗體的給藥途徑和濃度。The antibody of the present invention is preferably administered to a subject in the form of a pharmaceutical carrier. Generally, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of pharmaceutical carriers include physiological saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to 8, more preferably about 7 to 7.5. In addition, the carrier also includes sustained-release preparations, such as a semipermeable matrix of solid hydrophobic polymers containing antibodies, wherein the matrix is in the form of a tangible article, such as a film, liposome, or microparticle. Those skilled in the art are well aware that certain carriers may be more preferred, depending on, for example, the route of administration and concentration of the antibody.

該抗體可透過注射（如：靜脈內、腹腔內、皮下、肌肉內）或透過輸注等其他方法給予受試者、患者或細胞，確保其以有效的形式傳輸到血液中。這些抗體也可以透過瘤內或瘤周途徑給予，從而發揮局部和全身的治療作用。局部或靜脈注射為優選。The antibody can be administered to subjects, patients or cells by injection (such as intravenous, intraperitoneal, subcutaneous, intramuscular) or other methods such as infusion to ensure that it is effectively delivered to the bloodstream. These antibodies can also be administered via intratumoral or peritumoral routes to exert local and systemic therapeutic effects. Local or intravenous injection is preferred.

抗體給藥的有效劑量和時間表可根據經驗確定，並且作出此類決定屬本行業的技術範圍內。本行業的技術人員會明白，必須給予的抗體劑量根據以下因素會有所不同，例如：接受抗體的受試者、給藥途徑、使用的抗體以及其他正在使用的藥物的特定類型。單獨使用的抗體的通常日劑量可能為約 1 µg/kg 至最多 100 mg/kg 體重或更多，這取決於上述因素。給予抗體，優選為治療 CLL 後，治療抗體的療效可透過技術人員熟知的不同方法評估。例如：接受治療的受試者癌症的大小、數量和/或分佈可使用標準腫瘤成像技術進行監測。因治療而給予的抗體與不給予抗體時的病程相比，可阻止腫瘤生長、導致腫瘤縮小、和/或阻止新腫瘤的發展，這樣的抗體是一種有效治療癌症的抗體。The effective dose and schedule of antibody administration can be determined based on experience, and making such a decision is within the technical scope of the industry. Those skilled in the industry will understand that the dose of antibody that must be administered will vary based on factors such as the subject receiving the antibody, the route of administration, the antibody used, and the specific types of other drugs being used. The usual daily dose of antibodies used alone may be about 1 µg/kg up to 100 mg/kg body weight or more, depending on the factors mentioned above. After administration of the antibody, preferably for the treatment of CLL, the efficacy of the treatment antibody can be assessed by different methods well known to the skilled person. For example, the size, number, and/or distribution of cancers in subjects receiving treatment can be monitored using standard tumor imaging techniques. Antibodies given for treatment can prevent tumor growth, cause tumor shrinkage, and/or prevent the development of new tumors compared with the course of the disease when no antibody is given. Such antibodies are an effective treatment for cancer.

本發明的另一方面提出了製備識別特異性肽-MHC複合物的可溶性T細胞受體 (sTCR) 的一種方法。這種可溶性T細胞受體可從特異性 T細胞克隆中產生，並且它們的親和力可以透過互補決定區靶向誘變而增加。為了 T細胞受體選擇之目的，可以使用噬菌體展示（美國2010/0113300， (Liddy et al., 2012)）。為了在噬菌體展示期間以及實際使用為藥物時穩定 T細胞受體之目的，可透過非天然二硫鍵、其他共價鍵（單鏈 T細胞受體）或透過二聚化結構域連接 α和β鏈 (Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999)。T細胞受體可以連接到毒素、藥物、細胞因子（參見US 2013/0115191）、域招募效應細胞，如抗 CD3 域等，以便對靶細胞執行特定的功能。此外，它可能表達於用於過繼轉移的 T細胞。進一步的資訊可在 WO 2004/033685A1 和 WO 2004/074322A1 中找到。sTCR 的組合在 WO 2012/056407A1 中進行了描述。WO 2013/057586A1 中公開了製備的進一步的方法。Another aspect of the present invention provides a method for preparing a soluble T cell receptor (sTCR) that recognizes a specific peptide-MHC complex. Such soluble T cell receptors can be produced from specific T cell clones, and their affinity can be increased by targeted mutagenesis of complementarity determining regions. For the purpose of T cell receptor selection, phage display can be used (US 2010/0113300, (Liddy et al., 2012)). For the purpose of stabilizing T cell receptors during phage display and when actually used as drugs, α and β can be connected through unnatural disulfide bonds, other covalent bonds (single-chain T cell receptors) or through dimerization domains Chain (Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999). T cell receptors can be connected to toxins, drugs, cytokines (see US 2013/0115191), domains recruit effector cells, such as anti-CD3 domains, etc., in order to perform specific functions on target cells. In addition, it may be expressed on T cells used for adoptive transfer. Further information can be found in WO 2004/033685A1 and WO 2004/074322A1. The combination of sTCR is described in WO 2012/056407A1. A further method of preparation is disclosed in WO 2013/057586A1.

此外，可用本發明的肽和/或 TCR 或抗體或其他結合分子在活檢樣本的基礎上驗證病理師對癌症的診斷。In addition, the peptides and/or TCRs or antibodies or other binding molecules of the present invention can be used to verify the diagnosis of cancer by a pathologist on the basis of biopsy samples.

該抗體或 TCR 也可用於體內診斷實驗。一般來說，抗體用放射性核素標記（如：111In、99Tc、14C、131I、3H、32P 或 35S），從而可免疫閃爍掃描法使腫瘤局限化。在一實施方案中，其中的抗體或片段與兩個或兩個以上選自包括上述蛋白的組的蛋白質靶標的細胞外域結合，並且親和力值 (Kd) 低於 1 x 10µM。The antibody or TCR can also be used in in vivo diagnostic experiments. Generally speaking, antibodies are labeled with radionuclides (such as 111In, 99Tc, 14C, 131I, 3H, 32P, or 35S), which can localize tumors by immunoscintigraphy. In one embodiment, the antibody or fragment therein binds to two or more extracellular domains of protein targets selected from the group including the above-mentioned proteins, and the affinity value (Kd) is less than 1 x 10 µM.

診斷用抗體可透過各種影像學方法使用適合檢測的探針進行標記。探針檢測方法包括但不限於，螢光、光、共聚焦和電鏡方法；磁共振成像和光譜學技術；透視、電腦斷層掃描和正電子發射斷層掃描。合適的探針包括但不限於，螢光素、羅丹明、曙紅及其它螢光團、放射性同位素、黃金、釓和其他稀土、順磁鐵、氟-18 和其他正電子發射放射性核素。此外，探針可能是雙功能或多功能的，並且用一種以上的上述方法可進行檢測。這些抗體可用所述的探針直接或間接進行標記。抗體探針的連接，包括探針的共價連接、將探針融合入抗體、以及螯合化合物的共價連接從而結合探針、以及其他本行業熟知的方法。對於免疫組織化學方法，疾病組織樣本可能是新鮮或冷凍或可能包埋於石蠟中以及用福馬林等防腐劑固定。固定或包埋的切片包括與標記一抗和二抗接觸的樣本，其中該抗體用於檢測原位 蛋白的表達。Diagnostic antibodies can be labeled with probes suitable for detection through various imaging methods. Probe detection methods include, but are not limited to, fluorescence, light, confocal and electron microscopy methods; magnetic resonance imaging and spectroscopy techniques; fluoroscopy, computer tomography, and positron emission tomography. Suitable probes include, but are not limited to, luciferin, rhodamine, eosin and other fluorophores, radioisotopes, gold, gamma and other rare earths, paramagnets, fluorine-18 and other positron emitting radionuclides. In addition, the probe may be bifunctional or multifunctional, and can be detected by more than one of the above methods. These antibodies can be directly or indirectly labeled with the probes described. The connection of antibody probes includes covalent connection of probes, fusion of probes into antibodies, covalent connection of chelating compounds to bind probes, and other methods well known in the industry. For immunohistochemical methods, diseased tissue samples may be fresh or frozen or may be embedded in paraffin and fixed with preservatives such as formalin. The fixed or embedded sections include samples that are in contact with labeled primary and secondary antibodies, where the antibodies are used to detect in situ protein expression.

本發明的另一方面包括一種體外製備活化的T細胞的方法，該方法包括將T細胞與載有抗原的人MHC分子進行體外連接，這些分子在合適的抗原表現細胞表面表達足夠的一段時間從而以抗原特異性方式活化T細胞，其中所述抗原為根據本發明所述的一種肽。優選情況是足夠量的抗原與抗原表現細胞一同使用。Another aspect of the present invention includes a method for preparing activated T cells in vitro, the method comprising in vitro linking T cells with antigen-loaded human MHC molecules, and these molecules are expressed on the surface of suitable antigen-expressing cells for a sufficient period of time to T cells are activated in an antigen-specific manner, wherein the antigen is a peptide according to the present invention. Preferably, a sufficient amount of antigen is used together with antigen expressing cells.

優選情況是，哺乳動物細胞的TAP 肽轉運載體缺乏或水準下降或功能降低。缺乏 TAP 肽轉運載體的適合細胞包括 T2、RMA-S 和果蠅細胞。TAP 是與抗原加工相關的轉運載體。Preferably, the TAP peptide transporter of mammalian cells is lacking or the level is reduced or the function is reduced. Suitable cells that lack the TAP peptide transporter include T2, RMA-S, and Drosophila cells. TAP is a transporter associated with antigen processing.

人體肽載入的缺陷細胞株 T2 從屬美國菌種保藏中心（ATCC, 12301 Parklawn Drive, Rockville, Maryland 20852，美國）目錄號 CRL1992；果蠅細胞株 Schneider 2 號株從屬 ATCC 目錄 CRL 19863；小鼠 RMA-S 細胞株 Ljunggren 等人描述過 (Ljunggren and Karre, 1985)。The human peptide-loaded defective cell line T2 belongs to the American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, Maryland 20852, USA) catalog number CRL1992; Drosophila cell line Schneider 2 strain belongs to ATCC catalog CRL 19863; mouse RMA -S cell line described by Ljunggren et al. (Ljunggren and Karre, 1985).

優選情況是，宿主細胞在轉染前基本上不表達 MHC I類分子。刺激因子細胞還優選為表達對 T細胞共刺激信號起到重要作用的分子，如，B7.1、B7.2、ICAM-1 和 LFA 3 中的任一種分子。大量 MHC I類分子和共刺激分子的核酸序列可從 GenBank 和 EMBL 資料庫中公開獲得。Preferably, the host cell does not substantially express MHC class I molecules before transfection. The stimulator cell is also preferably a molecule that expresses an important role for T cell costimulatory signals, such as any one of B7.1, B7.2, ICAM-1 and LFA3. Nucleic acid sequences of a large number of MHC class I molecules and costimulatory molecules are publicly available from GenBank and EMBL databases.

當 MHC I類表位用作一種抗原時，T細胞為 CD8 陽性 T細胞。When MHC class I epitopes are used as an antigen, T cells are CD8-positive T cells.

如果抗原表現細胞受到轉染而表達這種表位，則優選的細胞包括一個表達載體，該載體有能力表達含SEQ ID NO:1至SEQ ID NO:385的肽或變體氨基酸序列。If the antigen expressing cell is transfected to express this epitope, the preferred cell includes an expression vector capable of expressing the amino acid sequence of the peptide or variant containing SEQ ID NO: 1 to SEQ ID NO: 385.

可使用其他一些方法來體外生成 T細胞。例如，自體腫瘤浸潤性淋巴細胞可用于生成 CTL。Plebanski 等人在 (Plebanski et al., 1995) 使用自體外周血淋巴細胞 (PLB) 制得 T細胞。另外，也可能用肽或多肽脈衝處理樹突狀細胞或透過與重組病毒感染而製成自體 T細胞。此外，B細胞可用於製備自體  T細胞。此外，用肽或多肽脈衝處理或用重組病毒感染的巨噬細胞可用於配製自體 T細胞。S. Walter 等人在 (Walter et al., 2003) 中描述了透過使用人工抗原表現細胞 (aAPC) 體外活化T細胞，這也是生成作用於所選肽的T細胞的一種合適方法。在本發明中，根據生物素：鏈黴素生物化學方法透過將預製的MHC：肽複合物耦合到聚苯乙烯顆粒（微球）而生成 aAPC。該系統實現了對 aAPC 上的 MHC 密度進行精確調節，這使得可以在血液樣本中選擇地引發高或低親合力的高效抗原特異性 T細胞反應。除了 MHC：肽複合物外，aAPC 還應攜運含共刺激活性的其他蛋白，如耦合至表面的抗-CD28 抗體。此外，此類基於 aAPC 的系統往往需要加入適當的可溶性因子，例如，諸如白細胞介素 12 的細胞因子。Several other methods can be used to generate T cells in vitro. For example, autologous tumor-infiltrating lymphocytes can be used to generate CTL. Plebanski et al. (Plebanski et al., 1995) used autologous peripheral blood lymphocytes (PLB) to prepare T cells. In addition, it is also possible to pulse dendritic cells with peptides or polypeptides or make autologous T cells through infection with recombinant viruses. In addition, B cells can be used to prepare autologous T cells. In addition, macrophages pulsed with peptides or polypeptides or infected with recombinant viruses can be used to formulate autologous T cells. S. Walter et al. (Walter et al., 2003) described in vitro activation of T cells through the use of artificial antigen expressing cells (aAPC), which is also a suitable method for generating T cells that act on selected peptides. In the present invention, aAPC is generated by coupling a prefabricated MHC: peptide complex to polystyrene particles (microspheres) according to the biotin:streptomycin biochemical method. The system achieves precise adjustment of the MHC density on aAPC, which makes it possible to selectively trigger high or low affinity high-efficiency antigen-specific T cell responses in blood samples. In addition to MHC: peptide complexes, aAPC should also carry other proteins with costimulatory activity, such as anti-CD28 antibodies coupled to the surface. In addition, such aAPC-based systems often require the addition of appropriate soluble factors, for example, cytokines such as interleukin-12.

也可用同種異體細胞制得 T細胞，在 WO 97/26328 中詳細描述了一種方法，以參考文獻方式併入本文。例如，除了果蠅細胞和 T2 細胞，也可用其他細胞來表現肽，如 CHO 細胞、杆狀病毒感染的昆蟲細胞、細菌、酵母、牛痘感染的靶細胞。此外，也可使用植物病毒（例如，參閱 Porta 等人在  (Porta et al., 1994) 中描述了將豇豆花葉病毒開發為一種表現外來肽的高產系統。T cells can also be prepared from allogeneic cells. A method is described in detail in WO 97/26328, which is incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells can also be used to express peptides, such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, and vaccinia-infected target cells. In addition, plant viruses can also be used (for example, see Porta et al. (Porta et al., 1994) describing the development of cowpea mosaic virus as a high-yield system for expressing foreign peptides.

被活化的T細胞直接針對本發明中的肽，有助於治療。因此，本發明的另一方面提出了用本發明前述方法制得的活化T細胞。The activated T cells directly target the peptides of the present invention, which is helpful for treatment. Therefore, another aspect of the present invention proposes activated T cells prepared by the aforementioned method of the present invention.

按上述方法製成的活化T細胞將會有選擇性地識別異常表達含SEQ ID NO:1至 SEQ ID NO 385 氨基酸序列的多肽。The activated T cells prepared by the above method will selectively recognize the abnormal expression of polypeptides containing the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 385.

優選情況是，T細胞透過與其含HLA/肽複合物的 TCR 相互作用（如，結合）而識別該細胞。T細胞是殺傷患者靶細胞方法中有用的細胞，其靶細胞異常表達含本發明中氨基酸序列的多肽。此類患者給予有效量的活化T細胞。給予患者的 T細胞可能源自該患者，並按上述方法活化（即，它們為自體T細胞）。或者，T細胞不是源自該患者，而是來自另一個人。當然，優選情況是該供體為健康人。發明人使用「健康個人」系指一個人一般狀況良好，優選為免疫系統合格，更優選為無任何可很容易測試或檢測到的疾病。Preferably, the T cell recognizes the cell by interacting (eg, binding) with its TCR containing the HLA/peptide complex. T cells are useful cells in the method of killing target cells of patients, and their target cells abnormally express the polypeptide containing the amino acid sequence of the present invention. Such patients are given an effective amount of activated T cells. The T cells given to the patient may be derived from the patient and activated as described above (ie, they are autologous T cells). Or, the T cells are not derived from the patient, but from another person. Of course, it is preferable that the donor is a healthy person. The inventors use a "healthy individual" to refer to a person who is generally in good condition, preferably has a qualified immune system, and more preferably does not have any diseases that can be easily tested or detected.

根據本發明，CD8-陽性 T細胞的體內靶細胞可為腫瘤細胞（有時表達 MHC-II類抗原）和/或腫瘤周圍的基質細胞（腫瘤細胞）（有時也表達 MHC-II類抗原； (Dengjel et al., 2006)）。According to the present invention, the in vivo target cells of CD8-positive T cells can be tumor cells (sometimes expressing MHC-II antigens) and/or stromal cells (tumor cells) surrounding tumors (sometimes expressing MHC-II antigens); (Dengjel et al., 2006)).

本發明所述的 T細胞可用作治療性組合物中的活性成分。因此，本發明也提出了一種殺傷患者靶細胞的方法，其中患者的靶細胞異常表達含本發明中氨基酸序列的多肽，該方法包括給予患者上述有效量的 T細胞。The T cells of the present invention can be used as active ingredients in therapeutic compositions. Therefore, the present invention also provides a method for killing target cells of a patient, wherein the target cells of the patient abnormally express the polypeptide containing the amino acid sequence of the present invention, and the method includes administering the above effective amount of T cells to the patient.

發明人所用的「異常表達」的意思還包括，與正常組織表達水準相比，多肽過量表達，或該基因在從腫瘤獲得的組織中未表達而在腫瘤中表達。「過量表達」系指多肽水準至少為正常組織中的 1.2 倍；優選為至少為正常組織中的 2 倍，更優選為至少 5 或 10 倍。The term "abnormal expression" as used by the inventors also includes that the polypeptide is overexpressed compared to the expression level of normal tissues, or the gene is not expressed in the tissues obtained from the tumor but is expressed in the tumor. "Overexpression" means that the polypeptide level is at least 1.2 times that of normal tissues; preferably, it is at least 2 times that of normal tissues, and more preferably at least 5 or 10 times.

T細胞可用本領域已知的方法制得（如，上述方法）。T cells can be prepared by methods known in the art (eg, the methods described above).

T細胞繼轉移方案為本領域所熟知的方案。綜述可發現於：Gattioni et al. 和 Morgan et al. (Gattinoni et al., 2006; Morgan et al., 2006)。The T cell subsequent transfer protocol is a well-known protocol in the art. A review can be found in: Gattioni et al. and Morgan et al. (Gattinoni et al., 2006; Morgan et al., 2006).

本發明的另一個方面包括使用與 MHC 複合的肽，以生成 T細胞受體，其核酸被克隆並被引入至宿主細胞，優選為 T細胞。然後，該透過基因工程改變的 T細胞可轉給患者用於癌症治療。Another aspect of the present invention includes the use of peptides complexed with MHC to generate T cell receptors, the nucleic acid of which is cloned and introduced into host cells, preferably T cells. Then, the T cells modified through genetic engineering can be transferred to patients for cancer treatment.

本發明的任一分子（即肽、核酸、抗體、表達載體、細胞，活化T細胞、T細胞受體或編碼核酸）都有益於治療疾病，其特點在於細胞逃避免疫反應的打擊。因此，本發明的任一分子都可用作藥劑或用於製造藥劑。這種分子可單獨使用也可與本發明中的其他分子或已知分子聯合使用。Any molecule of the present invention (ie, peptide, nucleic acid, antibody, expression vector, cell, activated T cell, T cell receptor or encoding nucleic acid) is beneficial to the treatment of diseases, and is characterized in that the cell evades the attack of the immune response. Therefore, any molecule of the present invention can be used as a medicament or for the manufacture of a medicament. This molecule can be used alone or in combination with other molecules in the present invention or known molecules.

本發明還涉及一種套件，其包括： (a) 一個容器，包含上述溶液或凍乾粉形式的藥物組合物； (b) 可選的第二個容器，其含有凍乾粉劑型的稀釋劑或重組溶液；和 (c) 可選的（i）溶液使用或（ii）重組和/或凍幹製劑使用的說明。The present invention also relates to a kit, which includes: (a) A container containing the above-mentioned pharmaceutical composition in the form of a solution or lyophilized powder; (b) An optional second container containing a diluent or reconstituted solution in a lyophilized powder dosage form; and (c) Optional instructions for (i) use of solutions or (ii) use of reconstituted and/or lyophilized preparations.

該套件還步包括一個或多個 (iii) 緩衝劑，(iv) 稀釋劑，(v) 過濾液，(vi) 針，或 (v) 注射器。容器最好是瓶子、小瓶、注射器或試管，可以為多用途容器。藥物組合物最好是凍幹的。The kit also includes one or more (iii) buffers, (iv) diluents, (v) filtrate, (vi) needles, or (v) syringes. The container is preferably a bottle, vial, syringe or test tube, and can be a multi-purpose container. The pharmaceutical composition is preferably lyophilized.

本發明中的套件優選包含一種置於合適容器中的凍幹製劑以及重組和/或使用說明。適當的容器包括，例如瓶子、西林瓶 (如雙室瓶)、注射器 (如雙室注射器) 和試管。該容器可能由多種材料製成，如玻璃或塑膠。試劑盒和/或容器最好有容器或關於容器的說明書，指明重組和/或使用的方向。例如，標籤可能表明凍幹劑型將重組為上述肽濃度。該標籤可進一步表明製劑用於皮下注射。The kit of the present invention preferably contains a lyophilized preparation placed in a suitable container and instructions for reconstitution and/or use. Suitable containers include, for example, bottles, vials (such as dual-chamber vials), syringes (such as dual-chamber syringes), and test tubes. The container may be made of multiple materials, such as glass or plastic. The kit and/or container preferably have a container or instructions on the container, indicating the direction of reconstitution and/or use. For example, the label may indicate that the lyophilized dosage form will be reconstituted to the peptide concentration described above. The label can further indicate that the formulation is for subcutaneous injection.

存放製劑的容器可使用多用途西林瓶，使得可重複給予（例如，2-6 次）重組劑型。該套件可進一步包括裝有合適稀釋劑（如碳酸氫鈉溶液）的第二個容器。A multi-purpose vial can be used as a container for the preparation, so that the reconstituted dosage form can be administered repeatedly (for example, 2-6 times). The kit may further include a second container filled with a suitable diluent (such as sodium bicarbonate solution).

稀釋液和凍幹製劑混合後，重組製劑中的肽終濃度優選為至少 0.15 mg/mL/肽 (=75µg)，不超過 3 mg/mL/肽 (=1500µg)。該套件還可包括商業和用戶角度來說可取的其他材料，包括其他緩衝劑、稀釋劑，過濾液、針頭、注射器和帶有使用說明書的包裝插頁。After mixing the diluent and the lyophilized preparation, the final concentration of the peptide in the recombinant preparation is preferably at least 0.15 mg/mL/peptide (=75µg) and not more than 3 mg/mL/peptide (=1500µg). The kit can also include other materials that are commercially and user-friendly, including other buffers, diluents, filtrate, needles, syringes, and package inserts with instructions for use.

本發明中的套件可能有一個單獨的容器，其中包含本發明所述的藥物組合物製劑，該製劑可有其他成分（例如，其他化合物或及其藥物組合物），也可無其他成分，或者每種成分都有其不同容器。The kit of the present invention may have a separate container, which contains the pharmaceutical composition preparation of the present invention, and the preparation may have other ingredients (for example, other compounds or pharmaceutical compositions thereof), or no other ingredients, or Each ingredient has its own different container.

優選情況是，本發明的套件包括與本發明的一種製劑，包裝後與第二種化合物（如佐劑（例如 GM-CSF）、化療藥物、天然產品、激素或拮抗劑、抗血管生成劑或抑制劑、凋亡誘導劑或螯合劑）或其藥物組合物聯合使用。該套件的成分可進行預絡合或每種成分在給予患者之前可放置於單獨的不同容器。該套件的成分可以是一種或多種溶液，優選為水溶液，更優選為無菌水溶液。該套件的成分也可為固體形式，加入合適的溶劑後轉換為液體，最好放置於另一個不同的容器中。Preferably, the kit of the present invention includes a preparation of the present invention, and after packaging, it is combined with a second compound (such as an adjuvant (such as GM-CSF), chemotherapeutics, natural products, hormones or antagonists, anti-angiogenesis agents or Inhibitors, apoptosis inducers or chelating agents) or their pharmaceutical compositions are used in combination. The components of the kit can be pre-complexed or each component can be placed in a separate container before being administered to the patient. The components of the kit can be one or more solutions, preferably an aqueous solution, more preferably a sterile aqueous solution. The components of the kit can also be in solid form, which can be converted to liquid after adding a suitable solvent, preferably in a different container.

治療套件的容器可能為西林瓶、試管、燒瓶、瓶子、注射器、或任何其他盛裝固體或液體的工具。通常，當成分不只一種時，套件將包含第二個西林瓶或其他容器，使之可以單獨定量。該套件還可能包含另一個裝載藥用液體的容器。優選情況是，治療套件將包含一個設備（如，一個或多個針頭、注射器、滴眼器、吸液管等），使得可注射本發明的藥物（本套件的組合物）。The container of the treatment kit may be a vial, test tube, flask, bottle, syringe, or any other tool for holding solids or liquids. Usually, when there is more than one ingredient, the kit will include a second vial or other container so that it can be quantified separately. The kit may also contain another container for medicinal liquids. Preferably, the treatment kit will include a device (eg, one or more needles, syringes, eye drops, pipettes, etc.) to allow injection of the drug of the present invention (composition of the kit).

本發明的藥物配方適合以任何可接受的途徑進行肽給藥，如口服（腸道）、鼻內、眼內、皮下、皮內、肌內，靜脈或經皮給藥。優選為皮下給藥，最優選為皮內給藥，也可透過輸液泵給藥。The pharmaceutical formulation of the present invention is suitable for peptide administration by any acceptable route, such as oral (intestinal), intranasal, intraocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal administration. It is preferably subcutaneous administration, most preferably intradermal administration, and can also be administered through an infusion pump.

由於本發明的肽從 CLL 中分離而得，因此，本發明的藥劑優選用於治療 CLL。Since the peptide of the present invention is isolated from CLL, the agent of the present invention is preferably used for the treatment of CLL.

本發明進一步涉及為個體患者製備個體化藥物的一種方法，其中包括：製造含選自預篩選 TUMAP 存儲庫至少一種肽的藥物組合物，其中藥物組合物中所用的至少一種肽選擇為適合於個體患者。在一項實施方案中，藥物組合物為一種疫苗。該方法也可以改動以產生下游應用的 T細胞克隆物，如：TCR 隔離物或可溶性抗體和其他治療選擇。The present invention further relates to a method for preparing a personalized medicine for an individual patient, which comprises: manufacturing a pharmaceutical composition containing at least one peptide selected from the pre-screened TUMAP repository, wherein the at least one peptide used in the pharmaceutical composition is selected to be suitable for the individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method can also be modified to generate T cell clones for downstream applications, such as TCR spacers or soluble antibodies and other treatment options.

「個體化藥物」系指專門針對個體患者的治療，將僅用於該等個體患者，包括個體化活性癌症疫苗以及使用自體組織的過繼細胞療法。"Individualized medicines" refer to treatments specifically targeted at individual patients and will only be used for such individual patients, including individualized active cancer vaccines and adoptive cell therapy using autologous tissues.

如本文所述，「存儲庫」應指已經接受免疫原性預篩查和/或在特定腫瘤類型中過量表現的一組或一系列肽。「存儲庫」一詞並不暗示，疫苗中包括的特定肽已預先製造並儲存於物理設備中，雖然預期有這種可能性。明確預期所述肽可以用於新製造每種個體化疫苗，也可能被預先製造和儲存。存儲庫（例如，資料庫形式）由腫瘤相關肽組成，其在各種 HLA-A HLA-B 和 HLA-C 等位元基因 CLL 患者的腫瘤組織中高度過度表達。其可能含有包括 MHC I類和 MHC II類肽或拉長的 MHC I類肽。除了從幾種 CLL 組織中採集的腫瘤相關肽外，存儲庫還可能包含 HLA-A*02 和 HLA-A*24 標記肽。這些肽可對 TUMAP 誘導的 T細胞免疫進行量化比較，從而可得出疫苗抗腫瘤反應能力的重要結論。其次，在沒有觀察到來自患者「自身」抗原 TUMAP 的任何疫苗誘導的 T細胞反應時，它們可作為來自「非自身」抗原的重要陽性對照肽。第三，它還可對患者的免疫功能狀態得出結論。As described herein, "repository" shall refer to a group or series of peptides that have undergone pre-screening for immunogenicity and/or overrepresented in a specific tumor type. The term "repository" does not imply that the specific peptides included in the vaccine have been pre-manufactured and stored in physical equipment, although this possibility is expected. It is clearly expected that the peptide can be used to newly manufacture each individualized vaccine, or it may be pre-manufactured and stored. The repository (for example, in the form of a database) is composed of tumor-associated peptides, which are highly overexpressed in the tumor tissues of patients with various HLA-A, HLA-B and HLA-C alleles CLL. It may contain MHC class I and MHC class II peptides or elongated MHC class I peptides. In addition to tumor-related peptides collected from several CLL tissues, the repository may also contain HLA-A*02 and HLA-A*24 labeled peptides. These peptides can be used to quantitatively compare the T cell immunity induced by TUMAP, and thus can draw important conclusions about the vaccine's anti-tumor response ability. Secondly, when no vaccine-induced T cell response from the patient's "self" antigen TUMAP is observed, they can be used as important positive control peptides from the "non-self" antigen. Third, it can also draw conclusions about the patient's immune function status.

存儲庫的 TUMAP 透過使用一種功能基因組學方法進行鑒定，該方法結合了基因表達分析、質譜法和 T細胞免疫學 (XPresident ®)。該方法確保了只選擇真實存在于高百分比腫瘤但在正常組織中不表達或僅很少量表達的 TUMAP 用於進一步分析。對於初始肽的選擇，患者 CLL 樣本和健康供體的血液以循序漸進的方法進行分析： 1. 惡性材料的 HLA  配體用質譜法確定 2. 使用全基因組信使核糖核酸 (mRNA) 表達分析法用於確定惡性腫瘤組織 (CLL) 與一系列正常器官和組織相比過度表達的基因。 3. 確定的 HLA 配體與基因表達資料進行比較。腫瘤組織上過度表現或選擇性表現的肽，優選為第 2 步中檢測到的選擇性表達或過量表達基因所編碼的考慮為多肽疫苗的合適候選 TUMAP。 4. 文獻檢索以確定更多證據以支持確認為 TUMP 的肽的相關性 5. 過度表達在 mRNA 水準的相關性由腫瘤組織第 3 步選定的 TUMAP 重新檢測而確定，並且在健康組織上缺乏（或不經常）檢測。 6. 為了評估透過選定的肽誘導體內 T細胞反應是否可行，使用健康供體以及 CLL 患者的人 T細胞進行體外免疫原性測定。The TUMAP of the repository is identified by using a functional genomics method that combines gene expression analysis, mass spectrometry, and T cell immunology (XPresident ®). This method ensures that only TUMAPs that are actually present in a high percentage of tumors but are not expressed in normal tissues or are expressed only in small amounts are selected for further analysis. For the selection of initial peptides, patients’ CLL samples and healthy donor blood are analyzed in a step-by-step method: 1. The HLA ligand of the malignant material is determined by mass spectrometry 2. Use genome-wide messenger ribonucleic acid (mRNA) expression analysis to identify genes that are overexpressed in malignant tumor tissue (CLL) compared to a range of normal organs and tissues. 3. The determined HLA ligand is compared with the gene expression data. The peptides overexpressed or selectively expressed on tumor tissues are preferably TUMAPs encoded by the selectively expressed or overexpressed genes detected in step 2 and are considered suitable candidates for peptide vaccines. 4. Literature search to determine more evidence to support the relevance of peptides identified as TUMP 5. The relevance of overexpression in mRNA levels is determined by the re-testing of TUMAP selected in step 3 of tumor tissues, and it is lacking (or infrequently) tested in healthy tissues. 6. In order to evaluate the feasibility of inducing T cell responses in vivo through selected peptides, human T cells from healthy donors and CLL patients were used for in vitro immunogenicity assays.

一方面，在將所述肽加入存儲庫之前，對其進行篩查以瞭解免疫原性。舉例來說（但不限於此），納入存儲庫的肽的免疫原性的確定方法包括體外 T細胞活化，具體為：用裝載肽/MHC 複合物和抗 CD28 抗體的人工抗原表現細胞反復刺激來自健康供體的 CD8+ T細胞。On the one hand, before adding the peptides to the reservoir, they are screened for immunogenicity. For example (but not limited to this), methods for determining the immunogenicity of peptides included in the repository include in vitro T cell activation, specifically: artificial antigen expression cells loaded with peptide/MHC complexes and anti-CD28 antibodies are repeatedly stimulated from CD8+ T cells from healthy donors.

這種方法優選用於罕見癌症以及有罕見表達譜的患者。與含目前開發為固定組分的多肽雞尾酒相反的是，存儲庫可將腫瘤中抗原的實際表達於疫苗進行更高程度的匹配。在多目標方法中，每名患者將使用幾種「現成」肽的選定單一肽或組合。理論上來說，基於從 50 抗原肽庫中選擇例如 5 種不同抗原肽的一種方法可提供大約 170萬 種可能的藥物產品 (DP) 組分。This method is preferably used for rare cancers and patients with rare expression profiles. In contrast to the peptide cocktails that are currently developed as fixed components, the repository can match the actual expression of the antigen in the tumor with the vaccine to a higher degree. In the multi-target approach, each patient will use a selected single peptide or combination of several "off-the-shelf" peptides. In theory, a method based on selecting, for example, 5 different antigen peptides from a library of 50 antigen peptides can provide approximately 1.7 million possible drug product (DP) components.

在一方面，選擇所述肽用於疫苗，其基於個體患者的適合性，並使用本發明此處或後文所述的方法。In one aspect, the peptides are selected for use in vaccines based on the suitability of the individual patient and using the methods of the present invention as described here or hereinafter.

HLA 表型、轉錄和肽組學資料從患者的腫瘤材料和血液樣本中收集，以確定最合適每名患者且含有「存儲庫」和患者獨特（即突變）TUMAP 的肽。將選擇的那些肽選擇性地或過度表達于患者腫瘤中，並且可能的情況下，如果用患者個體 PBMC 進行檢測，則表現出很強的體外免疫原性。HLA phenotype, transcription, and peptomic data are collected from patients’ tumor materials and blood samples to determine the most suitable peptides for each patient and contain a “repository” and the patient’s unique (ie, mutated) TUMAP. The selected peptides are selectively or overexpressed in the patient's tumor, and if possible, if the patient's individual PBMC is used for detection, it will show strong in vitro immunogenicity.

優選的情況是，疫苗所包括的肽的一種確定方法包括：(a) 識別由來自個體患者腫瘤樣本表現的腫瘤相關肽 (TUMAP)；(b) 將 (a) 中鑒定的肽與上述肽的存儲庫（資料庫）進行比對；且 (c) 從與患者中確定的腫瘤相關肽相關的存儲庫（資料庫）中選擇至少一種肽。例如，腫瘤樣本表現的 TUMAP 的鑒定方法有：(a1) 將來自腫瘤樣本的表達資料與所述腫瘤樣本組織類型相對應的正常組織樣本的表達資料相比對，以識別腫瘤組織中過量表達或異常表達的蛋白；以及 (a2) 將表達資料與結合到腫瘤樣本中 I類MHC 和/或 II 類分子的 MHC 配體序列想關聯，以確定來源於腫瘤過量表達或異常表達的蛋白質的 MHC 配體。優選情況是，MHC 配體的序列的確定方法是：洗脫來自腫瘤樣本分離的 MHC 分子結合肽，並測序洗脫配體。優選情況是，腫瘤樣本和正常組織從同一患者獲得。Preferably, a method for determining the peptides included in the vaccine includes: (a) identifying tumor-associated peptides (TUMAP) expressed by tumor samples from individual patients; (b) combining the peptides identified in (a) with the above-mentioned peptides The repository (database) is compared; and (c) at least one peptide is selected from the repository (database) related to the tumor-associated peptide identified in the patient. For example, the methods for identifying TUMAP expressed in tumor samples include: (a1) The expression data from the tumor sample is compared with the expression data of the normal tissue sample corresponding to the tumor sample tissue type to identify the overexpression or expression in the tumor tissue. Abnormally expressed protein; and (a2) Correlate the expression data with the MHC ligand sequence bound to the MHC class I and/or class II molecules in the tumor sample to determine the MHC ligand sequence derived from the overexpressed or abnormally expressed protein of the tumor. body. Preferably, the method for determining the sequence of the MHC ligand is: eluting the MHC molecule-binding peptide isolated from the tumor sample, and sequencing the eluted ligand. Preferably, the tumor sample and normal tissue are obtained from the same patient.

除了使用存儲庫（資料庫）模型選擇肽以外，或作為一種替代方法，TUMAP可能在新患者中進行鑒定，然後列入疫苗中。作為一種實施例，患者中的候選TUMAP可透過以下方法進行鑒定：(a1)將來自腫瘤樣本的表達資料與所述腫瘤樣本組織類型相對應的正常組織樣本的表達資料相比對，以識別腫瘤組織中過量表達或異常表達的蛋白；以及(a2)將表達資料與結合到腫瘤樣本中I類MHC和/或II類分子的MHC配體序列想關聯，以確定來源於腫瘤過量表達或異常表達的蛋白質的MHC配體。作為另一實施例，蛋白的鑒定方法為可包含突變，其對於腫瘤樣本相對于個體患者的相應正常組織是獨特的，並且TUMAP可透過特異性靶向作用於變異來鑒定。例如，腫瘤以及相應正常組織的基因組可透過全基因組測序方法進行測序：為了發現基因蛋白質編碼區域的非同義突變，從腫瘤組織中萃取基因組DNA和RNA，從外周血單核細胞(PBMC)中提取正常非突變基因組種系DNA。運用的NGS方法只限于蛋白編碼區的重測序（外顯子組重測序）。為了這一目的，使用供應商提供的靶序列富集試劑盒來捕獲來自人樣本的外顯子DNA，隨後使用HiSeq2000（Illumina公司）進行測序。此外，對腫瘤的mRNA進行測序，以直接定量基因表達，並確認突變基因在患者腫瘤中表達。得到的數以百萬計的序列讀數透過軟體演算法處理。輸出列表中包含突變和基因表達。腫瘤特異性體突變透過與PBMC衍生的種系變化比較來確定，並進行優化。然後，為了存儲庫可能測試新確定的肽瞭解如上所述的免疫原性，並且選擇具有合適免疫原性的候選TUMAP用於疫苗。In addition to using a repository (database) model to select peptides, or as an alternative method, TUMAP may be identified in new patients and then included in the vaccine. As an example, the candidate TUMAP in the patient can be identified by the following method: (a1) The expression data from the tumor sample is compared with the expression data of the normal tissue sample corresponding to the tissue type of the tumor sample to identify the tumor Overexpressed or abnormally expressed proteins in the tissue; and (a2) Correlate the expression data with the MHC ligand sequence bound to the MHC class I and/or class II molecules in the tumor sample to determine the overexpression or abnormal expression from the tumor The MHC ligand of the protein. As another example, the protein identification method may include mutations, which are unique to the tumor sample relative to the corresponding normal tissues of the individual patient, and TUMAP can be identified through specific targeting of mutations. For example, the genomes of tumors and corresponding normal tissues can be sequenced by whole-genome sequencing methods: in order to find non-synonymous mutations in gene protein coding regions, genomic DNA and RNA are extracted from tumor tissues, and extracted from peripheral blood mononuclear cells (PBMC) Normal non-mutated genomic germline DNA. The NGS method used is limited to the resequencing of protein coding regions (exome resequencing). For this purpose, the target sequence enrichment kit provided by the supplier was used to capture exon DNA from human samples, and then HiSeq2000 (Illumina) was used for sequencing. In addition, the tumor's mRNA is sequenced to directly quantify gene expression and confirm that the mutated gene is expressed in the patient's tumor. The millions of serial readings obtained are processed through software algorithms. The output list contains mutations and gene expression. Tumor-specific body mutations are determined by comparison with PBMC-derived germline changes and optimized. Then, for the repository, it is possible to test the newly determined peptides to understand the immunogenicity as described above, and to select candidate TUMAPs with suitable immunogenicity for the vaccine.

在一個示範實施方案中，疫苗中所含肽透過以下方法確定：(a)用上述方法識別由來自個體患者腫瘤樣本表現的腫瘤相關肽(TUMAP)；(b)將(a)中鑒定的肽與進行腫瘤（與相應的正常組織相比）免疫原性和過量表現預篩查肽的存儲庫進行比對；(c)從與患者中確定的腫瘤相關肽相關的存儲庫中選擇至少一種肽；及(d)可選地在(a)中選擇至少一種新確定的肽，確認其免疫原性。In an exemplary embodiment, the peptides contained in the vaccine are determined by the following method: (a) using the above method to identify tumor-associated peptides (TUMAP) expressed by tumor samples from individual patients; (b) combining the peptides identified in (a) Compare with the library of tumor (compared to the corresponding normal tissue) immunogenicity and overexpression of pre-screening peptides; (c) select at least one peptide from the library related to the tumor-associated peptides identified in the patient ; And (d) optionally selecting at least one newly identified peptide in (a) to confirm its immunogenicity.

在一個示範實施方案中，疫苗中所含肽透過以下方法確定：(a) 識別由來自個體患者腫瘤樣本表現的腫瘤相關肽 (TUMAP)；以及 (b) 在 (a) 中選擇至少一種新確定的肽，並確認其免疫原性。In an exemplary embodiment, the peptides contained in the vaccine are determined by: (a) identifying tumor-associated peptides (TUMAP) expressed by tumor samples from individual patients; and (b) selecting at least one new determination in (a) Peptides and confirm their immunogenicity.

一旦選定了用於個體化肽疫苗的肽時，則產生疫苗。該疫苗優選為一種液體製劑，包括溶解於 20-40% DMSO 之間，優選為約 30-35% DMSO，例如，約 33% DMSO 中的個體肽。Once the peptides for the personalized peptide vaccine are selected, the vaccine is produced. The vaccine is preferably a liquid formulation comprising individual peptides dissolved in 20-40% DMSO, preferably about 30-35% DMSO, for example, about 33% DMSO.

列入產品的每種肽都溶於 DMSO 中。單個肽溶液濃度的選擇取決於要列入產品中的肽的數量。單肽-DMSO 溶液均等混合，以實現一種溶液中包含所有的肽，且濃度為每肽~2.5 mg/ml。然後該混合溶液按照1：3比例用注射用水進行稀釋，以達到在 33% DMSO 中每肽 0.826 mg/ml 的濃度。稀釋的溶液透過 0.22 μm 無菌篩檢程序進行過濾。從而獲得最終本體溶液。Each peptide listed in the product is dissolved in DMSO. The choice of the concentration of a single peptide solution depends on the number of peptides to be included in the product. The single peptide-DMSO solution is evenly mixed to achieve a solution containing all the peptides, and the concentration is ~2.5 mg/ml per peptide. Then the mixed solution was diluted with water for injection in a ratio of 1:3 to achieve a concentration of 0.826 mg/ml per peptide in 33% DMSO. The diluted solution is filtered through a 0.22 μm sterile screening program. Thereby, the final bulk solution is obtained.

最終本體溶液填充到小瓶中，在使用前儲存於-20℃下。一個小瓶包含 700 μL 溶液，其中每種肽含有 0.578 mg。其中的 500 μL（每種肽約 400 μg）將用於皮內注射。The final bulk solution was filled into the vial and stored at -20°C before use. One vial contains 700 μL of solution, where each peptide contains 0.578 mg. 500 μL (approximately 400 μg for each peptide) will be used for intradermal injection.

本發明的肽除了用於治療癌症，也可用於診斷。由於肽由 CLL 細胞產生，並且已確定這些肽在正常組織中不存在或水準較低，因此這些肽可用於診斷癌症是否存在。The peptides of the present invention can also be used for diagnosis in addition to the treatment of cancer. Since the peptides are produced by CLL cells, and it has been determined that these peptides are absent or low in normal tissues, these peptides can be used to diagnose the presence of cancer.

血液樣本中組織活檢物含請求項的肽，可有助於病理師診斷癌症。用抗體、質譜或其他本領域內已知的方法檢測某些肽可使病理師判斷該組織樣本為惡性的還是炎症或一般病變，也可用作CLL的生物標誌物。肽基團的表現使得能對病變組織進行分類或進一步分成子類。The tissue biopsy in the blood sample contains the requested peptides, which can help pathologists diagnose cancer. Using antibodies, mass spectrometry, or other methods known in the art to detect certain peptides can enable pathologists to determine whether the tissue sample is malignant or inflammatory or general, and can also be used as a biomarker for CLL. The performance of the peptide group enables the classification or further sub-categories of diseased tissues.

對病變標本中肽的檢測使得能對免疫系統治療方法的利益進行判斷，特別是如果 T- 淋巴細胞已知或預計與作用機制有關。MHC 表達的缺失是一種機制，充分說明了哪些受感染的惡性細胞逃避了免疫監視。因此，肽的表現表明，分析過的細胞並沒有利用這種機制。The detection of peptides in diseased specimens makes it possible to judge the benefits of immune system treatments, especially if T-lymphocytes are known or expected to be related to the mechanism of action. The lack of MHC expression is a mechanism that fully illustrates which infected malignant cells evade immune surveillance. Therefore, the performance of the peptide indicated that the analyzed cells did not utilize this mechanism.

本發明的肽可用於分析淋巴細胞對肽的反應（如 T細胞反應），或抗體對肽或 MHC 分子絡合的肽發生的反應。這些淋巴細胞反應可以作為預後指標，決定是否採取進一步的治療。這些反應也可以用作免疫療法中的替代反應指標，旨在以不同方式誘導淋巴細胞反應，如接種蛋白疫苗、核酸、自體材料、淋巴細胞過繼轉移。基因治療中，淋巴細胞對肽發生的反應可以在副作用的評估中考慮。淋巴細胞反應監測也可能成為移植療法隨訪檢查中的一種有價值的工具，如，用於檢測移植物抗宿主和宿主抗移植物疾病。The peptides of the present invention can be used to analyze the response of lymphocytes to peptides (such as T cell responses), or the response of antibodies to peptides or peptides complexed with MHC molecules. These lymphocyte responses can be used as prognostic indicators to decide whether to take further treatment. These responses can also be used as alternative response indicators in immunotherapy, aiming to induce lymphocyte responses in different ways, such as protein vaccination, nucleic acid, autologous materials, and lymphocyte adoptive transfer. In gene therapy, the response of lymphocytes to peptides can be considered in the evaluation of side effects. Lymphocyte response monitoring may also become a valuable tool in the follow-up examination of transplantation therapy, for example, for the detection of graft-versus-host and host-versus-graft diseases.

下列描述優選方案的實施例將對本發明進行說明，並參照隨附圖表（但是不僅限於此）。考慮到本發明的目的，文中引用的所有參考文獻透過引用的方式併入在本文中。The following examples describing preferred solutions will illustrate the present invention, with reference to the accompanying chart (but not limited to this). In consideration of the purpose of the present invention, all references cited in the text are incorporated herein by reference.

實施例 實施例 1細胞表面表現的腫瘤相關肽的識別和定量 組織樣本 Examples Example 1 Recognition and quantification of tumor-associated peptides expressed on the surface of cells in tissue samples

患者的腫瘤組織獲得自：ProteoGenex Inc. (Culver City, CA, USA)、Tissue Solutions Ltd (Glasgow, UK)、波恩大學醫院 (Bonn, Germany)、蒂賓根大學醫院 (Tübingen, Germany)。The patient's tumor tissue was obtained from: ProteoGenex Inc. (Culver City, CA, USA), Tissue Solutions Ltd (Glasgow, UK), Bonn University Hospital (Bonn, Germany), Tübingen University Hospital (Tübingen, Germany).

正常組織獲得自 Asterand (Detroit, MI, USA & Royston, Herts, UK)、Bio-Options Inc. (Brea, CA, USA)、BioServe (Beltsville, MD, USA)、Capital BioScience Inc. (Rockville, MD, USA)、Geneticist Inc. (Glendale, CA, USA)、京都府立醫科大學 (KPUM) (Kyoto, Japan)、日內瓦大學醫院 (Geneva, Switzerland)、海德堡大學醫院 (Heidelberg, Germany)、慕尼克大學醫院 (Munich, Germany)、蒂賓根大學醫院 (Tübingen, Germany)。Normal tissues were obtained from Asterand (Detroit, MI, USA & Royston, Herts, UK), Bio-Options Inc. (Brea, CA, USA), BioServe (Beltsville, MD, USA), Capital BioScience Inc. (Rockville, MD, USA), Geneticist Inc. (Glendale, CA, USA), Kyoto Prefectural Medical University (KPUM) (Kyoto, Japan), Geneva University Hospital (Geneva, Switzerland), Heidelberg University Hospital (Heidelberg, Germany), Munich University Hospital ( Munich, Germany), Tübingen University Hospital (Tübingen, Germany).

所有患者在手術或屍檢前都獲得了書面知情同意。切除後組織立即進行冷休克處理，在分離 TUMAP 前儲存於 -70°C 或以下。從組織樣本中分離 HLA 肽 All patients obtained written informed consent before surgery or autopsy. The tissue is immediately subjected to cold shock treatment after excision, and stored at -70°C or below before separating TUMAP. Isolation of HLA peptides from tissue samples

根據方案 (Falk et al., 1991; Seeger et al., 1999) 略加修改，使用HLA-A*02特異性抗體BB7.2、HLA-A、HLA-B、HLA­C特異性抗體W6/32、CNBr活化的瓊脂糖凝膠、酸處理和超濾方法以免疫沉澱法從實體組織中獲得了冷凍組織樣本的 HLA 肽庫。質譜分析 According to the protocol (Falk et al., 1991; Seeger et al., 1999) slightly modified, using HLA-A*02 specific antibody BB7.2, HLA-A, HLA-B, HLAC specific antibody W6/32, CNBr-activated agarose gel, acid treatment and ultrafiltration methods were used to obtain HLA peptide libraries of frozen tissue samples from solid tissues by immunoprecipitation. Mass Spectrometry

獲得的 HLA 肽庫根據其疏水性用反相色譜 (nanoAcquity UPLC system, Waters) 分離，洗脫肽用裝有電噴霧源的 LTQ- velos 融合雜交質譜 (ThermoElectron) 進行了分析。肽庫被直接載入填充有 1.7 µm C18 反相材料 (Waters) 的分析用熔煉石英微毛細管柱（75 µm 內徑x 250 mm），應用流速為 400 nL 每分鐘。隨後，使用來自流速為 300 nL每分鐘、濃度為10%至33% 溶劑 B 中的兩步180分鐘二元梯度法對肽進行分離。梯度由溶劑 A（含0.1% 甲酸的水）和溶劑 B（含0.1 % 甲酸的乙腈）。金鍍膜玻璃毛細管 (PicoTip, New Objective) 用於引入到納升電噴霧源。使用前 5 (TOP5) 策略在資料依賴模式下操作 LTQ-Orbitrap 質譜儀。簡言之，首先以高精確品質完全掃描在 orbitrap 開始一個掃描週期 (R= 30 000)，之後用先前選定離子的動態排除技術在 orbitrap 中對 5 種含量最為豐富的前體離子進行 MS/MS 掃描 (R = 7500)。串聯質譜以 SEQUEST 和另一種手動控制器進行解讀。生成的自然肽破碎模式與合成序列相同參考肽的破碎模式進行比較後，確保了被識別的肽序列。The obtained HLA peptide library was separated by reversed-phase chromatography (nanoAcquity UPLC system, Waters) based on its hydrophobicity, and the eluted peptides were analyzed by LTQ-velos fusion hybrid mass spectrometry (ThermoElectron) equipped with an electrospray source. The peptide library was directly loaded into a fused silica microcapillary column (75 µm inner diameter x 250 mm) filled with 1.7 µm C18 reversed-phase material (Waters) for analysis, and the applied flow rate was 400 nL per minute. Subsequently, the peptides were separated using a two-step 180-minute binary gradient method from 10% to 33% solvent B at a flow rate of 300 nL per minute. The gradient consists of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). Gold-coated glass capillary (PicoTip, New Objective) is used to introduce into the nano-electrospray source. Use the top 5 (TOP5) strategy to operate the LTQ-Orbitrap mass spectrometer in data-dependent mode. In short, first complete scanning with high precision quality and start a scanning cycle (R=30 000) in orbitrap, and then use the dynamic exclusion technology of the previously selected ions to perform MS/MS on the five most abundant precursor ions in orbitrap Scan (R = 7500). The tandem mass spectrum is interpreted with SEQUEST and another manual controller. After comparing the fragmentation pattern of the generated natural peptide with the fragmentation pattern of the reference peptide with the same synthetic sequence, the identified peptide sequence is ensured.

無標記相對 LC-MS定量透過離子計數（即透過LC-MS功能提取和分析）來進行 (Mueller et al., 2007)。該方法假定肽的 LC-MS 信號區域與樣本中其豐度相關。提取的特徵透過充電狀態去卷積和保留時間校準進行進一步處理  (Mueller et al., 2008; Sturm et al., 2008)。最後，所有的 LC-MS 特徵與序列鑒定結果交叉引用，以將不同樣本和組織的定量資料與肽呈遞特徵結合。定量資料根據集中資料以兩層方式進行正態化處理，以說明技術和生物學複製變異。因此，每個被識別的肽均可與定量資料相關，從而可得出樣本和組織之間的相對定量。此外，對候選肽獲得的所有定量資料進行手動檢查，以確保資料的一致性，並驗證自動化分析的準確度。對於每種肽，計算了表現圖，其顯示樣本平均表現量以及複製變化。這些特徵使 CLL 樣本與正常組織樣本的基線值並列。示範性過度表現肽的表現譜示於圖 1 中。示範性肽的表現分數見表 8。Unlabeled relative LC-MS quantitative ion counting (ie extraction and analysis through LC-MS function) is performed (Mueller et al., 2007). This method assumes that the LC-MS signal area of the peptide is related to its abundance in the sample. The extracted features are further processed through state-of-charge deconvolution and retention time calibration (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MS features and sequence identification results are cross-referenced to combine quantitative data from different samples and tissues with peptide presentation features. Quantitative data are normalized in a two-tiered manner based on centralized data to illustrate technical and biological replication variation. Therefore, each identified peptide can be correlated with quantitative data, so that the relative quantification between the sample and the tissue can be obtained. In addition, all quantitative data obtained by candidate peptides are manually checked to ensure the consistency of the data and verify the accuracy of the automated analysis. For each peptide, a performance map was calculated, which shows the average performance of the sample and the change in replication. These characteristics align the baseline values of CLL samples with normal tissue samples. The performance profile of an exemplary over-expressing peptide is shown in Figure 1. The performance scores of the exemplary peptides are shown in Table 8.

表 8：表現分數。該表列出了與一系列正常組織相比在腫瘤上非常高度過量表現 (+++)、與一系列正常組織相比在腫瘤上高度過量表現 (++) 或與一系列正常組織相比在腫瘤上過量表現 (+) 的 HLA-A*02 肽。被認為與腫瘤比較相關的一系列正常組織組包括：脂肪組織、腎上腺、血細胞、血管、骨髓、腦、乳腺、食道、眼、膽囊、心臟、腎、大腸、肝、肺、淋巴結、神經、胰腺、甲狀旁腺、腹膜、垂體、胸膜、唾液腺、骨骼肌、皮膚、小腸、脾、胃、甲狀腺、氣管、輸尿管、膀胱。 序列ID號 序列 肽表現 1 AIPPSFASIFL +++ 2 ALHRPDVYL +++ 3 VIAELPPKV +++ 4 VIAELPPKVSV +++ 5 ALIFKIASA +++ 6 ALDTLEDDMTI +++ 7 ALLERTGYTL +++ 8 ALAASALPALV +++ 9 ALCDTLITV +++ 10 FVYGESVEL +++ 11 ALFTFJPLTV +++ 12 ALGEDEITL +++ 13 VVDGMPPGV ++ 14 ALLRLLPGL + 15 ALPEVSVEA + 16 ALPGGAAVAAV ++ 17 ALTKTNLQL +++ 18 LLGEFSIKM +++ 19 QVMEKLAAV + 20 ALVDPGPDFVV + 21 ALWAGLLTL +++ 22 ALWDPVIEL +++ 23 ALYLTEVFL +++ 24 AMAGDVVYA +++ 25 ATYSGLESQSV + 27 AVLGLVWLL ++ 28 AVLHLLLSV +++ 29 AVLQAVTAV +++ 30 ELLEGSEIYL + 31 ELMHGVAGV ++ 32 FIDKFTPPV +++ 33 FIINSSNIFL +++ 35 FILPSSLYL +++ 36 FILTHVDQL +++ 37 FIMEGGAMVL +++ 39 FLDALLTDV +++ 40 FLDEDDMSL + 41 FLDPSLDPLL +++ 43 FLLGPEALSFA +++ 45 FLPELPADLEA ++ 47 FLSDQPEPYL ++ 48 FLSPQQPPLL +++ 49 FLTDLFAQL + 50 FLFEPVVKAFL +++ 51 FLVEAPHDWDL ++ 52 FLVETGFLHV +++ 53 FLWQHVELVL +++ 54 FLYPFPLALF +++ 56 FVFEAPYTL +++ 57 GLSEISLRL +++ 58 YIQQGIFSV ++ 59 FVFGDENGTVSL +++ 60 FVLDHEDGLNL ++ 61 FVYFIVREV +++ 62 GIIDGSPRL + 63 SLAHVAGCEL +++ 64 KLLESVASA ++ 65 GLDDMKANL +++ 66 SLAGGLDDMKA +++ 68 GLDQQFAGLDL +++ 69 GLHQREIFL +++ 70 FVPDTPVGV ++ 71 GLKHDIARV +++ 72 GLLDAGKMYV +++ 73 GLLEVISALQL +++ 74 GLLRASFLL +++ 76 GLLRIIPYL ++ 77 GLLRLTWFL ++ 78 GLPSFLTEV +++ 79 GLQAKIQEA +++ 80 VLIEDELEEL +++ 81 WLVGQEFEL +++ 82 GLQSGVDIGV + 83 GQGEVLVYV + 86 HLYPGAVTI +++ 88 ILDEIGADVQA +++ 89 ILDFGTFQL +++ 90 VIADLGLIRV +++ 92 ILEPLNPLL +++ 93 ILFNTQINI +++ 94 ILFPLRFTL +++ 95 ILGYMAHEHKV +++ 96 ILIDKTSFV + 97 LLFATQITL + 98 SLIKYFLFV +++ 99 ILIFHSVAL +++ 100 ILNNEVFAI + 101 ILVVIEPLL +++ 102 IQDRAVPSL +++ 103 KLGGTPAPA ++ 104 KLILLDTPLFL +++ 105 KLMNDIADI +++ 106 FMASHLDYL +++ 107 ILYNLYDLL ++ 108 VIYTLIHYI + 109 KLWEGLTELV ++ 112 KTLDVDATYEI +++ 113 KVPAEEVLVAV + 114 LIPEGPPQV +++ 115 LLFDKLYLL +++ 116 LLIGATMQV +++ 117 LLILENILL ++ 118 RLLILENILL +++ 119 VLPAEFFEV +++ 121 RLYEITIEV ++ 122 LLIPVVPGV ++ 123 LLLAEAELLTL +++ 124 LLLEETEKQAV ++ 125 LLLEIGEVGKLFV ++ 126 LLPEGGITAI +++ 127 LLPTAPTTV +++ 128 LLSEEEYHL +++ 129 LLVGTLDVV + 130 LLVLIPVYL +++ 131 LQALEVLKI +++ 132 LVYEAIIMV +++ 133 YLLSGDISEA +++ 134 MLLEHGITLV +++ 135 MTAGFSTIAGSV +++ 137 NLIKTVIKL + 138 NLLDIDAPVTV ++ 139 NLTDVVEKL +++ 140 QIAELPATSV +++ 141 QILSEIVEA ++ 142 QLDEPAPQV +++ 144 QLPPFPREL +++ 145 SALDTITTV +++ 146 SIIEGPIIKL +++ 147 SILETVATL +++ 148 SIVASLITV +++ 149 SLDNGGYYI +++ 153 SLLAELHVL ++ 154 SLLAELHVLTV ++ 155 SLMLEVPAL +++ 157 SLNIRDFTM +++ 160 SLSFLVPSL +++ 162 SMKDDLENV +++ 163 SQLDISEPYKV + 164 SVHKGFAFV +++ 165 TLDDDLDTV +++ 166 TLDPNQVSL +++ 167 TLDTSKLYV +++ 168 YLLDQSFVM +++ 169 TLLLGLTEV +++ 170 TLTFRVETV +++ 171 TLVPPAALISI +++ 172 TLYDMLASI +++ 174 VLAELPIIVV +++ 175 FTVPRVVAV ++ 176 VLAEQNIIPSA +++ 177 VLDDRELLL +++ 178 VLFFNVQEV +++ 179 VLLGLEMTL +++ 181 VLLSIPFVSV + 182 VLLSVPGPPV +++ 185 VMDDQRDLI + 186 VMDPTKILI +++ 187 VMDTHLVNI +++ 189 VMLEMTPEL +++ 190 VVMGTVPRL +++ 191 YIFDGSDGGV +++ 192 YIQEYLTLL +++ 193 YLDLSNNRL +++ 194 YLDNVLAEL + 195 YLGGFALSV +++ 197 YLQEVPILTL +++ 198 YLTFLPAEV +++ 199 YLVELSSLL +++ 200 YMFEEVPIVI +++ 201 YQLELHGIEL +++ 202 YVDDVFLRV +++ 204 GLLQINDKIAL ++ 205 GLSQANFTL + 206 HMQDVRVLL +++ 208 ILLKTEGINL ++ 209 ILQAELPSL ++ 210 KLLVQDFFL +++ 211 LIDVKPLGV +++ 212 NIIEAINELLV ++ 213 RLLYQLVFL ++ 214 RLQELTEKL + 215 VMQDIVYKL + 217 ALDEPPYLTV + 218 ALGEEWKGYVV + 219 ALLNLLESA ++ 221 ALVSTIIMV + 225 ILQERELLPV + 226 AMNISVPQV + 227 FLAEASVMTQL ++ 228 FLGGLSPGV ++ 229 FLLNLQNCHL ++ 231 FLYIRQLAI + 232 FMHQIIDQV + 234 GLDDAEYAL + 235 GLDDLLLFL + 236 GLLESGRHYL +++ 237 GLQENLDVVV + 238 GLVETELQL + 240 ILARDILEI ++ 241 ILGDILLKV + 242 ILLGIQELL ++ 243 ILPTLEKELFL + 244 ILQALAVHL ++ 245 KIMDYSLLLGV + 248 KTVEPPISQV + 249 LLPTGVFQV + 251 LLYDNVPGA + 254 QLIPKLIFL +++ 255 YLFEEAISM + 258 RLDYITAEI ++ 259 RLLDEQFAVL + 260 SLDDVEGMSV ++ 261 SLVEAQGWLV ++ 263 SQWEDIHVV ++ 264 TILDYINVV +++ 266 TLLDQLDTQL + 267 TLLDWQDSL + 268 TLLQVFHLL ++ 270 TVLPVPPLSV +++ 271 VIRNIVEAA ++ 273 VLGEYSYLL + 275 VLLFIEHSV + 276 VLNDGAPNV + 277 VMILKLPFL + 278 YLDDLLPKL ++ 280 YTLDSLYWSV + 281 NLLDDRGMTAL +++ 282 LLRDGIELV +++ 283 ILQPMDIHV +++ 284 LLSAAEPVPA +++ 286 FLLEDLSQKL +++ 287 FLWEEKFNSL +++ 289 ILEEQPMDMLL +++ 290 LANPHELSL +++ 291 ILLNEDDLVTI +++ 292 AAALIIHHV + 294 ALLDQLHTLL + 297 FLVEPQEDTRL ++ 298 IILPVEVEV +++ 299 ILEENIPVL ++ 300 ILLNPAYDVYL ++ 302 ILQDLTFVHL + 304 LAIVPVNTL +++ 305 LLFPQIEGIKI + 307 LLLTKPTEA ++ 308 SLYDVSRMYV +++ 309 ILYGTQFVL + 310 LLSTLHLLV + 311 LLVDVEPKV + 312 LLYNSTDPTL +++ 314 LMKDCEAEV ++ 316 MLLEHGITL +++ 317 NLLAHIWAL + 318 NLQVTQPTV +++ 320 TIAPVTVAV + 322 SLASIHVPL +++ 323 SLDLFNCEV +++ 327 VLIKWFPEV +++ 328 VLLPQETAEIHL ++ 329 VLMDGSVKL + 330 VLMWEIYSL ++ 331 VLWELAHLPTL +++ 332 VMIQHVENL +++ 334 YLLEEKIASL +++ 336 YMAVTTQEV +++ 337 YMYEKESEL ++ 338 FLDMTNWNL + 339 GLWGTVVNI + 340 KLLEEICNL ++ 341 LLAELPASVHA ++ 342 SLITPLQAV +++ 343 TLLEALDCI ++ 344 VLAFENPQV ++ 345 YLIEPDVELQRI + 346 VLVQVSPSL +++ 347 YLGPVSPSL + 349 ALATHILSL ++ 350 ALEDRVWEL + 351 ALSEKLARL + 352 ALVFELHYV + 353 ATPMPTPSV ++ 354 FIMDDPAGNSYL + 355 FIWPMLIHI +++ 356 FLHDHQAEL + 357 FLIQEIKTL ++ 358 FLTDYLNDL + 359 FMQDPMEVFV ++ 360 HLIDTNKIQL + 362 ILTELGGFEV + 363 ITTEVVNELYV + 364 KMDWIFHTI + 365 LISPLLLPV ++ 367 NLWSLVAKV ++ 368 QLQPTDALLCV + 371 SLADDSVLERL + 372 SLFGPLPGPGPALV + 374 VLSVITEEL ++ 375 VLWFKPVEL ++ 377 VVDGTCVAV + 378 YILGKFFAL + 379 YLAELVTPIL + 380 YLDRKLLTL + 382 YLLPLLQRL +++ 383 YLLREWVNL +++ 384 YMIGSEVGNYL + 385 YTIPLAIKL +++ 實施例 2編碼本發明肽的基因的表達譜 Table 8: Performance scores. The table lists very high overrepresentation on tumors compared to a range of normal tissues (+++), high overrepresentation on tumors compared to a range of normal tissues (++), or compared to a range of normal tissues Overexpression of (+) HLA-A*02 peptides on tumors. A series of normal tissue groups considered to be more relevant to tumors include: adipose tissue, adrenal glands, blood cells, blood vessels, bone marrow, brain, breast, esophagus, eyes, gallbladder, heart, kidney, large intestine, liver, lung, lymph nodes, nerves, pancreas , Parathyroid glands, peritoneum, pituitary gland, pleura, salivary glands, skeletal muscle, skin, small intestine, spleen, stomach, thyroid, trachea, ureter, bladder. Serial ID number sequence Peptide performance 1 AIPPSFASIFL +++ 2 ALHRPDVYL +++ 3 VIAELPPKV +++ 4 VIAELPPKVSV +++ 5 ALIFKIASA +++ 6 ALDTLEDDMTI +++ 7 ALLERTGYTL +++ 8 ALAASALPALV +++ 9 ALCDTLITV +++ 10 FVYGESVEL +++ 11 ALFTFJPLTV +++ 12 ALGEDEITL +++ 13 VVDGMPPGV ++ 14 ALLRLLPGL + 15 ALPEVSVEA + 16 ALPGGAAVAAV ++ 17 ALTKTNLQL +++ 18 LLGEFSIKM +++ 19 QVMEKLAAV + 20 ALVDPGPDFVV + twenty one ALWAGLLTL +++ twenty two ALWDPVIEL +++ twenty three ALYLTEVFL +++ twenty four AMAGDVVYA +++ 25 ATYSGLESQSV + 27 AVLGLVWLL ++ 28 AVLHLLLSV +++ 29 AVLQAVTAV +++ 30 ELLEGSEIYL + 31 ELMHGVAGV ++ 32 FIDKFTPPV +++ 33 FIINSSNIFL +++ 35 FILPSSLYL +++ 36 FILTHVDQL +++ 37 FIMEGGAMVL +++ 39 FLDALLTDV +++ 40 FLDEDDMSL + 41 FLDPSLDPLL +++ 43 FLLGPEALSFA +++ 45 FLPELPADLEA ++ 47 FLSDQPEPYL ++ 48 FLSPQQPPLL +++ 49 FLTDLFAQL + 50 FLFEPVVKAFL +++ 51 FLVEAPHDWDL ++ 52 FLVETGFLHV +++ 53 FLWQHVELVL +++ 54 FLYPFPLALF +++ 56 FVFEAPYTL +++ 57 GLSEISLRL +++ 58 YIQQGIFSV ++ 59 FVFGDENGTVSL +++ 60 FVLDHEDGLNL ++ 61 FVYFIVREV +++ 62 GIIDGSPRL + 63 SLAHVAGCEL +++ 64 KLLESVASA ++ 65 GLDDMKANL +++ 66 SLAGGLDDMKA +++ 68 GLDQQFAGLDL +++ 69 GLHQREIFL +++ 70 FVPDTPVGV ++ 71 GLKHDIARV +++ 72 GLLDAGKMYV +++ 73 GLLEVISALQL +++ 74 GLLRASFLL +++ 76 GLLRIIPYL ++ 77 GLLRLTWFL ++ 78 GLPSFLTEV +++ 79 GLQAKIQEA +++ 80 VLIEDELEEL +++ 81 WLVGQEFEL +++ 82 GLQSGVDIGV + 83 GQGEVLVYV + 86 HLYPGAVTI +++ 88 ILDEIGADVQA +++ 89 ILDFGTFQL +++ 90 VIADLGLIRV +++ 92 ILEPLNPLL +++ 93 ILFNTQINI +++ 94 ILFPLRFTL +++ 95 ILGYMAHEHKV +++ 96 ILIDKTSFV + 97 LLFATQITL + 98 SLIKYFLFV +++ 99 ILIFHSVAL +++ 100 ILNNEVFAI + 101 ILVVIEPLL +++ 102 IQDRAVPSL +++ 103 KLGGTPAPA ++ 104 KLILLDTPLFL +++ 105 KLMNDIADI +++ 106 FMASHLDYL +++ 107 ILYNLYDLL ++ 108 VIYTLIHYI + 109 KLWEGLTELV ++ 112 KTLDVDATYEI +++ 113 KVPAEEVLVAV + 114 LIPEGPPQV +++ 115 LLFDKLYLL +++ 116 LLIGATMQV +++ 117 LLILENILL ++ 118 RLLILENILL +++ 119 VLPAEFFEV +++ 121 RLYEITIEV ++ 122 LLIPVVPGV ++ 123 LLLAEAELLTL +++ 124 LLLEETEKQAV ++ 125 LLLEIGEVGKLFV ++ 126 LLPEGGITAI +++ 127 LLPTAPTTV +++ 128 LLSEEEYHL +++ 129 LLVGTLDVV + 130 LLVLIPVYL +++ 131 LQALEVLKI +++ 132 LVYEAIIMV +++ 133 YLLSGDISEA +++ 134 MLLEHGITLV +++ 135 MTAGFSTIAGSV +++ 137 NLIKTVIKL + 138 NLLDIDAPVTV ++ 139 NLTDVVEKL +++ 140 QIAELPATSV +++ 141 QILSEIVEA ++ 142 QLDEPAPQV +++ 144 QLPPFPREL +++ 145 SALDTITTV +++ 146 SIIEGPIIKL +++ 147 SILETVATL +++ 148 SIVASLITV +++ 149 SLDNGGYYI +++ 153 SLLAELHVL ++ 154 SLLAELHVLTV ++ 155 SLMLEVPAL +++ 157 SLNIRDFTM +++ 160 SLSFLVPSL +++ 162 SMKDDLENV +++ 163 SQLDISEPYKV + 164 SVHKGFAFV +++ 165 TLDDDLDTV +++ 166 TLDPNQVSL +++ 167 TLDTSKLYV +++ 168 YLLDQSFVM +++ 169 TLLLGLTEV +++ 170 TLTFRVETV +++ 171 TLVPPAALISI +++ 172 TLYDMLASI +++ 174 VLAELPIIVV +++ 175 FTVPRVVAV ++ 176 VLAEQNIIPSA +++ 177 VLDDRELLL +++ 178 VLFFNVQEV +++ 179 VLLGLEMTL +++ 181 VLLSIPFVSV + 182 VLLSVPGPPV +++ 185 VMDDQRDLI + 186 VMDPTKILI +++ 187 VMDTHLVNI +++ 189 VMLEMTPEL +++ 190 VVMGTVPRL +++ 191 YIFDGSDGGV +++ 192 YIQEYLTLL +++ 193 YLDLSNNRL +++ 194 YLDNVLAEL + 195 YLGGFALSV +++ 197 YLQEVPILTL +++ 198 YLTFLPAEV +++ 199 YLVELSSLL +++ 200 YMFEEVPIVI +++ 201 YQLELHGIEL +++ 202 YVDDVFLRV +++ 204 GLLQINDKIAL ++ 205 GLSQANFTL + 206 HMQDVRVLL +++ 208 ILLKTEGINL ++ 209 ILQAELPSL ++ 210 KLLVQDFFL +++ 211 LIDVKPLGV +++ 212 NIIEAINELLV ++ 213 RLLYQLVFL ++ 214 RLQELTEKL + 215 VMQDIVYKL + 217 ALDEPPYLTV + 218 ALGEEWKGYVV + 219 ALLNLLESA ++ 221 ALVSTIIMV + 225 ILQERELLPV + 226 AMNISVPQV + 227 FLAEASVMTQL ++ 228 FLGGLSPGV ++ 229 FLLNLQNCHL ++ 231 FLYIRQLAI + 232 FMHQIIDQV + 234 GLDDAEYAL + 235 GLDDLLLFL + 236 GLLESGRHYL +++ 237 GLQENLDVVV + 238 GLVETELQL + 240 ILARDILEI ++ 241 ILGDILLKV + 242 ILLGIQELL ++ 243 ILPTLEKELFL + 244 ILQALAVHL ++ 245 KIMDYSLLLGV + 248 KTVEPPISQV + 249 LLPTGVFQV + 251 LLYDNVPGA + 254 QLIPKLIFL +++ 255 YLFEEAISM + 258 RLDYITAEI ++ 259 RLLDEQFAVL + 260 SLDDVEGMSV ++ 261 SLVEAQGWLV ++ 263 SQWEDIHVV ++ 264 TILDYINVV +++ 266 TLLDQLDTQL + 267 TLLDWQDSL + 268 TLLQVFHLL ++ 270 TVLPVPPLSV +++ 271 VIRNIVEAA ++ 273 VLGEYSYLL + 275 VLLFIEHSV + 276 VLNDGAPNV + 277 VMILKLPFL + 278 YLDDLLPKL ++ 280 YTLDSLYWSV + 281 NLLDDRGMTAL +++ 282 LLRDGIELV +++ 283 ILQPMDIHV +++ 284 LLSAAEPVPA +++ 286 FLLEDLSQKL +++ 287 FLWEEKFNSL +++ 289 ILEEQPMDMLL +++ 290 LANPHELSL +++ 291 ILLNEDDLVTI +++ 292 AAALIIHHV + 294 ALLDQLHTLL + 297 FLVEPQEDTRL ++ 298 IILPVEVEV +++ 299 ILEENIPVL ++ 300 ILLNPAYDVYL ++ 302 ILQDLTFVHL + 304 LAIVPVNTL +++ 305 LLFPQIEGIKI + 307 LLLTKPTEA ++ 308 SLYDVSRMYV +++ 309 ILYGTQFVL + 310 LLSTLHLLV + 311 LLVDVEPKV + 312 LLYNSTDPTL +++ 314 LMKDCEAEV ++ 316 MLLEHGITL +++ 317 NLLAHIWAL + 318 NLQVTQPTV +++ 320 TIAPVTVAV + 322 SLASIHVPL +++ 323 SLDLFNCEV +++ 327 VLIKWFPEV +++ 328 VLLPQETAEIHL ++ 329 VLMDGSVKL + 330 VLMWEIYSL ++ 331 VLWELAHLPTL +++ 332 VMIQHVENL +++ 334 YLLEEKIASL +++ 336 YMAVTTQEV +++ 337 YMYEKESEL ++ 338 FLDMTNWNL + 339 GLWGTVVNI + 340 KLLEEICNL ++ 341 LLAELPASVHA ++ 342 SLITPLQAV +++ 343 TLLEALDCI ++ 344 VLAFENPQV ++ 345 YLIEPDVELQRI + 346 VLVQVSPSL +++ 347 YLGPVSPSL + 349 ALATHILSL ++ 350 ALEDRVWEL + 351 ALSEKLARL + 352 ALVFELHYV + 353 ATPMPTPSV ++ 354 FIMDDPAGNSYL + 355 FIWPMLIHI +++ 356 FLHDHQAEL + 357 FLIQEIKTL ++ 358 FLTDYLNDL + 359 FMQDPMEVFV ++ 360 HLIDTNKIQL + 362 ILTELGGFEV + 363 ITTEVVNELYV + 364 KMDWIFHTI + 365 LISPLLLPV ++ 367 NLWSLVAKV ++ 368 QLQPTDALLCV + 371 SLADDSVLERL + 372 SLFGPLPGPGPALV + 374 VLSVITEEL ++ 375 VLWFKPVEL ++ 377 VVDGTCVAV + 378 YILGKFFAL + 379 YLAELVTPIL + 380 YLDRKLLTL + 382 YLLPLLQRL +++ 383 YLLREWVNL +++ 384 YMIGSEVGNYL + 385 YTIPLAIKL +++ Example 2 Expression profile of the gene encoding the peptide of the present invention

與正常細胞相比在腫瘤細胞上一種肽過度表現或特定表現足夠其在免疫治療中有效使用，一些肽為腫瘤特異性的，儘管存在其源蛋白也存在于正常組織中。但是，mRNA 表達譜增加了免疫治療目標肽選擇中其他級別的安全性。特別是對於具有高安全性風險的治療選擇，諸如親和力成熟的 TCR，理想的目標肽將來源於對該腫瘤獨一無二且不出現于正常組織中的蛋白。 RNA 來源與製備Compared with normal cells, a peptide overexpression or specific performance on tumor cells is sufficient for its effective use in immunotherapy. Some peptides are tumor-specific, although the source protein is also present in normal tissues. However, mRNA expression profiles increase other levels of safety in the selection of target peptides for immunotherapy. Especially for treatment options with high safety risks, such as affinity matured TCR, the ideal target peptide will be derived from a protein that is unique to the tumor and does not appear in normal tissues. RNA source and preparation

手術切除組織標本按如上所述（參見實施例 1）在獲得每名患者的書面知情同意後提供。手術後立即速凍腫瘤組織標本，之後在液態氮中用杵臼勻漿。使用 TRI 試劑（Ambion 公司, Darmstadt，德國）之後用 RNeasy（QIAGEN公司，Hilden，德國）清理從這些樣本中製備總 RNA；這兩種方法都根據製造商的方案進行。The surgically resected tissue specimens were provided after obtaining the written informed consent of each patient as described above (see Example 1). Immediately after the operation, the tumor tissue specimen was quick-frozen, and then homogenized with a pestle and mortar in liquid nitrogen. Prepare total RNA from these samples using TRI reagent (Ambion, Darmstadt, Germany) followed by RNeasy (QIAGEN, Hilden, Germany); both methods were performed according to the manufacturer's protocol.

用於 RNASeq 實驗來自健康人體組織的總 RNA 獲得自：Asterand (Detroit, MI, USA & Royston, Herts, UK)、BioCat GmbH (Heidelberg, Germany)、BioServe (Beltsville, MD, USA)、Geneticist Inc. (Glendale, CA, USA)、Istituto Nazionale Tumori "Pascale" (Naples, Italy)、ProteoGenex Inc. (Culver City, CA, USA)、海德堡大學醫院 (Heidelberg, Germany)。Total RNA from healthy human tissues used in RNASeq experiments was obtained from: Asterand (Detroit, MI, USA & Royston, Herts, UK), BioCat GmbH (Heidelberg, Germany), BioServe (Beltsville, MD, USA), Geneticist Inc. ( Glendale, CA, USA), Istituto Nazionale Tumori "Pascale" (Naples, Italy), ProteoGenex Inc. (Culver City, CA, USA), Heidelberg University Hospital (Heidelberg, Germany).

用於 RNASeq 實驗來自腫瘤組織的總 RNA 獲得自：Tissue Solutions Ltd（英國格拉斯哥）、波恩大學醫院（德國波恩）The total RNA from tumor tissue used in RNASeq experiments was obtained from: Tissue Solutions Ltd (Glasgow, UK), Bonn University Hospital (Bonn, Germany)

所有RNA樣品的質量和數量在Agilent 2100生物分析儀 (Agilent，Waldbronn，Germany) 上使用RNA 6000 Pico LabChip試劑盒 (Agilent) 評估。RNA 序列實驗 The quality and quantity of all RNA samples were evaluated on an Agilent 2100 bioanalyzer (Agilent, Waldbronn, Germany) using RNA 6000 Pico LabChip kit (Agilent). RNA sequence experiment

通過新一代測序技術 (RNAseq) 由 CeGaT (Tübingen, Germany) 對腫瘤和正常組織的 RNA 樣本進行基因表達分析。簡言之，根據供應商的方案 (Illumina Inc., San Diego, CA, USA)，其中包括 RNA 碎片化、cDNA 轉化和測序適配器的加入，利用 Illumina HiSeq v4 試劑盒準備測序文庫。從多個樣本獲得的文庫根據製造商的說明等摩爾混合並在 Illumina HiSeq 2500 定序器上測序，產生 50bp 的單端讀數。處理的讀數使用 STAR 軟體映射至人類基因組 (GRCh38)。根據 ENSEMBL 序列資料庫的說明 (Ensembl77)，表達資料在轉錄水準設置為 RPKM（每百萬映射讀數每千堿基讀數，由 Cufflinks 軟體生成）並在外顯子水準上設置（總讀數，由 Bedtools 軟體生成）。外顯子讀數被歸為外顯子長度和校準尺寸，以獲得 RPKM 值。本發明的代表性源基因在 CLL 中高度過量表達的表達譜如圖 3所示。進一步代表性基因的表達分數見表 9。Gene expression analysis of RNA samples from tumors and normal tissues was performed by CeGaT (Tübingen, Germany) using next-generation sequencing technology (RNAseq). In short, according to the supplier's plan (Illumina Inc., San Diego, CA, USA), which includes RNA fragmentation, cDNA conversion, and the addition of sequencing adapters, the Illumina HiSeq v4 kit was used to prepare the sequencing library. Libraries obtained from multiple samples were mixed equimolarly according to the manufacturer's instructions and sequenced on the Illumina HiSeq 2500 sequencer to generate single-ended reads of 50 bp. The processed reads are mapped to the human genome (GRCh38) using STAR software. According to the description of the ENSEMBL sequence database (Ensembl77), the expression data is set at the transcription level to RPKM (reads per million mapped readings per thousand base readings, generated by Cufflinks software) and set at the exon level (total readings, by Bedtools software) generate). Exon readings are classified as exon length and calibration size to obtain the RPKM value. The expression profile of the representative source gene of the present invention that is highly overexpressed in CLL is shown in Figure 3. See Table 9 for further expression scores of representative genes.

表9：表達分數。該表列出了與一系列正常組織相比在腫瘤上非常高度過量表達 (+++)、與一系列正常組織相比在腫瘤上高度過量表達 (++) 或與一系列正常組織相比在腫瘤上過量表達 (+) 的基因的肽。本基線得分根據以下相關正常組織的測量值計算：脂肪組織、腎上腺、動脈、血細胞、骨髓、腦、軟骨、結腸、食道、眼、膽囊、心臟、腎、肝、肺、淋巴結、胰腺、垂體、直腸、唾液腺、骨骼肌、皮膚、小腸、脾、胃、甲狀腺、氣管、膀胱、靜脈。如果獲得同一組織類型幾個樣本的表達資料，則使用各樣本的算術平均值進行計算。 序列ID號 序列 基因表達 1 AIPPSFASIFL ++ 2 ALHRPDVYL ++ 3 VIAELPPKV +++ 4 VIAELPPKVSV +++ 5 ALIFKIASA +++ 9 ALCDTLITV +++ 10 FVYGESVEL +++ 17 ALTKTNLQL +++ 18 LLGEFSIKM +++ 19 QVMEKLAAV +++ 21 ALWAGLLTL +++ 24 AMAGDVVYA +++ 31 ELMHGVAGV +++ 46 YIIDSAQAV ++ 62 GIIDGSPRL +++ 63 SLAHVAGCEL +++ 64 KLLESVASA +++ 65 GLDDMKANL +++ 66 SLAGGLDDMKA +++ 71 GLKHDIARV +++ 74 GLLRASFLL ++ 75 GLSIFAQDLRL ++ 85 HLMLHTAAL + 86 HLYPGAVTI +++ 91 ILDLNTYNV + 96 ILIDKTSFV +++ 97 LLFATQITL +++ 98 SLIKYFLFV +++ 105 KLMNDIADI ++ 106 FMASHLDYL + 107 ILYNLYDLL ++ 108 VIYTLIHYI + 113 KVPAEEVLVAV ++ 116 LLIGATMQV + 117 LLILENILL ++ 118 RLLILENILL ++ 119 VLPAEFFEV ++ 120 AIDAALTSV ++ 121 RLYEITIEV ++ 127 LLPTAPTTV + 137 NLIKTVIKL + 146 SIIEGPIIKL +++ 149 SLDNGGYYI +++ 150 SLFDQPLSII +++ 153 SLLAELHVL +++ 154 SLLAELHVLTV +++ 156 SLNIGDVQL +++ 163 SQLDISEPYKV +++ 177 VLDDRELLL ++ 189 VMLEMTPEL +++ 193 YLDLSNNRL + 195 YLGGFALSV ++ 205 GLSQANFTL +++ 206 HMQDVRVLL +++ 213 RLLYQLVFL +++ 220 ALPEILFAKV ++ 221 ALVSTIIMV + 223 ALWVSQPPEI ++ 235 GLDDLLLFL + 251 LLYDNVPGA +++ 254 QLIPKLIFL +++ 255 YLFEEAISM +++ 257 RIINGIIISV +++ 262 SLWNAGTSV ++ 275 VLLFIEHSV ++ 284 LLSAAEPVPA +++ 285 GVATAGCVNEV +++ 286 FLLEDLSQKL ++ 294 ALLDQLHTLL + 299 ILEENIPVL ++ 300 ILLNPAYDVYL ++ 301 AASPIITLV + 307 LLLTKPTEA ++ 308 SLYDVSRMYV + 309 ILYGTQFVL ++ 312 LLYNSTDPTL + 319 QVIPQLQTV ++ 320 TIAPVTVAV ++ 330 VLMWEIYSL ++ 332 VMIQHVENL +++ 337 YMYEKESEL +++ 352 ALVFELHYV +++ 356 FLHDHQAEL + 360 HLIDTNKIQL + 378 YILGKFFAL + 380 YLDRKLLTL ++ 實施例 3MHC-I 類表現肽的體外免疫原性 Table 9: Expression scores. The table lists very high overexpression on tumors compared to a range of normal tissues (+++), high overexpression on tumors compared to a range of normal tissues (++), or compared to a range of normal tissues Peptides of genes that are overexpressed (+) on tumors. This baseline score is calculated based on the measurement values of the following related normal tissues: adipose tissue, adrenal glands, arteries, blood cells, bone marrow, brain, cartilage, colon, esophagus, eyes, gallbladder, heart, kidney, liver, lung, lymph nodes, pancreas, pituitary, Rectum, salivary glands, skeletal muscle, skin, small intestine, spleen, stomach, thyroid, trachea, bladder, veins. If the expression data of several samples of the same tissue type are obtained, the arithmetic mean of each sample is used for calculation. Serial ID number sequence gene expression 1 AIPPSFASIFL ++ 2 ALHRPDVYL ++ 3 VIAELPPKV +++ 4 VIAELPPKVSV +++ 5 ALIFKIASA +++ 9 ALCDTLITV +++ 10 FVYGESVEL +++ 17 ALTKTNLQL +++ 18 LLGEFSIKM +++ 19 QVMEKLAAV +++ twenty one ALWAGLLTL +++ twenty four AMAGDVVYA +++ 31 ELMHGVAGV +++ 46 YIIDSAQAV ++ 62 GIIDGSPRL +++ 63 SLAHVAGCEL +++ 64 KLLESVASA +++ 65 GLDDMKANL +++ 66 SLAGGLDDMKA +++ 71 GLKHDIARV +++ 74 GLLRASFLL ++ 75 GLSIFAQDLRL ++ 85 HLMLHTAAL + 86 HLYPGAVTI +++ 91 ILDLNTYNV + 96 ILIDKTSFV +++ 97 LLFATQITL +++ 98 SLIKYFLFV +++ 105 KLMNDIADI ++ 106 FMASHLDYL + 107 ILYNLYDLL ++ 108 VIYTLIHYI + 113 KVPAEEVLVAV ++ 116 LLIGATMQV + 117 LLILENILL ++ 118 RLLILENILL ++ 119 VLPAEFFEV ++ 120 AIDAALTSV ++ 121 RLYEITIEV ++ 127 LLPTAPTTV + 137 NLIKTVIKL + 146 SIIEGPIIKL +++ 149 SLDNGGYYI +++ 150 SLFDQPLSII +++ 153 SLLAELHVL +++ 154 SLLAELHVLTV +++ 156 SLNIGDVQL +++ 163 SQLDISEPYKV +++ 177 VLDDRELLL ++ 189 VMLEMTPEL +++ 193 YLDLSNNRL + 195 YLGGFALSV ++ 205 GLSQANFTL +++ 206 HMQDVRVLL +++ 213 RLLYQLVFL +++ 220 ALPEILFAKV ++ 221 ALVSTIIMV + 223 ALWVSQPPEI ++ 235 GLDDLLLFL + 251 LLYDNVPGA +++ 254 QLIPKLIFL +++ 255 YLFEEAISM +++ 257 RIINGIIISV +++ 262 SLWNAGTSV ++ 275 VLLFIEHSV ++ 284 LLSAAEPVPA +++ 285 GVATAGCVNEV +++ 286 FLLEDLSQKL ++ 294 ALLDQLHTLL + 299 ILEENIPVL ++ 300 ILLNPAYDVYL ++ 301 AASPIITLV + 307 LLLTKPTEA ++ 308 SLYDVSRMYV + 309 ILYGTQFVL ++ 312 LLYNSTDPTL + 319 QVIPQLQTV ++ 320 TIAPVTVAV ++ 330 VLMWEIYSL ++ 332 VMIQHVENL +++ 337 YMYEKESEL +++ 352 ALVFELHYV +++ 356 FLHDHQAEL + 360 HLIDTNKIQL + 378 YILGKFFAL + 380 YLDRKLLTL ++ Example 3 In vitro immunogenicity of MHC-I peptides

為了獲得關於本發明 TUMAP 的免疫原性資訊，發明人使用體外 T細胞擴增分析方法進行了研究，其中該分析方法基於使用裝載肽/MHC 複合物和抗CD28 抗體的人工抗原表現細胞 (aAPC) 進行反復刺激。用這種方法，發明人可顯示，本發明的 HLA-A*0201 限制 TUMAP 具有免疫原性，這表明這些肽為對抗人 CD8+ 前體 T細胞的 T細胞表位（表 10）。CD8+ T 細胞體外活化 In order to obtain information on the immunogenicity of the TUMAP of the present invention, the inventors used an in vitro T cell expansion analysis method based on the use of artificial antigen expressing cells (aAPC) loaded with peptide/MHC complexes and anti-CD28 antibodies Perform repeated stimulation. In this way, the inventors can show that the HLA-A*0201 of the present invention restricts TUMAP to have immunogenicity, which indicates that these peptides are T cell epitopes against human CD8+ precursor T cells (Table 10). CD8+ T cell activation in vitro

為了用載有肽-MHC複合物(pMHC)和抗CD28抗體的人工抗原表現細胞進行體外刺激，發明人首先從University clinics Mannheim, Germany中獲取健康供體CD8微珠(Miltenyi Biotec, Bergisch-Gladbach, Germany)透過積極選擇白細胞清除術後新鮮HLA-A*02產物而分離出CD8+ T細胞。In order to stimulate in vitro with artificial antigen expressing cells loaded with peptide-MHC complex (pMHC) and anti-CD28 antibody, the inventors first obtained healthy donor CD8 microbeads from University clinics Mannheim, Germany (Miltenyi Biotec, Bergisch-Gladbach, Germany) CD8+ T cells were isolated by actively selecting fresh HLA-A*02 products after leukocyte depletion.

PBMC 和分離出的 CD8+ 淋巴細胞使用前在 T細胞培養基 (TCM) 中培養，培養基包括 RPMI- Glutamax （Invitrogen公司，Karlsruhe，德國）並補充 10% 熱滅活人 AB 血清（PAN-Biotech 公司，Aidenbach，德國）、100U/ml 青黴素/ 100 µg/ml 鏈黴素（Cambrex公司，Cologne，德國），1mM 丙酮酸鈉（CC Pro公司，Oberdorla，德國）和20 µg/ml  慶大黴素（Cambrex公司）。在此步驟，2.5 ng/ml 的 IL-7 （PromoCell公司，Heidelberg，德國） 和 10 U / ml 的 IL- 2（Novartis Pharma 公司，Nürnberg，德國）也加入 TCM。PBMC and the isolated CD8+ lymphocytes were cultured in T cell culture medium (TCM) before use. The medium included RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) and supplemented with 10% heat-inactivated human AB serum (PAN-Biotech, Aidenbach) , Germany), 100U/ml penicillin/ 100 µg/ml streptomycin (Cambrex, Cologne, Germany), 1mM sodium pyruvate (CC Pro, Oberdorla, Germany) and 20 µg/ml gentamicin (Cambrex) ). In this step, 2.5 ng/ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma, Nürnberg, Germany) were also added to TCM.

對於pMHC/抗-CD28 塗層珠的生成、T細胞的刺激和讀出，使用每刺激條件四個不同 pMHC 分子以及每個讀出條件 8 個不同的 pMHC 分子在高度限定的體外系統中進行。For the generation of pMHC/anti-CD28 coated beads, T cell stimulation and readout, four different pMHC molecules per stimulation condition and 8 different pMHC molecules per readout condition were used in a highly defined in vitro system.

純化的共刺激小鼠 IgG2a 抗人 CD28 抗體 9.3  (Jung et al., 1987) 使用製造商 （Perbio公司，波恩，德國）推薦的 N-羥基琥珀醯亞胺生物素進行化學生物素化處理。所用珠為 5.6 µm的鏈黴抗生物素蛋白包裹的多聚苯乙烯顆粒（Bangs Labooratories，伊利諾州，美國）。Purified co-stimulatory mouse IgG2a anti-human CD28 antibody 9.3 (Jung et al., 1987) The N-hydroxysuccinimidyl biotin recommended by the manufacturer (Perbio, Bonn, Germany) was used for chemical biotinylation. The beads used were 5.6 µm streptavidin-coated polystyrene particles (Bangs Labooratories, Illinois, USA).

用於陽性和陰性對照刺激物的 pMHC 分別為A*0201/MLA-001（從 Melan-A/MART-1中修飾制得的肽ELAGIGILTV (SEQ ID NO: 523)）和A*0201/DDX5-001（從 DDX5 中獲得的YLLPAIVHI (SEQ ID NO: 524)）。The pMHC used for the positive and negative control stimuli were A*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO: 523) modified from Melan-A/MART-1) and A*0201/DDX5- 001 (YLLPAIVHI (SEQ ID NO: 524) obtained from DDX5).

800.000 珠/200 µl 包裹於含有 4 x 12.5 ng 不同生物素-pMHC 的 96 孔板、進行洗滌，隨後加入體積為 200 µl 的 600 ng生物素抗-CD28。在 37℃ 下，在含 5 ng/ml IL-12 (PromoCell) 的 200 µl TCM 中共培養 1x106 CD8+T細胞與 2x105 的清洗塗層珠 3 天，從而啟動刺激。之後，一半培養基與補充 80 U/ml IL-2 的新鮮 TCM 進行交換，並且培養在 37℃ 下持續 4 天。這種刺激性週期總共進行 3 次。對於使用每條件 8 種不同 pMHC 分子的 pMHC 多聚體讀出，二維組合編碼方法如前述使用 (Andersen et al., 2012)，稍作修飾，涵蓋耦合至 5 種不同的螢光染料。最後，用 Live/dead near IR 染料（Invitrogen公司，Karlsruhe，德國）、CD8-FITC  抗體克隆 SK1（BD公司，Heidelberg，德國）和螢光 pMHC多聚體而執行多聚體分析。對於分析，使用了配有合適鐳射儀和篩檢程序的 BD LSRII SORP 細胞儀。肽特異性細胞以占總 CD8+ 細胞的百分比形式進行計算。多聚體分析結果使用 FlowJo 軟體 (Tree Star 公司，Oregon，美國) 進行評估。特定多聚體+ CD8+淋巴細胞的體外填裝用與陰性對照刺激組比較而進行檢測。如果健康供體中的至少一個可評價的體外刺激孔在體外刺激後發現含有特異性 CD8+ T細胞株（即該孔包含至少 1% 特定多聚體+ CD8+ T細胞，並且特定多聚體+的百分比至少為陰性對照刺激中位數的 10 倍），則檢測給定抗原的免疫原性。CLL 肽體外免疫原性 800.000 beads/200 µl were packaged in a 96-well plate containing 4 x 12.5 ng different biotin-pMHC, washed, and then 600 ng biotin anti-CD28 in a volume of 200 µl was added. In 200 µl TCM containing 5 ng/ml IL-12 (PromoCell), 1x106 CD8+ T cells and 2x105 washed coated beads were co-cultured at 37°C for 3 days to initiate stimulation. After that, half of the medium was exchanged with fresh TCM supplemented with 80 U/ml IL-2, and the culture was continued at 37°C for 4 days. This stimulation cycle is performed 3 times in total. For the readout of pMHC multimers using 8 different pMHC molecules per condition, the two-dimensional combinatorial encoding method was used as described above (Andersen et al., 2012), with a slight modification to cover coupling to 5 different fluorescent dyes. Finally, the multimer analysis was performed with Live/dead near IR dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany) and fluorescent pMHC multimer. For the analysis, a BD LSRII SORP cytometer equipped with a suitable laser and screening program was used. Peptide-specific cells are calculated as a percentage of total CD8+ cells. The results of polymer analysis were evaluated using FlowJo software (Tree Star Company, Oregon, USA). The in vitro packing of specific polymer + CD8 + lymphocytes was tested by comparing with the negative control stimulation group. If at least one evaluable in vitro stimulation hole in a healthy donor is found to contain a specific CD8+ T cell strain after in vitro stimulation (that is, the hole contains at least 1% specific multimer + CD8 + T cells, and specific multimer + The percentage is at least 10 times the median stimulus of the negative control), then the immunogenicity of the given antigen is tested. CLL peptide immunogenicity in vitro

對於受到測試的 HLA-I 類肽，可透過肽特異性 T細胞株的生成證明其體外免疫原性。TUMAP 特異性多聚體對本發明的 2 種肽染色後流式細胞儀檢測的典型結果如圖 3 所示，同時也含有相應的陰性對照資訊。本發明 2 種肽的結果匯總於表 10A，其他的結果列於 10B。 表 10A：本發明中 HLA I 類肽的體外免疫原性For the tested HLA-I peptides, the in vitro immunogenicity can be proved through the generation of peptide-specific T cell lines. The typical results of flow cytometry detection of the two peptides of the present invention after staining with TUMAP-specific multimers are shown in Figure 3, which also contains corresponding negative control information. The results of the two peptides of the present invention are summarized in Table 10A, and the other results are listed in Table 10B. Table 10A: In vitro immunogenicity of HLA class I peptides of the present invention

申請人對本發明的肽所做的體外免疫原性實驗的示例性結果。＜20 % = +; 20 % - 49 % = ++; 50 % - 69 %= +++; ＞= 70 % = ++++ Seq ID NO 序列 孔 427 LLYDAVHIV ++ 521 YLYGQTTTYL ++ 表 10B ：本發明額外 HLA  I 類肽的體外免疫原性Exemplary results of in vitro immunogenicity experiments conducted by the applicant on the peptides of the present invention. ＜20% = +; 20%-49% = ++; 50%-69% = +++; ＞= 70% = ++++ Seq ID NO sequence hole 427 LLYDAVHIV ++ 521 YLYGQTTTYL ++ Table 10B: In vitro immunogenicity of additional HLA class I peptides of the invention

申請人對本發明的 HLA-A*02 限制肽所做的體外免疫原性實驗的示例性結果。提示了體外免疫原性實驗的結果。陽性孔和供體（其他可評價）的百分比概括為 ＜20 % = +；20 % - 49 % = ++；50 % - 69 %= +++；＞= 70 %= ++++ Seq ID No 序列 陽性孔 [%] 1 AIPPSFASIFL "+++" 2 ALHRPDVYL "+" 5 ALIFKIASA "+++" 17 ALTKTNLQL "+" 26 AVLLVLPLV "+" 32 FIDKFTPPV "++++" 49 FLTDLFAQL "++" 54 FLYPFPLALF "+" 56 FVFEAPYTL "++" 57 GLSEISLRL "+" 69 GLHQREIFL "+" 81 WLVGQEFEL "+" 86 HLYPGAVTI "+" 96 ILIDKTSFV "+" 100 ILNNEVFAI "++" 101 ILVVIEPLL "+" 102 IQDRAVPSL "+" 112 KTLDVDATYEI "+" 114 LIPEGPPQV "+" 154 SLLAELHVLTV "+" 192 YIQEYLTLL "+" 197 YLQEVPILTL "+" 200 YMFEEVPIVI "+" 201 YQLELHGIEL "+" 202 YVDDVFLRV "++" 220 ALPEILFAKV "++" 240 ILARDILEI "+" 281 NLLDDRGMTAL "+" 282 LLRDGIELV "++" 307 LLLTKPTEA "+" 320 TIAPVTVAV "+" 322 SLASIHVPL "+" 323 SLDLFNCEV "+" 329 VLMDGSVKL "+++" 332 VMIQHVENL "+" 380 YLDRKLLTL "++++" 實施例 4肽的合成 Exemplary results of the in vitro immunogenicity experiment conducted by the applicant on the HLA-A*02 restricted peptide of the present invention. The results of in vitro immunogenicity experiments are suggested. The percentages of positive wells and donors (other evaluable) are summarized as <20% = +; 20%-49% = ++; 50%-69% = +++; >= 70% = ++++ Seq ID No sequence Positive hole [%] 1 AIPPSFASIFL "+++" 2 ALHRPDVYL "+" 5 ALIFKIASA "+++" 17 ALTKTNLQL "+" 26 AVLLVLPLV "+" 32 FIDKFTPPV "++++" 49 FLTDLFAQL "++" 54 FLYPFPLALF "+" 56 FVFEAPYTL "++" 57 GLSEISLRL "+" 69 GLHQREIFL "+" 81 WLVGQEFEL "+" 86 HLYPGAVTI "+" 96 ILIDKTSFV "+" 100 ILNNEVFAI "++" 101 ILVVIEPLL "+" 102 IQDRAVPSL "+" 112 KTLDVDATYEI "+" 114 LIPEGPPQV "+" 154 SLLAELHVLTV "+" 192 YIQEYLTLL "+" 197 YLQEVPILTL "+" 200 YMFEEVPIVI "+" 201 YQLELHGIEL "+" 202 YVDDVFLRV "++" 220 ALPEILFAKV "++" 240 ILARDILEI "+" 281 NLLDDRGMTAL "+" 282 LLRDGIELV "++" 307 LLLTKPTEA "+" 320 TIAPVTVAV "+" 322 SLASIHVPL "+" 323 SLDLFNCEV "+" 329 VLMDGSVKL "+++" 332 VMIQHVENL "+" 380 YLDRKLLTL "++++" Example 4 Synthesis of peptide

所有的肽透過使用 Fmoc 策略以標準、廣為接受的固相肽合成法合成。每個肽的身份和純度已使用質譜和 RP-HPLC 分析法確定。用凍幹法（三氟乙酸鹽）獲得白色至類白色的肽，純度為 ＞50%。所有的 TUMAP 優選作為三氟乙酸鹽或乙酸鹽進行給藥，其他藥用鹽形式也可以。 實施例 5MHC 結合測定 All peptides were synthesized using the standard and widely accepted solid phase peptide synthesis method using the Fmoc strategy. The identity and purity of each peptide has been determined using mass spectrometry and RP-HPLC analysis methods. The white to off-white peptide was obtained by freeze-drying method (trifluoroacetate) with a purity of >50%. All TUMAPs are preferably administered as trifluoroacetate or acetate, but other pharmaceutically acceptable salt forms are also possible. Example 5 MHC binding assay

本發明基於 T細胞療法的候選肽進一步測試其 MHC 結合能力（親和性）。單個肽-MHC 複合體透過 UV-配體交換產生，其中，紫外線敏感肽經紫外線照射後裂解，與分析的相關肽交換。只有能夠有效地結合並穩定肽接受 MHC 分子的候選肽才能阻止 MHC 複合物的解離。為了確定交換反應的產率，將基於穩定 MHC 複合物輕鏈 (β2m) 的檢測結果進行 ELISA 測定。檢測總體上按照 Rodenko 等人在 (Rodenko et al., 2006) 中描述的方法進行。The candidate peptides of the present invention based on T cell therapy are further tested for their MHC binding ability (affinity). A single peptide-MHC complex is produced through UV-ligand exchange, where the UV-sensitive peptide is cleaved after being irradiated with UV light, and exchanged with the analyzed related peptide. Only candidate peptides that can effectively bind and stabilize the peptide accepting MHC molecules can prevent the dissociation of the MHC complex. In order to determine the yield of the exchange reaction, an ELISA measurement will be performed based on the detection result of the stable MHC complex light chain (β2m). The detection is generally carried out according to the method described by Rodenko et al. (Rodenko et al., 2006).

96 孔 Maxisorp 板 (NUNC) 在室溫下在 PBS 中以 2ug/ml 鏈黴包被過夜，用 4 倍洗滌並在37°C 下在含封閉緩衝液的 2% BSA 中封閉 1 小時。折疊的 HLA-A*02:01/MLA-001 單體作為標準品，涵蓋 15-500ng/ml 的範圍。紫外線交換反應的肽-MHC 單體在封閉緩衝液中稀釋100倍。樣本在 37°C下孵育 1 小時，洗滌四次，在 37°C 下以 2ug/ml HRP 綴合抗-β2m 溫育 1 小時，再次洗滌，並以 NH2SO4 封堵的 TMB 溶液進行檢測。在 450nm 處測量吸收。在生成和產生抗體或其片段時和/或 T細胞受體或其片段時，通常優選顯示為高交換產率（優選為高於50%，最優選為高於75%）的候選肽，這是因為它們對MHC分子表現出足夠的親合力，並能防止 MHC 複合物的解離。96-well Maxisorp plates (NUNC) were coated with 2ug/ml Streptomyces in PBS overnight at room temperature, washed 4 times and blocked in 2% BSA with blocking buffer at 37°C for 1 hour. The folded HLA-A*02:01/MLA-001 monomer is used as a standard product, covering the range of 15-500ng/ml. The peptide-MHC monomer for UV exchange reaction is diluted 100 times in blocking buffer. The sample was incubated at 37°C for 1 hour, washed four times, incubated with 2ug/ml HRP-conjugated anti-β2m at 37°C for 1 hour, washed again, and tested with NH2SO4 blocked TMB solution. The absorption is measured at 450nm. In the production and production of antibodies or fragments thereof and/or T cell receptors or fragments thereof, it is generally preferred to show candidate peptides with high exchange yields (preferably higher than 50%, most preferably higher than 75%). It is because they show sufficient affinity for MHC molecules and can prevent the dissociation of MHC complexes.

表 11：MHC-I類結合分數。HLA-I 類限制肽與 HLA-A*02:01 的結合根據肽交換產量評估範圍：＞10% = +; ＞20% = ++; ＞50 = +++; ＞ 75% = ++++ 序列 ID 肽代碼 肽交換 1 AIPPSFASIFL "+++" 2 ALHRPDVYL "+++" 3 VIAELPPKV "++" 4 VIAELPPKVSV "+++" 5 ALIFKIASA "+++" 6 ALDTLEDDMTI "+++" 7 ALLERTGYTL "+++" 8 ALAASALPALV "+++" 9 ALCDTLITV "+++" 10 FVYGESVEL "+++" 11 ALFTFJPLTV "+++" 12 ALGEDEITL "+++" 13 VVDGMPPGV "+++" 14 ALLRLLPGL "+++" 15 ALPEVSVEA "+++" 16 ALPGGAAVAAV "++++" 17 ALTKTNLQL "+++" 18 LLGEFSIKM "+++" 19 QVMEKLAAV "+++" 20 ALVDPGPDFVV "+++" 21 ALWAGLLTL "+++" 22 ALWDPVIEL "+++" 23 ALYLTEVFL "++++" 24 AMAGDVVYA "+++" 25 ATYSGLESQSV "++" 26 AVLLVLPLV "+++" 27 AVLGLVWLL "+++" 28 AVLHLLLSV "++" 29 AVLQAVTAV "+++" 30 ELLEGSEIYL "+++" 31 ELMHGVAGV "+++" 32 FIDKFTPPV "++++" 34 YLPYIFPNI "+++" 35 FILPSSLYL "+++" 36 FILTHVDQL "++++" 37 FIMEGGAMVL "++++" 38 FIMEGGAMV "+++" 39 FLDALLTDV "+++" 40 FLDEDDMSL "++" 41 FLDPSLDPLL "+++" 42 FLEEGGVVTV "+++" 43 FLLGPEALSFA "++++" 44 FLLSINDFL "+++" 45 FLPELPADLEA "+++" 46 YIIDSAQAV "+++" 47 FLSDQPEPYL "++" 48 FLSPQQPPLL "+++" 49 FLTDLFAQL "++++" 50 FLFEPVVKAFL "+++" 51 FLVEAPHDWDL "++++" 52 FLVETGFLHV "++++" 53 FLWQHVELVL "+++" 54 FLYPFPLALF "+++" 55 FMEPTLLML "+++" 56 FVFEAPYTL "+++" 57 GLSEISLRL "++++" 58 YIQQGIFSV "+++" 59 FVFGDENGTVSL "+++" 60 FVLDHEDGLNL "+++" 61 FVYFIVREV "+++" 62 GIIDGSPRL "+++" 63 SLAHVAGCEL "+++" 64 KLLESVASA "+++" 65 GLDDMKANL "++" 66 SLAGGLDDMKA "+++" 67 GLDDVTVEV "++" 68 GLDQQFAGLDL "+" 69 GLHQREIFL "+++" 70 FVPDTPVGV "+++" 71 GLKHDIARV "+++" 72 GLLDAGKMYV "+++" 73 GLLEVISALQL "+++" 74 GLLRASFLL "++" 75 GLSIFAQDLRL "++" 76 GLLRIIPYL "+++" 78 GLPSFLTEV "+++" 79 GLQAKIQEA "+++" 80 VLIEDELEEL "++" 81 WLVGQEFEL "+++" 82 GLQSGVDIGV "+++" 83 GQGEVLVYV "+++" 84 GVMDVNTAL "++" 85 HLMLHTAAL "++++" 86 HLYPGAVTI "+++" 87 HQIEAVDGEEL "++" 88 ILDEIGADVQA "+++" 89 ILDFGTFQL "+++" 90 VIADLGLIRV "+++" 91 ILDLNTYNV "+++" 92 ILEPLNPLL "+++" 93 ILFNTQINI "+++" 94 ILFPLRFTL "+++" 95 ILGYMAHEHKV "++" 96 ILIDKTSFV "+++" 97 LLFATQITL "++" 98 SLIKYFLFV "+" 99 ILIFHSVAL "+++" 100 ILNNEVFAI "+++" 101 ILVVIEPLL "++++" 102 IQDRAVPSL "++" 103 KLGGTPAPA "++" 104 KLILLDTPLFL "+++" 105 KLMNDIADI "++" 106 FMASHLDYL "+++" 107 ILYNLYDLL "+++" 108 VIYTLIHYI "+++" 109 KLWEGLTELV "+++" 110 LLFDHLEPMEL "+++" 111 KLWNVAAPLYL "+++" 112 KTLDVDATYEI "+++" 113 KVPAEEVLVAV "+++" 114 LIPEGPPQV "+++" 115 LLFDKLYLL "+++" 116 LLIGATMQV "+++" 117 LLILENILL "+++" 118 RLLILENILL "+++" 119 VLPAEFFEV "+++" 120 AIDAALTSV "+++" 121 RLYEITIEV "++++" 122 LLIPVVPGV "+++" 123 LLLAEAELLTL "++" 124 LLLEETEKQAV "+++" 125 LLLEIGEVGKLFV "+++" 126 LLPEGGITAI "+++" 127 LLPTAPTTV "+++" 128 LLSEEEYHL "+++" 130 LLVLIPVYL "+" 131 LQALEVLKI "+" 132 LVYEAIIMV "+++" 133 YLLSGDISEA "+++" 134 MLLEHGITLV "++++" 135 MTAGFSTIAGSV "+" 136 NLDKLWTLV "++" 137 NLIKTVIKL "+++" 138 NLLDIDAPVTV "+++" 139 NLTDVVEKL "+++" 140 QIAELPATSV "+++" 141 QILSEIVEA "++" 142 QLDEPAPQV "++" 143 QLLDTYFTL "+++" 145 SALDTITTV "+++" 146 SIIEGPIIKL "+++" 147 SILETVATL "+++" 148 SIVASLITV "+++" 149 SLDNGGYYI "+++" 150 SLFDQPLSII "+++" 151 SLFDSAYGA "+++" 152 SLIRILQTI "+++" 153 SLLAELHVL "+++" 154 SLLAELHVLTV "++++" 155 SLMLEVPAL "+++" 156 SLNIGDVQL "+++" 157 SLNIRDFTM "+++" 158 SLPEAPLDV "+++" 159 SLQEEKLIYV "+++" 160 SLSFLVPSL "+++" 161 SMDDGMINV "+++" 162 SMKDDLENV "++" 163 SQLDISEPYKV "+++" 164 SVHKGFAFV "+++" 165 TLDDDLDTV "++" 166 TLDPNQVSL "++" 167 TLDTSKLYV "+++" 168 YLLDQSFVM "++++" 169 TLLLGLTEV "+++" 170 TLTFRVETV "+++" 171 TLVPPAALISI "+++" 172 TLYDMLASI "+++" 173 TVIENIHTI "++" 174 VLAELPIIVV "+++" 175 FTVPRVVAV "+++" 176 VLAEQNIIPSA "+++" 177 VLDDRELLL "+++" 178 VLFFNVQEV "+++" 179 VLLGLEMTL "+++" 180 LLKDGPEIGL "++" 181 VLLSIPFVSV "+++" 182 VLLSVPGPPV "+++" 183 VLMPTVYQQGV "+++" 184 VLSHNLYTV "+++" 185 VMDDQRDLI "++" 186 VMDPTKILI "++++" 187 VMDTHLVNI "++++" 188 VMGDIPAAV "++" 189 VMLEMTPEL "+++" 190 VVMGTVPRL "+++" 191 YIFDGSDGGV "+++" 192 YIQEYLTLL "+++" 193 YLDLSNNRL "+++" 194 YLDNVLAEL "++++" 195 YLGGFALSV "++++" 196 YLLLQTYVL "+" 197 YLQEVPILTL "++++" 198 YLTFLPAEV "+++" 199 YLVELSSLL "++++" 200 YMFEEVPIVI "++++" 201 YQLELHGIEL "++++" 202 YVDDVFLRV "++++" 203 ALLSSQLAL "+++" 204 GLLQINDKIAL "+++" 205 GLSQANFTL "+++" 206 HMQDVRVLL "+++" 207 IIADLDTTIMFA "+++" 208 ILLKTEGINL "+++" 209 ILQAELPSL "+++" 210 KLLVQDFFL "+++" 211 LIDVKPLGV "+++" 214 RLQELTEKL "+++" 215 VMQDIVYKL "+++" 216 WLAGDVPAA "+++" 217 ALDEPPYLTV "+++" 218 ALGEEWKGYVV "++" 219 ALLNLLESA "+++" 220 ALPEILFAKV "+++" 221 ALVSTIIMV "+" 222 ALWELSLKI "+++" 223 ALWVSQPPEI "+++" 224 AMEALVVEV "+++" 225 ILQERELLPV "+++" 226 AMNISVPQV "+++" 227 FLAEASVMTQL "+++" 228 FLGGLSPGV "+++" 229 FLLNLQNCHL "++++" 230 FLQDSKVIFV "+++" 231 FLYIRQLAI "+++" 232 FMHQIIDQV "++++" 233 GIIDINVRL "++" 234 GLDDAEYAL "++" 235 GLDDLLLFL "+++" 236 GLLESGRHYL "+++" 237 GLQENLDVVV "++++" 238 GLVETELQL "+++" 239 ILAGEMLSV "+++" 240 ILARDILEI "+++" 241 ILGDILLKV "+++" 242 ILLGIQELL "+++" 243 ILPTLEKELFL "+++" 244 ILQALAVHL "+++" 245 KIMDYSLLLGV "++++" 246 KLDETGVAL "++" 247 KLKDRLPSI "++" 248 KTVEPPISQV "++" 249 LLPTGVFQV "+++" 250 LLVQEPDGLMV "+++" 251 LLYDNVPGA "+++" 252 NLLDPGSSYLL "+++" 253 NLWSVDGEVTV "+++" 254 QLIPKLIFL "+++" 255 YLFEEAISM "+++" 256 QLLPVSNVVSV "+++" 258 RLDYITAEI "+++" 259 RLLDEQFAVL "+++" 260 SLDDVEGMSV "+++" 261 SLVEAQGWLV "+++" 262 SLWNAGTSV "+++" 263 SQWEDIHVV "+++" 264 TILDYINVV "+++" 265 TLLADDLEIKL "+++" 266 TLLDQLDTQL "++" 267 TLLDWQDSL "+++" 268 TLLQVFHLL "+++" 269 TLTDEQFLV "++" 270 TVLPVPPLSV "+++" 271 VIRNIVEAA "++" 272 VLDELPPLI "+++" 273 VLGEYSYLL "+++" 274 VLLEYHIAYL "+++" 275 VLLFIEHSV "+++" 276 VLNDGAPNV "+++" 277 VMILKLPFL "++" 278 YLDDLLPKL "+++" 279 YMAPEVVEA "+++" 280 YTLDSLYWSV "+++" 281 NLLDDRGMTAL "+++" 282 LLRDGIELV "+++" 283 ILQPMDIHV "+++" 284 LLSAAEPVPA "++" 285 GVATAGCVNEV "++++" 286 FLLEDLSQKL "+++" 287 FLWEEKFNSL "++++" 288 GLAESTGLLAV "+++" 289 ILEEQPMDMLL "+++" 291 ILLNEDDLVTI "+++" 292 AAALIIHHV "+++" 293 ALDIMIPMV "+++" 294 ALLDQLHTLL "++++" 295 ALLQKLQQL "+++" 296 FIAPTGHSL "++" 297 FLVEPQEDTRL "+++" 298 IILPVEVEV "+++" 299 ILEENIPVL "+++" 300 ILLNPAYDVYL "+++" 301 AASPIITLV "+++" 302 ILQDLTFVHL "+++" 303 ILSQPTPSL "+++" 304 LAIVPVNTL "+" 305 LLFPQIEGIKI "+++" 306 VVAEELENV "++" 307 LLLTKPTEA "+++" 308 SLYDVSRMYV "++++" 309 ILYGTQFVL "+++" 310 LLSTLHLLV "+++" 311 LLVDVEPKV "+++" 312 LLYNSTDPTL "+++" 313 LMADLEGLHL "+++" 314 LMKDCEAEV "+++" 315 LVYEAPETV "+++" 316 MLLEHGITL "+++" 317 NLLAHIWAL "+++" 318 NLQVTQPTV "+++" 319 QVIPQLQTV "+++" 320 TIAPVTVAV "++++" 321 RLLEFELAQL "+++" 322 SLASIHVPL "+++" 323 SLDLFNCEV "++++" 324 SLYSALQQA "+++" 325 TLENGVPCV "+++" 326 VLAFLVHEL "++++" 327 VLIKWFPEV "++++" 328 VLLPQETAEIHL "++++" 329 VLMDGSVKL "++++" 330 VLMWEIYSL "+++" 331 VLWELAHLPTL "+++" 332 VMIQHVENL "+++" 333 VTLEFPQLIRV "+++" 334 YLLEEKIASL "+++" 335 YLYQEQYFI "+++" 336 YMAVTTQEV "++++" 337 YMYEKESEL "++" 338 FLDMTNWNL "++++" 339 GLWGTVVNI "+++" 340 KLLEEICNL "++++" 341 LLAELPASVHA "+++" 342 SLITPLQAV "+++" 344 VLAFENPQV "+++" 345 YLIEPDVELQRI "+++" 346 VLVQVSPSL "+++" 347 YLGPVSPSL "+++" 348 ALAKPPVVSV "+++" 349 ALATHILSL "+++" 350 ALEDRVWEL "+++" 351 ALSEKLARL "+++" 352 ALVFELHYV "+++" 353 ATPMPTPSV "++" 354 FIMDDPAGNSYL "+++" 355 FIWPMLIHI "+++" 356 FLHDHQAEL "++" 357 FLIQEIKTL "+++" 358 FLTDYLNDL "+++" 359 FMQDPMEVFV "+++" 360 HLIDTNKIQL "+++" 361 ILQEFESKL "+++" 362 ILTELGGFEV "+++" 363 ITTEVVNELYV "+++" 364 KMDWIFHTI "+++" 365 LISPLLLPV "+++" 367 NLWSLVAKV "+++" 369 RLLDLENSLLGL "+++" 370 SIFASPESV "+++" 371 SLADDSVLERL "+++" 372 SLFGPLPGPGPALV "+++" 373 TLLADQGEIRV "++" 374 VLSVITEEL "+++" 375 VLWFKPVEL "+++" 376 VLYNQRVEEI "+++" 377 VVDGTCVAV "+++" 378 YILGKFFAL "+++" 379 YLAELVTPIL "+++" 380 YLDRKLLTL "+++" 381 YLLEENKIKL "+++" 382 YLLPLLQRL "++++" 383 YLLREWVNL "++++" 384 YMIGSEVGNYL "++++" 385 YTIPLAIKL "+++" 實施例 6 細胞表面表現的腫瘤相關肽的絕對定量。Table 11: MHC class I binding scores. The binding of HLA-I restricted peptides to HLA-A*02:01 is evaluated based on the peptide exchange yield: ＞10% = +; ＞20% = ++; ＞50 = +++; ＞75% = +++ + Serial ID Peptide code Peptide exchange 1 AIPPSFASIFL "+++" 2 ALHRPDVYL "+++" 3 VIAELPPKV "++" 4 VIAELPPKVSV "+++" 5 ALIFKIASA "+++" 6 ALDTLEDDMTI "+++" 7 ALLERTGYTL "+++" 8 ALAASALPALV "+++" 9 ALCDTLITV "+++" 10 FVYGESVEL "+++" 11 ALFTFJPLTV "+++" 12 ALGEDEITL "+++" 13 VVDGMPPGV "+++" 14 ALLRLLPGL "+++" 15 ALPEVSVEA "+++" 16 ALPGGAAVAAV "++++" 17 ALTKTNLQL "+++" 18 LLGEFSIKM "+++" 19 QVMEKLAAV "+++" 20 ALVDPGPDFVV "+++" twenty one ALWAGLLTL "+++" twenty two ALWDPVIEL "+++" twenty three ALYLTEVFL "++++" twenty four AMAGDVVYA "+++" 25 ATYSGLESQSV "++" 26 AVLLVLPLV "+++" 27 AVLGLVWLL "+++" 28 AVLHLLLSV "++" 29 AVLQAVTAV "+++" 30 ELLEGSEIYL "+++" 31 ELMHGVAGV "+++" 32 FIDKFTPPV "++++" 34 YLPYIFPNI "+++" 35 FILPSSLYL "+++" 36 FILTHVDQL "++++" 37 FIMEGGAMVL "++++" 38 FIMEGGAMV "+++" 39 FLDALLTDV "+++" 40 FLDEDDMSL "++" 41 FLDPSLDPLL "+++" 42 FLEEGGVVTV "+++" 43 FLLGPEALSFA "++++" 44 FLLSINDFL "+++" 45 FLPELPADLEA "+++" 46 YIIDSAQAV "+++" 47 FLSDQPEPYL "++" 48 FLSPQQPPLL "+++" 49 FLTDLFAQL "++++" 50 FLFEPVVKAFL "+++" 51 FLVEAPHDWDL "++++" 52 FLVETGFLHV "++++" 53 FLWQHVELVL "+++" 54 FLYPFPLALF "+++" 55 FMEPTLLML "+++" 56 FVFEAPYTL "+++" 57 GLSEISLRL "++++" 58 YIQQGIFSV "+++" 59 FVFGDENGTVSL "+++" 60 FVLDHEDGLNL "+++" 61 FVYFIVREV "+++" 62 GIIDGSPRL "+++" 63 SLAHVAGCEL "+++" 64 KLLESVASA "+++" 65 GLDDMKANL "++" 66 SLAGGLDDMKA "+++" 67 GLDDVTVEV "++" 68 GLDQQFAGLDL "+" 69 GLHQREIFL "+++" 70 FVPDTPVGV "+++" 71 GLKHDIARV "+++" 72 GLLDAGKMYV "+++" 73 GLLEVISALQL "+++" 74 GLLRASFLL "++" 75 GLSIFAQDLRL "++" 76 GLLRIIPYL "+++" 78 GLPSFLTEV "+++" 79 GLQAKIQEA "+++" 80 VLIEDELEEL "++" 81 WLVGQEFEL "+++" 82 GLQSGVDIGV "+++" 83 GQGEVLVYV "+++" 84 GVMDVNTAL "++" 85 HLMLHTAAL "++++" 86 HLYPGAVTI "+++" 87 HQIEAVDGEEL "++" 88 ILDEIGADVQA "+++" 89 ILDFGTFQL "+++" 90 VIADLGLIRV "+++" 91 ILDLNTYNV "+++" 92 ILEPLNPLL "+++" 93 ILFNTQINI "+++" 94 ILFPLRFTL "+++" 95 ILGYMAHEHKV "++" 96 ILIDKTSFV "+++" 97 LLFATQITL "++" 98 SLIKYFLFV "+" 99 ILIFHSVAL "+++" 100 ILNNEVFAI "+++" 101 ILVVIEPLL "++++" 102 IQDRAVPSL "++" 103 KLGGTPAPA "++" 104 KLILLDTPLFL "+++" 105 KLMNDIADI "++" 106 FMASHLDYL "+++" 107 ILYNLYDLL "+++" 108 VIYTLIHYI "+++" 109 KLWEGLTELV "+++" 110 LLFDHLEPMEL "+++" 111 KLWNVAAPLYL "+++" 112 KTLDVDATYEI "+++" 113 KVPAEEVLVAV "+++" 114 LIPEGPPQV "+++" 115 LLFDKLYLL "+++" 116 LLIGATMQV "+++" 117 LLILENILL "+++" 118 RLLILENILL "+++" 119 VLPAEFFEV "+++" 120 AIDAALTSV "+++" 121 RLYEITIEV "++++" 122 LLIPVVPGV "+++" 123 LLLAEAELLTL "++" 124 LLLEETEKQAV "+++" 125 LLLEIGEVGKLFV "+++" 126 LLPEGGITAI "+++" 127 LLPTAPTTV "+++" 128 LLSEEEYHL "+++" 130 LLVLIPVYL "+" 131 LQALEVLKI "+" 132 LVYEAIIMV "+++" 133 YLLSGDISEA "+++" 134 MLLEHGITLV "++++" 135 MTAGFSTIAGSV "+" 136 NLDKLWTLV "++" 137 NLIKTVIKL "+++" 138 NLLDIDAPVTV "+++" 139 NLTDVVEKL "+++" 140 QIAELPATSV "+++" 141 QILSEIVEA "++" 142 QLDEPAPQV "++" 143 QLLDTYFTL "+++" 145 SALDTITTV "+++" 146 SIIEGPIIKL "+++" 147 SILETVATL "+++" 148 SIVASLITV "+++" 149 SLDNGGYYI "+++" 150 SLFDQPLSII "+++" 151 SLFDSAYGA "+++" 152 SLIRILQTI "+++" 153 SLLAELHVL "+++" 154 SLLAELHVLTV "++++" 155 SLMLEVPAL "+++" 156 SLNIGDVQL "+++" 157 SLNIRDFTM "+++" 158 SLPEAPLDV "+++" 159 SLQEEKLIYV "+++" 160 SLSFLVPSL "+++" 161 SMDDGMINV "+++" 162 SMKDDLENV "++" 163 SQLDISEPYKV "+++" 164 SVHKGFAFV "+++" 165 TLDDDLDTV "++" 166 TLDPNQVSL "++" 167 TLDTSKLYV "+++" 168 YLLDQSFVM "++++" 169 TLLLGLTEV "+++" 170 TLTFRVETV "+++" 171 TLVPPAALISI "+++" 172 TLYDMLASI "+++" 173 TVIENIHTI "++" 174 VLAELPIIVV "+++" 175 FTVPRVVAV "+++" 176 VLAEQNIIPSA "+++" 177 VLDDRELLL "+++" 178 VLFFNVQEV "+++" 179 VLLGLEMTL "+++" 180 LLKDGPEIGL "++" 181 VLLSIPFVSV "+++" 182 VLLSVPGPPV "+++" 183 VLMPTVYQQGV "+++" 184 VLSHNLYTV "+++" 185 VMDDQRDLI "++" 186 VMDPTKILI "++++" 187 VMDTHLVNI "++++" 188 VMGDIPAAV "++" 189 VMLEMTPEL "+++" 190 VVMGTVPRL "+++" 191 YIFDGSDGGV "+++" 192 YIQEYLTLL "+++" 193 YLDLSNNRL "+++" 194 YLDNVLAEL "++++" 195 YLGGFALSV "++++" 196 YLLLQTYVL "+" 197 YLQEVPILTL "++++" 198 YLTFLPAEV "+++" 199 YLVELSSLL "++++" 200 YMFEEVPIVI "++++" 201 YQLELHGIEL "++++" 202 YVDDVFLRV "++++" 203 ALLSSQLAL "+++" 204 GLLQINDKIAL "+++" 205 GLSQANFTL "+++" 206 HMQDVRVLL "+++" 207 IIADLDTTIMFA "+++" 208 ILLKTEGINL "+++" 209 ILQAELPSL "+++" 210 KLLVQDFFL "+++" 211 LIDVKPLGV "+++" 214 RLQELTEKL "+++" 215 VMQDIVYKL "+++" 216 WLAGDVPAA "+++" 217 ALDEPPYLTV "+++" 218 ALGEEWKGYVV "++" 219 ALLNLLESA "+++" 220 ALPEILFAKV "+++" 221 ALVSTIIMV "+" 222 ALWELSLKI "+++" 223 ALWVSQPPEI "+++" 224 AMEALVVEV "+++" 225 ILQERELLPV "+++" 226 AMNISVPQV "+++" 227 FLAEASVMTQL "+++" 228 FLGGLSPGV "+++" 229 FLLNLQNCHL "++++" 230 FLQDSKVIFV "+++" 231 FLYIRQLAI "+++" 232 FMHQIIDQV "++++" 233 GIIDINVRL "++" 234 GLDDAEYAL "++" 235 GLDDLLLFL "+++" 236 GLLESGRHYL "+++" 237 GLQENLDVVV "++++" 238 GLVETELQL "+++" 239 ILAGEMLSV "+++" 240 ILARDILEI "+++" 241 ILGDILLKV "+++" 242 ILLGIQELL "+++" 243 ILPTLEKELFL "+++" 244 ILQALAVHL "+++" 245 KIMDYSLLLGV "++++" 246 KLDETGVAL "++" 247 KLKDRLPSI "++" 248 KTVEPPISQV "++" 249 LLPTGVFQV "+++" 250 LLVQEPDGLMV "+++" 251 LLYDNVPGA "+++" 252 NLLDPGSSYLL "+++" 253 NLWSVDGEVTV "+++" 254 QLIPKLIFL "+++" 255 YLFEEAISM "+++" 256 QLLPVSNVVSV "+++" 258 RLDYITAEI "+++" 259 RLLDEQFAVL "+++" 260 SLDDVEGMSV "+++" 261 SLVEAQGWLV "+++" 262 SLWNAGTSV "+++" 263 SQWEDIHVV "+++" 264 TILDYINVV "+++" 265 TLLADDLEIKL "+++" 266 TLLDQLDTQL "++" 267 TLLDWQDSL "+++" 268 TLLQVFHLL "+++" 269 TLTDEQFLV "++" 270 TVLPVPPLSV "+++" 271 VIRNIVEAA "++" 272 VLDELPPLI "+++" 273 VLGEYSYLL "+++" 274 VLLEYHIAYL "+++" 275 VLLFIEHSV "+++" 276 VLNDGAPNV "+++" 277 VMILKLPFL "++" 278 YLDDLLPKL "+++" 279 YMAPEVVEA "+++" 280 YTLDSLYWSV "+++" 281 NLLDDRGMTAL "+++" 282 LLRDGIELV "+++" 283 ILQPMDIHV "+++" 284 LLSAAEPVPA "++" 285 GVATAGCVNEV "++++" 286 FLLEDLSQKL "+++" 287 FLWEEKFNSL "++++" 288 GLAESTGLLAV "+++" 289 ILEEQPMDMLL "+++" 291 ILLNEDDLVTI "+++" 292 AAALIIHHV "+++" 293 ALDIMIPMV "+++" 294 ALLDQLHTLL "++++" 295 ALLQKLQQL "+++" 296 FIAPTGHSL "++" 297 FLVEPQEDTRL "+++" 298 IILPVEVEV "+++" 299 ILEENIPVL "+++" 300 ILLNPAYDVYL "+++" 301 AASPIITLV "+++" 302 ILQDLTFVHL "+++" 303 ILSQPTPSL "+++" 304 LAIVPVNTL "+" 305 LLFPQIEGIKI "+++" 306 VVAEELENV "++" 307 LLLTKPTEA "+++" 308 SLYDVSRMYV "++++" 309 ILYGTQFVL "+++" 310 LLSTLHLLV "+++" 311 LLVDVEPKV "+++" 312 LLYNSTDPTL "+++" 313 LMADLEGLHL "+++" 314 LMKDCEAEV "+++" 315 LVYEAPETV "+++" 316 MLLEHGITL "+++" 317 NLLAHIWAL "+++" 318 NLQVTQPTV "+++" 319 QVIPQLQTV "+++" 320 TIAPVTVAV "++++" 321 RLLEFELAQL "+++" 322 SLASIHVPL "+++" 323 SLDLFNCEV "++++" 324 SLYSALQQA "+++" 325 TLENGVPCV "+++" 326 VLAFLVHEL "++++" 327 VLIKWFPEV "++++" 328 VLLPQETAEIHL "++++" 329 VLMDGSVKL "++++" 330 VLMWEIYSL "+++" 331 VLWELAHLPTL "+++" 332 VMIQHVENL "+++" 333 VTLEFPQLIRV "+++" 334 YLLEEKIASL "+++" 335 YLYQEQYFI "+++" 336 YMAVTTQEV "++++" 337 YMYEKESEL "++" 338 FLDMTNWNL "++++" 339 GLWGTVVNI "+++" 340 KLLEEICNL "++++" 341 LLAELPASVHA "+++" 342 SLITPLQAV "+++" 344 VLAFENPQV "+++" 345 YLIEPDVELQRI "+++" 346 VLVQVSPSL "+++" 347 YLGPVSPSL "+++" 348 ALAKPPVVSV "+++" 349 ALATHILSL "+++" 350 ALEDRVWEL "+++" 351 ALSEKLARL "+++" 352 ALVFELHYV "+++" 353 ATPMPTPSV "++" 354 FIMDDPAGNSYL "+++" 355 FIWPMLIHI "+++" 356 FLHDHQAEL "++" 357 FLIQEIKTL "+++" 358 FLTDYLNDL "+++" 359 FMQDPMEVFV "+++" 360 HLIDTNKIQL "+++" 361 ILQEFESKL "+++" 362 ILTELGGFEV "+++" 363 ITTEVVNELYV "+++" 364 KMDWIFHTI "+++" 365 LISPLLLPV "+++" 367 NLWSLVAKV "+++" 369 RLLDLENSLLGL "+++" 370 SIFASPESV "+++" 371 SLADDSVLERL "+++" 372 SLFGPLPGPGPALV "+++" 373 TLLADQGEIRV "++" 374 VLSVITEEL "+++" 375 VLWFKPVEL "+++" 376 VLYNQRVEEI "+++" 377 VVDGTCVAV "+++" 378 YILGKFFAL "+++" 379 YLAELVTPIL "+++" 380 YLDRKLLTL "+++" 381 YLLEENKIKL "+++" 382 YLLPLLQRL "++++" 383 YLLREWVNL "++++" 384 YMIGSEVGNYL "++++" 385 YTIPLAIKL "+++" Example 6 Absolute quantification of tumor-associated peptides expressed on the surface of cells.

粘合劑例如抗體和/或 TCR 的產生是一個費力的過程，其可以僅針對一些選定靶標進行。在腫瘤相關和特異性肽的情況下，選擇標準包括但不限於排除表現於細胞表面上肽的表現和濃度。除了如實施例 1 中所述肽的分離和相對定量，發明人也分析了每個細胞的絕對肽拷貝數，如 x 發明所述。實體腫瘤樣本中每個細胞的 TUMAP拷貝數定量需要分離TUMAP的絕對定量、TUMAP分離效率和分析的組織樣本細胞計數。我們的實驗方法概述在圖4中給出，實驗步驟描述如下。 nanoLC-MS/MS 肽定量The production of adhesives such as antibodies and/or TCR is a laborious process, which can be performed only against some selected targets. In the case of tumor-related and specific peptides, the selection criteria include, but are not limited to, exclusion of the expression and concentration of the peptide on the cell surface. In addition to the separation and relative quantification of peptides as described in Example 1, the inventors also analyzed the absolute peptide copy number of each cell, as described in the x invention. The quantification of the TUMAP copy number of each cell in a solid tumor sample requires the absolute quantification of the separated TUMAP, the separation efficiency of TUMAP, and the cell count of the analyzed tissue sample. An overview of our experimental method is given in Figure 4, and the experimental steps are described as follows. nanoLC-MS/MS peptide quantification

對於通過質譜法對肽的準確定量，使用內標法生成每種肽的校準曲線。內標是每種肽的雙同位素標記的變體，即，TUMAP合成中納入2個同位素標記的氨基酸。它與腫瘤相關肽僅在品質上不同，但在其他的物理化學性質方面無差異(Anderson et al., 2012)。內標被摻入到每個MS樣本，所有MS信號均標準化為內標MS信號，以平衡MS實驗之間潛在的技術差異。校準曲線用至少三種不同的矩陣繪製，即，來自于類似於常規MS樣本的天然樣本的HLA肽洗脫液，並且每個制備品以重複MS運行進行測量。對於評價，MS信號被標準化為內標信號，校準曲線通過logistic回歸計算。對於來自組織樣本的腫瘤相關肽的定量，各樣本也摻有內標；MS信號標準化為內標並使用該肽校正曲線進行定量。 肽/MHC 分離的效率For accurate quantification of peptides by mass spectrometry, an internal standard method was used to generate a calibration curve for each peptide. The internal standard is a double-isotope-labeled variant of each peptide, that is, two isotopically-labeled amino acids are included in the synthesis of TUMAP. It differs from tumor-associated peptides only in quality, but no difference in other physical and chemical properties (Anderson et al., 2012). An internal standard is incorporated into each MS sample, and all MS signals are normalized to internal standard MS signals to balance potential technical differences between MS experiments. The calibration curve is drawn with at least three different matrices, namely, HLA peptide eluate from natural samples similar to conventional MS samples, and each preparation is measured with repeated MS runs. For the evaluation, the MS signal was normalized to the internal standard signal, and the calibration curve was calculated by logistic regression. For the quantification of tumor-associated peptides from tissue samples, each sample is also spiked with an internal standard; the MS signal is normalized to the internal standard and the peptide calibration curve is used for quantification. Efficiency of peptide/MHC separation

對於任何蛋白質純化過程，來自組織樣本蛋白的分離與相關蛋白的一定損失相關聯。為了確定TUMAP分離的效率，針對選定為絕對定量的所有TUMAP產生了肽/MHC複合體。為了能夠天然肽MHC/複合體與加樣物，使用了單同位素標記版本的TUMAP，即TUMAP合成期間納入1個同位素標記的氨基酸。這些複合物被摻入新製備的組織裂解物中，例如，在TUMAP分離過程中最早可能時間點，然後在之後的親和純化中像天然肽MHC/複合物被獲取。因此，測量單標記TUMAP的恢復可得到個體TUMAP分離效率相關的結論。For any protein purification process, the separation of proteins from tissue samples is associated with a certain loss of related proteins. To determine the efficiency of TUMAP separation, peptide/MHC complexes were generated for all TUMAPs selected as absolute quantification. In order to be able to use the natural peptide MHC/complex and sample, a monoisotope-labeled version of TUMAP is used, that is, an isotope-labeled amino acid is included during the synthesis of TUMAP. These complexes are incorporated into newly prepared tissue lysates, for example, at the earliest possible time point in the TUMAP separation process, and then obtained as natural peptide MHC/complexes in subsequent affinity purification. Therefore, measuring the recovery of single-labeled TUMAP can draw conclusions about the separation efficiency of individual TUMAP.

分離效率使用少量樣本進行分析，且這些組織樣本可比較。與此相反，個體肽之間的分離效率不同。這表明，分離效率雖然只在有限數量的樣本中進行測定，但可外推至任何其他組織制備品中。但是，由於分離效率不能從一種肽外推至其他肽，因此，有必要單獨分析每個TUMAP。 固體、冷凍組織中細胞計數的測定The separation efficiency uses a small number of samples for analysis, and these tissue samples are comparable. In contrast, the separation efficiency differs between individual peptides. This shows that although the separation efficiency is only measured in a limited number of samples, it can be extrapolated to any other tissue preparation. However, since the separation efficiency cannot be extrapolated from one peptide to other peptides, it is necessary to analyze each TUMAP separately. Determination of cell count in solid and frozen tissues

為了確定經過絕對肽定量的組織樣本的細胞數，發明人採用了DNA含量分析。此方法適用于不同來源的廣泛樣本，最重要的是，冷凍樣本(Alcoser et al., 2011; Forsey and Chaudhuri, 2009; Silva et al., 2013)。在肽分離方案期間，組織樣本被加工為均勻的裂解物，從中取一小等份裂解物。樣本等分為三份，從中分離 DNA被分離（QiaAmp DNA Mini Kit, Qiagen, Hilden，德國）。每次 DNA 分離的總 DNA 含量至少重複兩次使用基於螢光的 DNA 定量測定法（Qubit dsDNA HS Assay Kit, Life Technologies, Darmstadt，德國）進行定量。In order to determine the number of cells in a tissue sample subjected to absolute peptide quantification, the inventors used DNA content analysis. This method is suitable for a wide range of samples from different sources, most importantly, frozen samples (Alcoser et al., 2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During the peptide isolation protocol, the tissue sample is processed into a homogeneous lysate from which a small aliquot of the lysate is taken. The sample is divided into three equal parts, and the DNA is separated from it (QiaAmp DNA Mini Kit, Qiagen, Hilden, Germany). The total DNA content of each DNA separation was repeated at least twice using fluorescence-based DNA quantification (Qubit dsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) for quantification.

為了計算細胞數，生成了來自單個健康血細胞等分試樣的DNA標準曲線，使用一系列指定的細胞數。標準曲線用於計算每次 DNA 分離總 DNA 含量的總細胞含量。用於肽分離的組織樣本的平均總細胞計數，在考慮裂解物等份的已知體積和總裂解物體積的情況下進行推算。 每細胞的肽拷貝數To calculate the number of cells, a standard curve of DNA from a single aliquot of healthy blood cells was generated, using a series of designated cell numbers. The standard curve is used to calculate the total cell content of the total DNA content of each DNA separation. The average total cell count of the tissue sample used for peptide separation is extrapolated considering the known volume of the lysate aliquot and the total lysate volume. Number of peptide copies per cell

使用前述實驗的資料，發明人以總肽量除以樣本總細胞計數計算得出每個細胞的 TUMAP 拷貝數，隨後除以分離效率。選定肽的細胞拷貝數如表 12 所示。Using the data from the aforementioned experiment, the inventors calculated the number of copies of TUMAP per cell by dividing the total peptide amount by the total cell count of the sample, and then dividing by the separation efficiency. The cell copy numbers of the selected peptides are shown in Table 12.

表 12：絕對拷貝數。該表列出了 NSCLC  腫瘤樣本中絕對肽定量的結果。針對每種肽，每個細胞的中位元拷貝數表示：＜100 = +; ＞=100 = ++; ＞=1,000 +++; ＞=10,000 = ++++。提示樣本數量，其中提供評估的高品質 MS 數據。 序列號 序列 拷貝數分類 可定量的樣本數量 62 GIIDGSPRL  + 17 153 SLLAELHVL  ++ 17 Table 12: Absolute copy number. This table lists the results of absolute peptide quantification in NSCLC tumor samples. For each peptide, the median copy number of each cell is expressed as: <100 = +; >=100 = ++; >=1,000 +++; >=10,000 = ++++. Prompt the sample size, which provides high-quality MS data for evaluation. serial number sequence Copy number classification The number of samples that can be quantified 62 GIIDGSPRL + 17 153 SLLAELHVL ++ 17

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E. et al., Current Protocols in Protein Science (1995) Colombetti, S. et al., J Immunol. 176 (2006): 2730-2738 Dengjel, J. et al., Clin Cancer Res 12 (2006): 4163-4170 Denkberg, G. et al., J Immunol 171 (2003): 2197-2207 Falk, K. et al., Nature 351 (1991): 290-296 Fong, L. et al., Proc.Natl.Acad.Sci.U.S.A 98 (2001): 8809-8814 Gabrilovich, D. I. et al., Nat Med. 2 (1996): 1096-1103 Gattinoni, L. et al., Nat Rev.Immunol 6 (2006): 383-393 Gnjatic, S. et al., Proc Natl.Acad.Sci.U.S.A 100 (2003): 8862-8867 Godkin, A. et al., Int.Immunol 9 (1997): 905-911 Green, M. R. et al., Molecular Cloning, A Laboratory Manual 4th (2012) Greenfield, E. A., Antibodies: A Laboratory Manual 2nd (2014) Hwang, M. L. et al., J Immunol. 179 (2007): 5829-5838 Jung, G. et al., Proc Natl Acad Sci U S A 84 (1987): 4611-4615 Kibbe, A. H., Handbook of Pharmaceutical Excipients rd (2000) Krieg, A. M., Nat Rev.Drug Discov. 5 (2006): 471-484 Liddy, N. et al., Nat Med. 18 (2012): 980-987 Ljunggren, H. G. et al., J Exp.Med. 162 (1985): 1745-1759 Longenecker, B. M. et al., Ann N.Y.Acad.Sci. 690 (1993): 276-291 Lonsdale, J., Nat.Genet. 45 (2013): 580-585 Lukas, T. J. et al., Proc.Natl.Acad.Sci.U.S.A 78 (1981): 2791-2795 Lundblad, R. L., Chemical Reagents for Protein Modification 3rd (2004) Meziere, C. et al., J Immunol 159 (1997): 3230-3237 Morgan, R. A. et al., Science 314 (2006): 126-129 Mori, M. et al., Transplantation 64 (1997): 1017-1027 Mortara, L. et al., Clin Cancer Res. 12 (2006): 3435-3443 Mueller, L. N. et al., J Proteome.Res 7 (2008): 51-61 Mueller, L. N. et al., Proteomics. 7 (2007): 3470-3480 Mumberg, D. et al., Proc.Natl.Acad.Sci.U.S.A 96 (1999): 8633-8638 Pinheiro, J. et al., nlme: Linear and Nonlinear Mixed Effects Models; August 19, 2015. Version 3.1-122. (2015) Plebanski, M. et al., Eur.J Immunol 25 (1995): 1783-1787 Porta, C. et al., Virology 202 (1994): 949-955 Rammensee, H. G. et al., Immunogenetics 50 (1999): 213-219 Rini, B. I. et al., Cancer 107 (2006): 67-74 Rock, K. L. et al., Science 249 (1990): 918-921 Rodenko, B. et al., Nat Protoc. 1 (2006): 1120-1132 Saiki, R. K. et al., Science 239 (1988): 487-491 Seeger, F. H. et al., Immunogenetics 49 (1999): 571-576 Sherman, F. et al., Laboratory Course Manual for Methods in Yeast Genetics (1986) Singh-Jasuja, H. et al., Cancer Immunol.Immunother. 53 (2004): 187-195 Small, E. J. et al., J Clin Oncol. 24 (2006): 3089-3094 Sturm, M. et al., BMC.Bioinformatics. 9 (2008): 163 Teufel, R. et al., Cell Mol Life Sci. 62 (2005): 1755-1762 Tran, E. et al., Science 344 (2014): 641-645 Walter, S. et al., J Immunol 171 (2003): 4974-4978 Walter, S. et al., Nat Med. 18 (2012): 1254-1261 Willcox, B. E. et al., Protein Sci. 8 (1999): 2418-2423 Zaremba, S. et al., Cancer Res. 57 (1997): 4570-4577Reference list Allison, J. P. et al., Science 270 (1995): 932-933 Andersen, R. S. et al., Nat.Protoc. 7 (2012): 891-902 Appay, V. et al., Eur. J Immunol. 36 (2006): 1805-1814 Banchereau, J. et al., Cell 106 (2001): 271-274 Beatty, G. et al., J Immunol 166 (2001): 2276-2282 Beggs, J. D., Nature 275 (1978): 104-109 Benjamini, Y. et al., Journal of the Royal Statistical Society.Series B (Methodological), Vol.57 (1995): 289-300 Boulter, J. M. et al., Protein Eng 16 (2003): 707-711 Braumuller, H. et al., Nature (2013) Brossart, P. et al., Blood 90 (1997): 1594-1599 Bruckdorfer, T. et al., Curr.Pharm.Biotechnol. 5 (2004): 29-43 Card, K. F. et al., Cancer Immunol Immunother. 53 (2004): 345-357 Chanock, S. J. et al., Hum. Immunol. 65 (2004): 1211-1223 Cohen, C. J. et al., J Mol Recognit. 16 (2003a): 324-332 Cohen, C. J. et al., J Immunol 170 (2003b): 4349-4361 Cohen, S. N. et al., Proc.Natl.Acad.Sci.U.S.A 69 (1972): 2110-2114 Coligan, J. E. et al., Current Protocols in Protein Science (1995) Colombetti, S. et al., J Immunol. 176 (2006): 2730-2738 Dengjel, J. et al., Clin Cancer Res 12 (2006): 4163-4170 Denkberg, G. et al., J Immunol 171 (2003): 2197-2207 Falk, K. et al., Nature 351 (1991): 290-296 Fong, L. et al., Proc.Natl.Acad.Sci.U.S.A 98 (2001): 8809-8814 Gabrilovich, D. I. et al., Nat Med. 2 (1996): 1096-1103 Gattinoni, L. et al., Nat Rev. Immunol 6 (2006): 383-393 Gnjatic, S. et al., Proc Natl.Acad.Sci.U.S.A 100 (2003): 8862-8867 Godkin, A. et al., Int. Immunol 9 (1997): 905-911 Green, M. R. et al., Molecular Cloning, A Laboratory Manual 4th (2012) Greenfield, E. A., Antibodies: A Laboratory Manual 2nd (2014) Hwang, M. L. et al., J Immunol. 179 (2007): 5829-5838 Jung, G. et al., Proc Natl Acad Sci U S A 84 (1987): 4611-4615 Kibbe, A. H., Handbook of Pharmaceutical Excipients rd (2000) Krieg, A. M., Nat Rev. Drug Discov. 5 (2006): 471-484 Liddy, N. et al., Nat Med. 18 (2012): 980-987 Ljunggren, H. G. et al., J Exp. Med. 162 (1985): 1745-1759 Longenecker, B. M. et al., Ann N.Y.Acad.Sci. 690 (1993): 276-291 Lonsdale, J., Nat.Genet. 45 (2013): 580-585 Lukas, T. J. et al., Proc.Natl.Acad.Sci.U.S.A 78 (1981): 2791-2795 Lundblad, R. L., Chemical Reagents for Protein Modification 3rd (2004) Meziere, C. et al., J Immunol 159 (1997): 3230-3237 Morgan, R. A. et al., Science 314 (2006): 126-129 Mori, M. et al., Transplantation 64 (1997): 1017-1027 Mortara, L. et al., Clin Cancer Res. 12 (2006): 3435-3443 Mueller, L. N. et al., J Proteome.Res 7 (2008): 51-61 Mueller, L. N. et al., Proteomics. 7 (2007): 3470-3480 Mumberg, D. et al., Proc.Natl.Acad.Sci.U.S.A 96 (1999): 8633-8638 Pinheiro, J. et al., nlme: Linear and Nonlinear Mixed Effects Models; August 19, 2015. Version 3.1-122. (2015) Plebanski, M. et al., Eur. J Immunol 25 (1995): 1783-1787 Porta, C. et al., Virology 202 (1994): 949-955 Rammensee, H. G. et al., Immunogenetics 50 (1999): 213-219 Rini, B. I. et al., Cancer 107 (2006): 67-74 Rock, K. L. et al., Science 249 (1990): 918-921 Rodenko, B. et al., Nat Protoc. 1 (2006): 1120-1132 Saiki, R. K. et al., Science 239 (1988): 487-491 Seeger, F. H. et al., Immunogenetics 49 (1999): 571-576 Sherman, F. et al., Laboratory Course Manual for Methods in Yeast Genetics (1986) Singh-Jasuja, H. et al., Cancer Immunol.Immunother. 53 (2004): 187-195 Small, E. J. et al., J Clin Oncol. 24 (2006): 3089-3094 Sturm, M. et al., BMC.Bioinformatics. 9 (2008): 163 Teufel, R. et al., Cell Mol Life Sci. 62 (2005): 1755-1762 Tran, E. et al., Science 344 (2014): 641-645 Walter, S. et al., J Immunol 171 (2003): 4974-4978 Walter, S. et al., Nat Med. 18 (2012): 1254-1261 Willcox, B. E. et al., Protein Sci. 8 (1999): 2418-2423 Zaremba, S. et al., Cancer Res. 57 (1997): 4570-4577

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圖 1A至V 顯示了正常組織（白色柱）和 CLL（黑色柱）中各種肽的過量表現。圖 1A）基因：IGHM，肽：ALHRPDVYL (SEQ ID NO.:2)。從左到右的組織：2脂肪組織，3腎上腺，4血細胞，10血管，6骨髓，7腦，6乳腺，2軟骨，2眼，3膽囊，6心臟，14腎臟，19大腸，20肝臟，45肺，6淋巴結，7神經，3卵巢，10胰腺，3甲狀旁腺，1腹膜，5腦垂體，6胎盤，3胸膜，3攝護腺，7唾液腺，5骨骼肌，6皮膚，4小腸，9脾，5胃，6睾丸，2胸腺，3甲狀腺，9氣管，3輸尿管，6膀胱，2子宮，6食道，17慢性淋巴細胞白血病樣本。該肽還在 2/21 非霍奇金淋巴瘤樣本中檢測出。圖 1B）基因：IGHM，肽：VIAELPPKV (SEQ ID NO.:3)。從左到右的組織：2脂肪組織，3腎上腺，4血細胞，10血管，6骨髓，7腦，6乳腺，2軟骨，2眼，3膽囊，6心臟，14腎臟，19大腸，20肝臟，45肺，6淋巴結，7神經，3卵巢，10胰腺，3甲狀旁腺，1腹膜，5腦垂體，6胎盤，3胸膜，3攝護腺，7唾液腺，5骨骼肌，6皮膚，4小腸，9脾，5胃，6睾丸，2胸腺，3甲狀腺，9氣管，3輸尿管，6膀胱，2子宮，6食道，17慢性淋巴細胞白血病樣本。該肽還在 5/21 非霍奇金淋巴瘤樣本和 1/90 肺癌中檢測出。圖 1C），基因：IGHM，肽：VIAELPPKVSV (SEQ ID No.:4)，從左到右的組織：2脂肪組織，3腎上腺，4血細胞，10血管，6骨髓，7腦，6乳腺，2軟骨，2眼，3膽囊，6心臟，14腎臟，19大腸，20肝臟，45肺，6淋巴結，7神經，3卵巢，10胰腺，3甲狀旁腺，1腹膜，5腦垂體，6胎盤，3胸膜，3攝護腺，7唾液腺，5骨骼肌，6皮膚，4小腸，9脾，5胃，6睾丸，2胸腺，3甲狀腺，9氣管，3輸尿管，6膀胱，2子宮，6食道，17慢性淋巴細胞白血病樣本。圖 1D），基因：NUP210，肽：RLYEITIEV (SEQ ID No.:121)。從左到右的樣本：1 PBMC培養物，1良性攝護腺，8正常組織（3肺，4脾，1氣管），49癌組織（2腦癌，2乳腺癌，1結腸癌，13白細胞白血病，13肺癌，8淋巴結癌，1骨髓細胞癌，4卵巢癌，2皮膚癌，1膀胱癌，2子宮癌）。圖 1D 和表 4 之間的腫瘤類型相關的差異可能是由於表 4 採用更嚴格的選擇標準所致（詳情請參照表 4）。測試的正常組織系列、癌細胞系和異種移植體與 圖 1A-C 中相同。圖 1E），基因：COBRA1，肽：ALLRLLPGL (SEQ ID No.:14)。從左到右的樣本：1細胞系，11癌組織（1乳腺癌，1結腸癌，1頭頸癌，2白細胞性白血病，1肝癌，1肺癌，2淋巴結癌，1髓樣細胞癌，1皮膚癌）。圖 1F），基因：KIAA0226L，肽：GIIDGSPRL (SEQ ID No.:62)。從左到右的樣本：2正常組織（1淋巴結，1脾），18癌組織（2結腸癌，9白細胞白血病，6淋巴結癌，1直腸癌）。圖 1G），基因：DDX3X，肽：GLDQQFAGLDL (SEQ ID No.:68)。從左到右的樣本：1細胞系，2原代培養物，21癌組織（1膽管癌，1腦癌，1乳腺癌，1頭頸癌，2腎癌，3白細胞性白血病，2肝癌，2肺癌，2淋巴結癌，3卵巢癌，1直腸癌，1皮膚癌，1子宮癌）。圖 1H），基因：LIG1，肽：KTLDVDATYEI (SEQ ID No.:112)。從左到右的樣本：2細胞系，1原代培養物，13癌組織（1食管癌，2白細胞白血病，1肝癌，3肺癌，2淋巴結癌，2卵巢癌，1皮膚癌，1子宮癌）。圖 1I），基因：SMCHD1，肽：SIIEGPIIKL (SEQ ID No.:146)。從左到右的樣本：14癌組織（1頭頸癌，3白細胞白血病，6肺癌，1淋巴結癌，1卵巢癌，2膀胱癌）。圖 1J），基因：MMS22L，肽：SILETVATL (SEQ ID No.:147)。從左到右的樣本：1原代培養物，11癌組織（1結腸癌，4白細胞性白血病，1肺癌，3淋巴結癌，1骨髓細胞癌，1皮膚癌）。圖 1K），基因：FCRL3，肽：SLLAELHVL (SEQ ID No.:153)。從左到右的樣本：1細胞系，9正常組織（1淋巴結，1直腸，7脾），27癌組織（1乳腺癌，1腎癌，10白細胞性白血病，14淋巴結癌，1皮膚癌）。圖 1L），基因：FAM126A，肽：SLQEEKLIYV (SEQ ID No.:159)。從左到右的樣本：2細胞系，23癌症組織（5腦癌，1乳腺癌，1食管癌，3頭頸癌，2白細胞白血病，5肺癌，3淋巴結癌，1皮膚癌，2膀胱癌）。圖 1M），基因：HNRNPC，肽：SVHKGFAFV (SEQ ID No.:164)。從左到右的樣本：10細胞系，1原代培養物，1正常組織（1腎上腺），36癌組織（12腦癌，1結腸癌，2頭頸癌，2腎癌，3白細胞白血病，1肝癌，5肺癌，1骨髓細胞癌，1卵巢癌，1直腸癌，1皮膚癌，2胃癌，2膀胱癌，2子宮癌）。圖 1N），基因：RASGRF1，肽：TLDTSKLYV (SEQ ID No.:167)。從左到右的樣本：13癌組織（1腦癌，8白細胞白血病，4淋巴結癌）。圖 1O），基因：RASGRF1，肽：YLLDQSFVM (SEQ ID No.:168)。從左到右的樣本：16癌組織（10白細胞白血病，6淋巴結癌）。圖 1P），基因：RNF213，肽：YIQEYLTLL (SEQ ID No.:192)。從左到右的樣本：2細胞系，18癌組織（1結腸癌，1頭頸癌，1腎癌，4白細胞白血病，5肺癌，3淋巴結癌，1骨髓細胞癌，1胃癌，1膀胱癌）。圖 1Q），基因：DCK，肽：YLQEVPILTL (SEQ ID No.:197)。從左到右的樣本：11癌組織（5白細胞白血病，4淋巴結癌，1皮膚癌，1子宮癌）。圖 1R），基因：EIF3H，肽：YMFEEVPIVI (SEQ ID No.:200)。從左到右的樣本：4細胞系，2原代培養物，34癌組織（1膽管癌，2乳腺癌，1結腸癌，1食管癌，1膽囊癌，5頭頸癌，2白細胞白血病，2肝癌，9肺癌，4淋巴結癌，2卵巢癌，1直腸癌，2皮膚癌，1膀胱癌）。圖 1S），基因：UBLCP1，肽：LIDVKPLGV (SEQ ID No.:211)。從左到右的樣本：3細胞系，23癌組織（5腦癌，1乳腺癌，1食管癌，1頭頸癌，1腎癌，3白細胞性白血病，2肝癌，2肺癌，4淋巴結癌，1卵巢癌，1攝護腺癌，1皮膚癌）。圖 1T），基因：TMCO6，肽：QLLPVSNVVSV (SEQ ID No.:256)。從左到右的樣本：2原代培養物，13癌組織（2頭頸癌，3白細胞白血病，1肝癌，2肺癌，4淋巴結癌，1卵巢癌）。圖 1U），基因：MYO1G，肽：LLYNSTDPTL (SEQ ID No.:312)。從左到右的樣本：1細胞系，2原代培養物，14癌組織（1結腸癌，4白細胞白血病，2肺癌，5淋巴結癌，1卵巢癌，1膀胱癌）。圖 1V），基因：PIM1，肽：VLLPQETAEIHL (SEQ ID No.:328)。從左到右的樣本：3原代培養物，27癌組織（1乳腺癌，1結腸癌，5白細胞白血病，5肺癌，7淋巴結癌，2卵巢癌，2皮膚癌，1胃癌，3尿膀胱癌）。Figures 1A to V show the overrepresentation of various peptides in normal tissues (white bars) and CLL (black bars). Figure 1A) Gene: IGHM, peptide: ALHRPDVYL (SEQ ID NO.: 2). Tissues from left to right: 2 adipose tissue, 3 adrenal glands, 4 blood cells, 10 blood vessels, 6 bone marrow, 7 brains, 6 breasts, 2 cartilage, 2 eyes, 3 gallbladder, 6 heart, 14 kidneys, 19 large intestine, 20 liver, 45 lungs, 6 lymph nodes, 7 nerves, 3 ovaries, 10 pancreas, 3 parathyroid glands, 1 peritoneum, 5 pituitary gland, 6 placenta, 3 pleura, 3 prostate, 7 salivary glands, 5 skeletal muscle, 6 skin, 4 Small intestine, 9 spleen, 5 stomach, 6 testes, 2 thymus, 3 thyroid, 9 trachea, 3 ureter, 6 bladder, 2 uterus, 6 esophagus, 17 chronic lymphocytic leukemia samples. The peptide was also detected in 2/21 non-Hodgkin's lymphoma samples. Figure 1B) Gene: IGHM, peptide: VIAELPPKV (SEQ ID NO.: 3). Tissues from left to right: 2 adipose tissue, 3 adrenal glands, 4 blood cells, 10 blood vessels, 6 bone marrow, 7 brains, 6 breasts, 2 cartilage, 2 eyes, 3 gallbladder, 6 heart, 14 kidneys, 19 large intestine, 20 liver, 45 lungs, 6 lymph nodes, 7 nerves, 3 ovaries, 10 pancreas, 3 parathyroid glands, 1 peritoneum, 5 pituitary gland, 6 placenta, 3 pleura, 3 prostate, 7 salivary glands, 5 skeletal muscle, 6 skin, 4 Small intestine, 9 spleen, 5 stomach, 6 testes, 2 thymus, 3 thyroid, 9 trachea, 3 ureter, 6 bladder, 2 uterus, 6 esophagus, 17 chronic lymphocytic leukemia samples. The peptide was also detected in 5/21 non-Hodgkin’s lymphoma samples and 1/90 lung cancer. Figure 1C), gene: IGHM, peptide: VIAELPPKVSV (SEQ ID No.: 4), tissue from left to right: 2 adipose tissue, 3 adrenal glands, 4 blood cells, 10 blood vessels, 6 bone marrow, 7 brain, 6 breast, 2 Cartilage, 2 eyes, 3 gallbladder, 6 heart, 14 kidneys, 19 large intestine, 20 liver, 45 lungs, 6 lymph nodes, 7 nerves, 3 ovaries, 10 pancreas, 3 parathyroid glands, 1 peritoneum, 5 pituitary gland, 6 placenta , 3 pleura, 3 prostate, 7 salivary gland, 5 skeletal muscle, 6 skin, 4 small intestine, 9 spleen, 5 stomach, 6 testis, 2 thymus, 3 thyroid, 9 trachea, 3 ureter, 6 bladder, 2 uterus, 6 Esophagus, 17 samples of chronic lymphocytic leukemia. Figure 1D), gene: NUP210, peptide: RLYEITIEV (SEQ ID No.: 121). Samples from left to right: 1 PBMC culture, 1 benign prostate, 8 normal tissues (3 lungs, 4 spleen, 1 trachea), 49 cancerous tissues (2 brain cancer, 2 breast cancer, 1 colon cancer, 13 white blood cells) Leukemia, 13 lung cancer, 8 lymph node cancer, 1 bone marrow cell carcinoma, 4 ovarian cancer, 2 skin cancer, 1 bladder cancer, 2 uterine cancer). The tumor type-related differences between Figure 1D and Table 4 may be due to the stricter selection criteria used in Table 4 (see Table 4 for details). The normal tissue series, cancer cell lines and xenografts tested are the same as in Figure 1A-C. Figure 1E), gene: COBRA1, peptide: ALLRLLPGL (SEQ ID No.: 14). Samples from left to right: 1 cell line, 11 cancer tissue (1 breast cancer, 1 colon cancer, 1 head and neck cancer, 2 leukemia, 1 liver cancer, 1 lung cancer, 2 lymph node cancer, 1 medullary cell carcinoma, 1 skin cancer). Figure 1F), gene: KIAA0226L, peptide: GIDGSPRL (SEQ ID No.: 62). Samples from left to right: 2 normal tissues (1 lymph node, 1 spleen), 18 cancer tissues (2 colon cancer, 9 leukemia, 6 lymph node cancer, 1 rectal cancer). Figure 1G), gene: DDX3X, peptide: GLDQQFAGLDL (SEQ ID No.: 68). Samples from left to right: 1 cell line, 2 primary cultures, 21 cancer tissues (1 cholangiocarcinoma, 1 brain cancer, 1 breast cancer, 1 head and neck cancer, 2 kidney cancer, 3 leukemia, 2 liver cancer, 2 Lung cancer, 2 lymph node cancer, 3 ovarian cancer, 1 rectal cancer, 1 skin cancer, 1 uterine cancer). Figure 1H), gene: LIG1, peptide: KTLDVDATYEI (SEQ ID No.: 112). Samples from left to right: 2 cell lines, 1 primary culture, 13 cancer tissues (1 esophageal cancer, 2 leukemia, 1 liver cancer, 3 lung cancer, 2 lymph node cancer, 2 ovarian cancer, 1 skin cancer, 1 uterine cancer ). Figure 1I), gene: SMCHD1, peptide: SIIEGPIIKL (SEQ ID No.: 146). Samples from left to right: 14 cancer tissues (1 head and neck cancer, 3 leukemia, 6 lung cancer, 1 lymph node cancer, 1 ovarian cancer, 2 bladder cancer). Figure 1J), gene: MMS22L, peptide: SILETVATL (SEQ ID No.: 147). Samples from left to right: 1 primary culture, 11 cancer tissues (1 colon cancer, 4 leukemia, 1 lung cancer, 3 lymph node cancer, 1 bone marrow cell cancer, 1 skin cancer). Figure 1K), gene: FCRL3, peptide: SLLAELHVL (SEQ ID No.: 153). Samples from left to right: 1 cell line, 9 normal tissues (1 lymph node, 1 rectum, 7 spleen), 27 cancer tissues (1 breast cancer, 1 kidney cancer, 10 leukemia, 14 lymph node cancer, 1 skin cancer) . Figure 1L), gene: FAM126A, peptide: SLQEEKLIYV (SEQ ID No.: 159). Samples from left to right: 2 cell lines, 23 cancer tissues (5 brain cancer, 1 breast cancer, 1 esophageal cancer, 3 head and neck cancer, 2 leukemia, 5 lung cancer, 3 lymph node cancer, 1 skin cancer, 2 bladder cancer) . Figure 1M), gene: HNRNPC, peptide: SVHKGFAFV (SEQ ID No.: 164). Samples from left to right: 10 cell lines, 1 primary culture, 1 normal tissue (1 adrenal gland), 36 cancerous tissue (12 brain cancer, 1 colon cancer, 2 head and neck cancer, 2 kidney cancer, 3 leukemia, 1 Liver cancer, 5 lung cancer, 1 bone marrow cell cancer, 1 ovarian cancer, 1 rectal cancer, 1 skin cancer, 2 stomach cancer, 2 bladder cancer, 2 uterine cancer). Figure 1N), gene: RASGRF1, peptide: TLDTSKLYV (SEQ ID No.: 167). Samples from left to right: 13 cancer tissues (1 brain cancer, 8 leukemia, 4 lymph node cancer). Figure 10), gene: RASGRF1, peptide: YLLDQSFVM (SEQ ID No.: 168). Samples from left to right: 16 cancerous tissues (10 leukemia, 6 lymph node cancer). Figure 1P), gene: RNF213, peptide: YIQEYLTLL (SEQ ID No.: 192). Samples from left to right: 2 cell lines, 18 cancer tissues (1 colon cancer, 1 head and neck cancer, 1 kidney cancer, 4 leukemia, 5 lung cancer, 3 lymph node cancer, 1 bone marrow cell cancer, 1 gastric cancer, 1 bladder cancer) . Figure 1Q), gene: DCK, peptide: YLQEVPILTL (SEQ ID No.: 197). Samples from left to right: 11 cancer tissues (5 leukemia, 4 lymph node cancer, 1 skin cancer, 1 uterine cancer). Figure 1R), gene: EIF3H, peptide: YMFEEVPIVI (SEQ ID No.: 200). Samples from left to right: 4 cell lines, 2 primary cultures, 34 cancer tissues (1 cholangiocarcinoma, 2 breast cancer, 1 colon cancer, 1 esophageal cancer, 1 gallbladder cancer, 5 head and neck cancer, 2 leukemia, 2 Liver cancer, 9 lung cancer, 4 lymph node cancer, 2 ovarian cancer, 1 rectal cancer, 2 skin cancer, 1 bladder cancer). Figure 1S), gene: UBLCP1, peptide: LIDVKPLGV (SEQ ID No.: 211). Samples from left to right: 3 cell lines, 23 cancer tissues (5 brain cancer, 1 breast cancer, 1 esophageal cancer, 1 head and neck cancer, 1 kidney cancer, 3 leukemia, 2 liver cancer, 2 lung cancer, 4 lymph node cancer, 1 ovarian cancer, 1 prostate cancer, 1 skin cancer). Figure 1T), gene: TMCO6, peptide: QLLPVSNVVSV (SEQ ID No.: 256). Samples from left to right: 2 primary cultures, 13 cancer tissues (2 head and neck cancer, 3 leukemia, 1 liver cancer, 2 lung cancer, 4 lymph node cancer, 1 ovarian cancer). Figure 1U), gene: MYO1G, peptide: LLYNSTDPTL (SEQ ID No.: 312). Samples from left to right: 1 cell line, 2 primary cultures, 14 cancer tissues (1 colon cancer, 4 leukemia, 2 lung cancer, 5 lymph node cancer, 1 ovarian cancer, 1 bladder cancer). Figure 1V), gene: PIM1, peptide: VLLPQETAEIHL (SEQ ID No.: 328). Samples from left to right: 3 primary cultures, 27 cancer tissues (1 breast cancer, 1 colon cancer, 5 leukemia, 5 lung cancer, 7 lymph node cancer, 2 ovarian cancer, 2 skin cancer, 1 gastric cancer, 3 urine bladder cancer).

圖2A至D顯示了本發明的源基因的代表性表達特徵，這些基因在一系列正常組織（白色柱）的 CLL 中以及17個CLL樣本（黑色柱）中高度過度表達或專門表達。從左到右的組織：6動脈，1血細胞，1腦，1心臟，2肝臟，2肺，2靜脈，1脂肪組織，1腎上腺，6骨髓，1軟骨，1結腸，1食管，2眼，2膽囊，1腎，6淋巴結，5胰腺，2腦垂體，1直腸，1唾液腺，1骨骼肌，1皮膚，1小腸，1脾，1胃，1甲狀腺，7氣管，1膀胱，1乳腺，5卵巢，3胎盤，1攝護腺，1睾丸，1胸腺，1子宮，10個慢性淋巴細胞白血病樣本。圖 2A）基因符號：FCER2；圖 2B）基因符號：KIAA0226L；圖 2C）基因符號：PAX5；圖 2D）基因符號：CLEC17A。Figures 2A to D show the representative expression characteristics of the source genes of the present invention, which are highly overexpressed or exclusively expressed in a series of CLLs of normal tissues (white bars) and 17 CLL samples (black bars). Tissues from left to right: 6 arteries, 1 blood cells, 1 brain, 1 heart, 2 liver, 2 lungs, 2 veins, 1 adipose tissue, 1 adrenal gland, 6 bone marrow, 1 cartilage, 1 colon, 1 esophagus, 2 eyes, 2 gallbladder, 1 kidney, 6 lymph nodes, 5 pancreas, 2 pituitary gland, 1 rectum, 1 salivary gland, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thyroid, 7 trachea, 1 bladder, 1 breast, 5 ovaries, 3 placenta, 1 prostate, 1 testis, 1 thymus, 1 uterus, 10 chronic lymphocytic leukemia samples. Figure 2A) Gene symbol: FCER2; Figure 2B) Gene symbol: KIAA0226L; Figure 2C) Gene symbol: PAX5; Figure 2D) Gene symbol: CLEC17A.

圖3顯示了示例性的免疫原性資料：肽特定多聚體染色(multimer staining)後流式細胞儀結果。使用披覆有抗-CD28 mAB和HLA-A*02的人造APC來引發(prime) CD8+ T細胞，其中HLA-A*02分別與SeqID No 1胜肽(C，左圖)、SeqID No 5胜肽(D，左圖)、SeqID No 32胜肽(E，左圖)及SeqID No 220胜肽(F、左圖)複合。在三個循環的刺激之後，藉由2D多聚體染色(2D multimer staining)進行胜肽-反應性細胞的偵測，所述2D多聚體染色伴隨著A*02/SeqID No 1 (C)、A*02/SeqID No 5 (D)、A*02/SeqID No 32 (E)或A*02/SeqID No 220 (F)。右圖(C、D、E及F)顯示以不相關的A*02/胜肽複合物刺激的細胞之對照染色。針對CD8+淋巴細胞閘管(gate)可用的單態細胞(singlet cell)。布林閘(Boolean gates)有助於排除以對不同胜肽具特異性的多聚體所檢測到的假陽性事件。CD8+淋巴細胞中之特異性多聚體+細胞的頻率被指出。Figure 3 shows exemplary immunogenicity data: flow cytometry results after peptide-specific multimer staining. Use artificial APC coated with anti-CD28 mAB and HLA-A*02 to prime CD8+ T cells, among which HLA-A*02 is compatible with SeqID No 1 peptide (C, left picture) and SeqID No 5 respectively. Peptide (D, left picture), SeqID No 32 peptide (E, left picture) and SeqID No 220 peptide (F, left picture) are compounded. After three cycles of stimulation, the peptide-reactive cells were detected by 2D multimer staining, which was accompanied by A*02/SeqID No 1 (C) , A*02/SeqID No 5 (D), A*02/SeqID No 32 (E) or A*02/SeqID No 220 (F). The right panels (C, D, E, and F) show control staining of cells stimulated with the irrelevant A*02/peptide complex. Singlet cell available for CD8+ lymphocyte gate. Boolean gates help eliminate false positive events detected with polymers specific to different peptides. The frequency of specific multimer+ cells in CD8+ lymphocytes is indicated.

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Claims (22)

一種肽或其藥用鹽，其中該肽由選自SEQ ID NO:10與SEQ ID NO:1至SEQ ID NO:8與SEQ ID NO:11至SEQ ID NO:85與SEQ ID NO:87至SEQ ID NO:385所構成之群組的氨基酸序列所組成。A peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is selected from SEQ ID NO: 10 and SEQ ID NO: 1 to SEQ ID NO: 8 and SEQ ID NO: 11 to SEQ ID NO: 85 and SEQ ID NO: 87 to SEQ ID NO: 385 is composed of the amino acid sequence of the group. 根據請求項1所述的肽，其中所述肽有能力與一MHC-I類分子結合，且其中所述肽與MHC結合時能夠被CD8 T細胞識別。The peptide according to claim 1, wherein the peptide is capable of binding to an MHC class I molecule, and wherein the peptide is capable of being recognized by CD8 T cells when bound to MHC. 根據請求項1或2所述的肽，其中所述肽包含非肽鍵。The peptide according to claim 1 or 2, wherein the peptide comprises a non-peptide bond. 根據請求項1或2所述的肽，其中所述肽為一融合蛋白的部分，包含 HLA-DR抗原相關不變鏈 (Ii)的N-端氨基酸。The peptide according to claim 1 or 2, wherein the peptide is a part of a fusion protein and comprises the N-terminal amino acid of the HLA-DR antigen-related invariant chain (Ii). 一種核酸，編碼根據請求項1至4任一項所述的肽。A nucleic acid encoding the peptide according to any one of claims 1 to 4. 一種表達載體，可表達根據請求項5所述的核酸。An expression vector capable of expressing the nucleic acid according to claim 5. 一種重組宿主細胞，其包括根據請求項1至4任一項所述的肽、根據請求項5所述的核酸或根據請求項6所述的表達載體，其中所述宿主細胞非為人類胚胎幹細胞。A recombinant host cell, comprising the peptide according to any one of claims 1 to 4, the nucleic acid according to claim 5, or the expression vector according to claim 6, wherein the host cell is not a human embryonic stem cell . 根據請求項7所述的宿主細胞，其中所述細胞為一抗原提呈細胞。The host cell according to claim 7, wherein the cell is an antigen presenting cell. 根據請求項7或8所述的宿主細胞，其中所述細胞為一樹突狀細胞。The host cell according to claim 7 or 8, wherein the cell is a dendritic cell. 一種製備根據請求項1至4任一項所述的肽的方法，該方法包括培養根據請求項7至9任一項所述的宿主細胞，其提呈根據請求項1至4任一項所述的肽或表達根據請求項5所述的核酸或包含根據請求項6所述的表達載體，以及從該宿主細胞或其培養基中分離出所述肽。A method for preparing the peptide according to any one of claims 1 to 4, the method comprising culturing the host cell according to any one of claims 7 to 9, and submitting it according to any one of claims 1 to 4 The peptide or the expression of the nucleic acid according to claim 5 or the expression vector according to claim 6, and the peptide is isolated from the host cell or its culture medium. 一種體外製備活化的T淋巴細胞的方法，該方法包括將T細胞與載有抗原的人I類MHC分子進行體外接觸足夠的一段時間從而以抗原特異性方式活化T細胞，這些人I類MHC分子在合適的抗原提呈細胞表面或人工類比的抗原提呈細胞結構表面上表達，其中所述抗原為請求項1至4任一項所述的肽。A method for preparing activated T lymphocytes in vitro, which comprises contacting T cells with human class I MHC molecules carrying antigens in vitro for a sufficient period of time to activate T cells in an antigen-specific manner. These human class I MHC molecules It is expressed on the surface of a suitable antigen-presenting cell or an artificial analogous antigen-presenting cell structure, wherein the antigen is the peptide according to any one of claims 1 to 4. 一種根據請求項11所述的方法製成的活化T淋巴細胞，其有選擇性地識別提呈一種包含請求項1至4任一項給定氨基酸序列的多肽的細胞。An activated T lymphocyte prepared according to the method of claim 11, which selectively recognizes and presents a cell containing a polypeptide containing a given amino acid sequence of any one of claims 1 to 4. 一種根據請求項12所述活化毒性T淋巴細胞在製備用於殺滅患者體內靶向細胞之藥物的用途，其中該些靶向細胞異常地表現和/或提呈一種包含請求項1至4任一項給定的氨基酸序列的多肽。A use of the activated toxic T lymphocytes according to claim 12 in the preparation of a drug for killing targeted cells in a patient, wherein the targeted cells abnormally behave and/or present a product containing any of claims 1 to 4 A polypeptide of a given amino acid sequence. 一種根據請求項1至4任一項所述的肽用於產生與開發抗體的非治療性用途，該些抗體特異性地識別該肽。A non-therapeutic use of the peptide according to any one of claims 1 to 4 for the production and development of antibodies, and these antibodies specifically recognize the peptide. 一種抗體，特異性結合至一種請求項1至4任一項所述的肽。An antibody that specifically binds to a peptide according to any one of claims 1 to 4. 根據請求項15所述的抗體，其中該抗體係一單株抗體。The antibody according to claim 15, wherein the antibody system is a monoclonal antibody. 一種根據請求項1至4任一項所述的肽、根據請求項5所述的核酸、根據請求項6所述的表達載體、根據請求項7至9任一項所述的細胞、根據請求項12所述的活化T淋巴細胞或根據請求項15或16所述的抗體在製備一種藥劑的用途，其中該藥劑具有抗癌活性。A peptide according to any one of claims 1 to 4, a nucleic acid according to claim 5, an expression vector according to claim 6, a cell according to any one of claims 7 to 9, upon request Use of the activated T lymphocyte according to item 12 or the antibody according to claim 15 or 16 in the preparation of a medicament, wherein the medicament has anticancer activity. 一種根據請求項1至4任一項所述的肽鹽、根據請求項5所述的核酸、根據請求項6所述的表達載體、根據請求項7至9任一項所述的細胞、根據請求項12所述的活化T淋巴細胞或根據請求項15或16所述的抗體在製備一種藥劑的用途，其中該藥劑具有抵抗一種選自下列所構成之群組的癌症的活性：急性骨髓性白血病、膽管癌、腦癌、乳腺癌、結直腸癌、食管癌、膽囊癌、胃癌、肝細胞癌、梅克爾細胞癌、黑色素瘤、非霍奇金淋巴瘤、非小細胞肺癌、卵巢癌、胰腺癌、攝護腺癌、腎細胞癌、小細胞肺癌、膀胱癌、子宮癌、CLL和其他腫瘤，這些腫瘤過度表達自其衍生選自SEQ ID NO: 10與SEQ ID NO:1至SEQ ID NO:8與SEQ ID NO:11至SEQ ID NO:85與SEQ ID NO:87至SEQ ID NO:385所構成之群組的肽的蛋白。A peptide salt according to any one of claims 1 to 4, a nucleic acid according to claim 5, an expression vector according to claim 6, a cell according to any one of claims 7 to 9, according to Use of the activated T lymphocyte according to claim 12 or the antibody according to claim 15 or 16 in the preparation of a medicament, wherein the medicament has activity against a cancer selected from the group consisting of: acute myeloid Leukemia, cholangiocarcinoma, brain cancer, breast cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, hepatocellular carcinoma, Merkel cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, Pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine cancer, CLL and other tumors, these tumors are overexpressed from which are derived from SEQ ID NO: 10 and SEQ ID NO: 1 to SEQ ID NO: 8 and SEQ ID NO: 11 to SEQ ID NO: 85 and SEQ ID NO: 87 to SEQ ID NO: 385 are the peptides of the group. 一種藥盒套件，包括： (a)    一容器，包含一藥物組合物，其含有根據請求項1至4任一項所述的肽、根據請求項5所述的核酸、根據請求項6所述的表達載體、根據請求項7至9任一項所述的細胞、根據請求項12所述的活化T淋巴細胞或根據請求項15或16所述的抗體之溶液或凍幹形式； (b)    一第二容器，其含有凍乾粉劑型的稀釋劑或重組溶液；和/或 (c)     (i)使用溶液或(ii)重組和/或使用凍乾粉劑型的說明書。A kit of medicine boxes, including: (a) A container containing a pharmaceutical composition containing the peptide according to any one of claims 1 to 4, the nucleic acid according to claim 5, the expression vector according to claim 6, and The cell according to any one of 7 to 9, the activated T lymphocyte according to claim 12, or the antibody according to claim 15 or 16 in solution or lyophilized form; (b) A second container containing a diluent or reconstituted solution in the form of a lyophilized powder; and/or (c) (i) Instructions for use of solution or (ii) reconstitution and/or use of lyophilized powder dosage form. 根據請求項19所述的藥盒套件，進一步包括一個或多個(iii)緩衝劑、(iv)稀釋劑、(v)過濾件、(vi)針、或(v)注射器。The kit kit according to claim 19, further comprising one or more of (iii) buffer, (iv) diluent, (v) filter, (vi) needle, or (v) syringe. 一種可溶性或膜結合T細胞受體，其與HLA配體反應，其中所述配體相同於選自SEQ ID NO: 10與SEQ ID NO:1至SEQ ID NO:8與SEQ ID NO:11至SEQ ID NO:85與SEQ ID NO:87至SEQ ID NO:385所構成之群組的氨基酸序列。A soluble or membrane-bound T cell receptor that reacts with HLA ligands, wherein the ligands are the same as those selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 1 to SEQ ID NO: 8 and SEQ ID NO: 11 to The amino acid sequence of the group consisting of SEQ ID NO:85 and SEQ ID NO:87 to SEQ ID NO:385. 一種藥物組合物，其包括至少一種活性成分，該成分選自下列組成的群組： a)  根據請求項1至4任一項所述的肽； b) 根據請求項5所述的核酸； c)  根據請求項6所述的表達載體； d) 根據請求項7至9任一項所述的重組宿主細胞； e)  根據請求項12所述的活化T淋巴細胞； f)  根據請求項15或16所述的抗體。A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of: a) The peptide according to any one of claims 1 to 4; b) The nucleic acid according to claim 5; c) According to the expression vector described in claim 6; d) The recombinant host cell according to any one of claims 7 to 9; e) Activate T lymphocytes according to claim 12; f) The antibody according to claim 15 or 16.

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