CN110662553A - 转基因巨噬细胞、嵌合抗原受体和相关方法 - Google Patents
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Abstract
本文描述了嵌合受体。嵌合受体包含细胞质结构域、跨膜结构域和细胞外结构域。在实施方案中，所述细胞质结构域包含受体的细胞质部分，所述细胞质部分在活化时使巨噬细胞极化。在另外的实施方案中，包含所述细胞质部分的野生型蛋白质不包含所述嵌合受体的所述细胞外结构域。在实施方案中，配体与所述嵌合受体的所述细胞外结构域的结合活化所述嵌合受体的所述细胞内部分。所述嵌合受体的所述细胞内部分的活化可使所述巨噬细胞极化成M1或M2巨噬细胞。
Description
技术领域
本公开整体涉及生物技术。更具体地，本公开涉及嵌合抗原受体、编码嵌合抗原受体的核酸、携带嵌合抗原受体和/或编码核酸的巨噬细胞，以及相关方法。
背景技术
癌症包括一组疾病，这些疾病涉及不受调控的细胞生长和死亡、基因组不稳定性和突变、肿瘤促进炎症、血管生成诱导、免疫系统逃避、代谢途径失调、永生细胞复制和转移性组织入侵[1]。癌症是美国仅次于心脏病的第二大死亡原因[2]。预计每年将诊断出超过160万新的癌症病例，预期将有超过58万美国人死亡(每天约1600例癌症死亡)，占美国人死亡总数的近四分之一[2,3]。
免疫系统在癌症的发展和进展中起着重要作用。免疫细胞向肿瘤部位的浸润可对恶性肿瘤的进展和转移产生不利影响[4,5]。据显示，在某些乳腺癌病例中，巨噬细胞向肿瘤部位的浸润占肿瘤质量的50％以上，这表明巨噬细胞在肿瘤进展中具有重要作用[6-8]。
巨噬细胞是来源于髓系并属于先天免疫系统的细胞。它们来源于迁移到组织中的血液单核细胞。它们的主要功能之一是吞噬微生物并清除细胞碎片。它们在炎症的引发和消退中也起着重要的作用[9,10]。此外，巨噬细胞可表现出不同的反应，范围从促炎到抗炎，这取决于它们从周围微环境中接收到的刺激类型[11]。已经提出了两种主要的巨噬细胞表型，它们与极端巨噬细胞反应有关：M1和M2。
M1促炎巨噬细胞在与某些分子接触诸如脂多糖(LPS)、IFN-γ、IL-1β、TNF-α和Toll样受体结合时被活化。M1巨噬细胞构成免疫系统的为抵抗感染而部署的强有力武器。它们能够直接(病原体模式识别受体)或间接(Fc受体、补体受体)识别病原体。它们还具备产生反应性氧物质(ROS)的能力，作为帮助杀死病原体的手段。此外，M1巨噬细胞分泌促炎细胞因子和趋化因子，吸引其他类型的免疫细胞并整合/协调免疫反应。M1活化由IFN-g、TNFa、GM-CSF、LPS和其他toll样受体(TLR)配体诱导。
相比之下，M2抗炎巨噬细胞(也称为替代性活化的巨噬细胞)通过抗炎分子诸如IL-4、IL-13和IL-10活化[12,13]。M2巨噬细胞表现出免疫调节、组织修复和血管生成特性，使它们能够将调节性T细胞募集到炎症部位。M2巨噬细胞并不构成均质群体，并且通常被进一步细分为M2a类别、M2b类别和M2c类别。所有三个亚群的共同特性是IL-10产量高，而IL-12产量低。它们的特征之一是产生精氨酸酶-1，该酶耗尽L-精氨酸，从而抑制T细胞反应并剥夺其底物的iNOS。
由于巨噬细胞在细胞微环境中所经历的各种信号，因此巨噬细胞极化的体内分子机制的表征较差[10,14]。近年来，在诸如个体发育、妊娠的生理条件和诸如过敏、慢性炎症和癌症的病理条件下，在鉴定体内巨噬细胞极化方面已取得进展。然而，我们确实知道，体外巨噬细胞的极化是具有可塑性的，在细胞因子的帮助下，巨噬细胞可来回极化为任一表型[15,16]。干扰素γ(IFN-γ)和IL-4是两种可分别将巨噬细胞极化为M1表型和M2表型的细胞因子[15]。
巨噬细胞的存在对于肿瘤的进展和生长至关重要，并且对确定预后具有重要意义[17,18]。由于巨噬细胞可表现出促炎和抗炎特性，因此了解它们的极化和在肿瘤进展和转移中的功能是很重要的。
巨噬细胞极化
肿瘤微环境可影响巨噬细胞极化。由于IL-10、糖皮质激素、凋亡细胞和免疫复合物的不利环境会干扰先天免疫细胞的功能，因此极化过程可能是多种多样且复杂的[11,19]。极化的机制尚不清楚，但我们知道它们涉及转录调节。例如，暴露于LPS或IFN-γ的巨噬细胞将向M1表型极化，而暴露于IL-4或IL-13的巨噬细胞将向M2表型极化。LPS或IFN-γ可与巨噬细胞表面上的Toll样受体4(TLR4)相互作用，诱导Trif和MyD88途径，诱导转录因子IRF3、AP-1和NFκB的活化，从而活化在M1促炎巨噬细胞反应中必需的TNF基因、干扰素基因、CXCL10、NOS2、IL-12等[20]。类似地，IL-4和IL-13与IL-4R结合，活化Jak/Stat6途径，从而调节CCL17、ARG1、IRF4、IL-10、SOCS3等的表达，这些是与抗炎反应(M2反应)相关的基因。
巨噬细胞极化的附加机制包括微RNA(miRNA)微管理。miRNA是长度为22个核苷酸、在转录后调节基因表达的小的非编码RNA，因为它们影响mRNA降解的速率。几种miRNA已被证明在极化巨噬细胞中高度表达，尤其是miRNA-155、miRNA-125、miRNA-378(M1极化)和miRNA let-7c、miRNA-9、miRNA-21、miRNA-146、miRNA147、miRNA-187(M2极化)[21]。
巨噬细胞极化是复杂的过程，在这个过程中，巨噬细胞会根据微环境刺激表现并引起不同的反应。因此，巨噬细胞极化更好地由连续的活化状态表示，其中M1表型和M2表型是极端状态。近年来，关于巨噬细胞活化和巨噬细胞极化的定义/描述一直存在很大争议。Murray等人最近发表了一篇论文，其中描述了将考虑用于巨噬细胞活化、极化、活化剂和标记物的共有定义/描述的一组标准。该论文的发表对于活化/极化的巨噬细胞的定义和表征而言是非常必要的[22]。
M1表型
M1促炎巨噬细胞或经典活化巨噬细胞具有攻击性、高度吞噬性，并产生大量反应性氧和氮物质，从而促进Th1反应[11]。M1巨噬细胞分泌高水平的两种重要的炎性细胞因子IL-12和IL-23。IL-12诱导Th17细胞的活化和克隆扩增，该细胞分泌大量的IL-17，从而导致炎症[23]。这些特征允许M1巨噬细胞控制转移、抑制肿瘤生长和控制微生物感染[24]。此外，M1巨噬细胞向肿瘤部位的浸润和募集与实体瘤患者的更好预后和更高总生存率相关[17,18,25-28]。
巨噬细胞向M1表型的极化在体外受炎症信号诸如IFN-γ、TNF-α、IL-1β和LPS以及转录因子和miRNA的调节[29,30]。经典活化的巨噬细胞引发靶向CXCL9、CXCL10(也称为IP-10)、IFN调节因子-1和细胞因子信号传导-1的抑制因子的STAT1转录因子的诱导[31]。细胞因子信号传导-1蛋白在细胞因子受体下游发挥作用，并参与负反馈回路以减弱细胞因子信号传导。在肿瘤微环境中，Notch信号传导在M1巨噬细胞的极化中起重要作用，因为它允许转录因子RBP-J调节经典活化。Notch信号传导缺失的巨噬细胞表达M2表型，而与其他外在诱导物无关[32]。当巨噬细胞从M2转变至M1时，一种关键的miRNA即miRNA-155被上调；过表达miRNA-155的M1巨噬细胞通常更具攻击性，并且与肿瘤的减少有关[33]。此外，已发现miRNA-342-5p通过靶向小鼠中的Akt1而在巨噬细胞中促进更大的炎性反应。该miRNA还促进Nos2和IL-6的上调，这两者均充当巨噬细胞的炎症信号[34]。其他miRNA诸如miRNA-125和miRNA-378也被证明参与巨噬细胞(M1)的经典活化途径[35]。
经典活化的巨噬细胞被认为在癌细胞的识别和破坏中起重要作用，因为它们的存在通常表明预后良好。识别后，恶性肿瘤细胞可通过几种机制被M1巨噬细胞破坏，这些机制包括接触依赖性吞噬作用和细胞毒性(即，细胞因子释放，诸如TNF-α)[24]。然而，诸如肿瘤微环境或组织驻留细胞的环境信号可使M1巨噬细胞极化为M2巨噬细胞。小鼠巨噬细胞的体内研究已表明，巨噬细胞在其细胞因子和表面标记物表达方面具有可塑性，并且在癌症存在下将巨噬细胞重新极化为M1表型可帮助免疫系统排斥肿瘤[19]。
M2表型
M2巨噬细胞具有抗炎作用，有助于血管生成和组织修复的过程。它们表达清道夫受体并产生大量的IL-10和其他抗炎细胞因子[33,36]。通过M2巨噬细胞表达IL-10促进了Th2反应。因此，Th2细胞上调IL-3和IL-4的产生。IL-3结合其他细胞因子，例如促红细胞生成素(EPO)、粒细胞巨噬细胞集落刺激因子(GM-CSF)和IL-6，刺激髓系中的所有细胞(粒细胞、单核细胞和树突状细胞)的增殖。IL-4是愈合过程中的一种重要细胞因子，因为它有助于细胞外基质的产生[23]。M2巨噬细胞通过允许血管滋养恶性肿瘤细胞从而促进它们的生长，显示出可能有助于肿瘤进展的功能。大多数实体瘤中巨噬细胞(被认为是M2)的存在与治疗成功和更长的生存率负相关[37]。另外，M2巨噬细胞的存在已与乳腺癌的转移潜力相关。Lin和同事发现，小鼠乳腺肿瘤部位巨噬细胞的早期募集会增加血管生成和恶性肿瘤的发生率[38]。据认为，肿瘤微环境有助于巨噬细胞维持M2表型[23,39]。肿瘤微环境中存在的抗炎信号，诸如脂联素和IL-10，可增强M2反应[41]。
肿瘤相关巨噬细胞(TAM)
暴露于肿瘤微环境的细胞表现不同。例如，在实体瘤周围发现的肿瘤相关巨噬细胞被认为有助于促进肿瘤的生长和转移，并具有M2样表型[42]。肿瘤相关巨噬细胞可以是组织驻留巨噬细胞，也可以是来源于骨髓的募集的巨噬细胞(从单核细胞分化为巨噬细胞并迁移到组织中的巨噬细胞)。Cortez-Retamozo进行的一项研究发现，脾中大量的TAM前体迁移到肿瘤基质中，这表明该器官也是TAM的储库[43]。在脾中发现的TAM前体被发现通过其CCR2趋化因子受体开始迁移[43]。最近的研究发现，CSF-1是将巨噬细胞吸引到肿瘤外周的主要因素，并且由癌细胞产生的CSF-1预测存活率较低，这表明预后总体较差[44-46]。其他细胞因子诸如TNF-α和IL-6也与巨噬细胞向肿瘤外周的聚集/募集有关[45]。
据认为，在肿瘤边界周围募集的巨噬细胞由在肿瘤中活化的“血管生成转换”调节。血管生成转换被定义为肿瘤形成高密度血管网络的过程，这种高密度血管网络有可能使肿瘤转移，并且是恶性转变所必需的。在乳腺癌小鼠模型中，观察到巨噬细胞的存在是完全血管生成转换所必需的。当巨噬细胞在肿瘤周围的成熟、迁移和聚集延迟时，血管生成转换也延迟，这表明血管生成转换不会在不存在巨噬细胞的情况下发生，并且巨噬细胞的存在是恶性肿瘤的进展所必需的[47]。此外，肿瘤基质细胞产生趋化因子，诸如CSF1、CCL2、CCL3、CCL5和胎盘生长因子，它们将巨噬细胞募集到肿瘤周围。这些趋化因子为巨噬细胞活化血管生成转换提供了环境，其中巨噬细胞将产生高水平的IL-10、TGF-β、ARG-1和低水平的IL-12、TNF-α和IL-6。这些细胞因子的表达水平表明巨噬细胞调节免疫逃逸。重要的是应注意，巨噬细胞被吸引到缺氧肿瘤环境中，并且将通过产生缺氧诱导因子-1α(HIF-1α)和HIF-2α来反应，这些因子调节与血管生成相关的基因的转录。在血管生成转换期间，巨噬细胞还可分泌VEGF(受NF-κB途径刺激)，从而促进血管成熟和血管通透性[48]。
人们认为，肿瘤相关巨噬细胞能够通过接收来自恶性肿瘤细胞诸如IL-1R和MyD88的极化信号来维持其M2样表型，该极化信号通过IkB激酶β和NF-kB信号级联进行介导。抑制TAM中的NF-kB会促进经典活化[40]。此外，另一项研究表明，p50 NF-kB亚基参与了M1巨噬细胞的抑制，并且炎症的减少促进了肿瘤的生长。Saccani等人培育的p50 NF-κB敲除小鼠表明，在p50 NF-kB敲除后，M1的攻击性恢复，降低了肿瘤存活率[49]。
由于肿瘤块包含大量M2样巨噬细胞，因此TAM可用作癌症治疗的靶标。减少TAM的数量或使它们朝向M1表型极化可有助于破坏癌细胞并减少肿瘤生长[50-52]。Luo和同事使用了抗天冬酰胺内肽酶的疫苗，天冬酰胺内肽酶是一种半胱氨酸蛋白酶和应激蛋白，在TAM中上调，被认为是潜在的肿瘤靶标[52]。当将抗天冬酰胺内肽酶的疫苗施用给小鼠时，控制血管生成的基因被下调，并且肿瘤生长停止[52]。
代谢和活化途径
肿瘤细胞中存在的代谢变化由产生癌症的相同基因突变控制[53]。作为这些代谢变化的结果，癌细胞能够产生可改变巨噬细胞极化并促进肿瘤生长的信号[54,55]。
M1和M2巨噬细胞展示出不同的代谢模式，反映出它们的不同行为[56]。M1表型增加了糖酵解，并使葡萄糖代谢偏向氧化戊糖磷酸途径，从而减少了耗氧量，并因此产生了大量的自由基氧和氮物质以及炎性细胞因子，诸如TNF-α、IL-12和IL-6[56,57]。M2表型增加了脂肪酸的摄入和氧化，从而降低了通向戊糖磷酸途径的通量，同时提高了整个细胞的氧化还原电位，因此上调了清道夫受体和免疫调节细胞因子诸如IL-10和TGF-β[56]。
多种代谢途径在巨噬细胞极化中起重要作用。蛋白激酶诸如Akt1和Akt2通过允许癌细胞存活、增殖和利用中间代谢来改变巨噬细胞极化[58]。其他蛋白激酶可通过增加糖酵解和减少耗氧量以通过葡萄糖代谢引导巨噬细胞极化[57,59]。Shu和同事率先使用PET扫描和葡萄糖类似物使体内巨噬细胞代谢和免疫反应可视化[60]。
L-精氨酸代谢还表现出对巨噬细胞中细胞因子表达具有重要意义的离散转移，并举例说明了改变TAM-肿瘤细胞相互作用的不同代谢途径[61]。经典活化的(M1)巨噬细胞有利于诱导型一氧化氮合酶(iNOS)。iNOS途径产生细胞毒性的一氧化氮(NO)，因此表现出抗肿瘤行为。替代性活化的(M2)巨噬细胞已被证明有利于精氨酸酶途径，并产生尿和l-鸟氨酸，这有助于进行性肿瘤细胞生长[61,62]。
代谢途径的直接操纵可改变巨噬细胞极化。在葡萄糖代谢中起作用的碳水化合物激酶样蛋白(CARKL)蛋白质已被用于改变巨噬细胞的细胞因子特征[56,57]。当CARKL被RNAi敲除时，巨噬细胞趋于采用M1样代谢途径(代谢偏向于糖酵解并减少耗氧量)。当CARKL过表达时，巨噬细胞采用M2样代谢(糖酵解通量减少，耗氧量增加)[56]。当巨噬细胞通过LPS/TLR4结合而采用M1样代谢状态时，CARKL水平降低，由NFκB途径控制的基因被活化(TNF-α、IL-12和IL-6)，并且细胞氧化还原电位由于NADH:NAD+和GSH:GSSSG复合物浓度的增加而升高。在M2样代谢状态期间，巨噬细胞上调CARKL和受STAT6/IL-4调节的基因(IL-10和TGF-β)。
肥胖症还可影响巨噬细胞极化。肥胖症与慢性炎症的状态有关，慢性炎症是驱动IL4/STAT6途径活化NKT细胞的环境，而NKT细胞则使巨噬细胞趋向于M2反应。在后期饮食引起的肥胖症中，巨噬细胞迁移到脂肪组织，免疫细胞改变脂肪组织中TH1或TH2细胞因子的表达水平，引起M2表型偏倚并可能增加胰岛素敏感性[63]。
通过靶向TAMS中的代谢途径的M1表型偏倚可能提供减少肿瘤生长和转移的替代方法。
对抗癌症的巨噬细胞免疫治疗方法
癌症免疫治疗的作用是刺激免疫系统识别、排斥并破坏癌细胞。用单核细胞/巨噬细胞进行的癌症免疫治疗的目标是使巨噬细胞朝向促炎反应(M1)极化，从而允许巨噬细胞和其他免疫细胞破坏肿瘤。尽管体内的副作用通常过于严重，但许多细胞因子和细菌化合物均可在体外达到这一目标。关键是寻找患者副作用最小或易于控制的化合物。在过去的几十年中，已使用单核细胞/巨噬细胞进行免疫治疗，并且每年都在开发新的方法[64,65]。早期免疫治疗为更好的癌症治疗和提高接受免疫治疗的患者的生存率奠定了良好的基础[66]。
癌症免疫治疗的一些方法包括使用细胞因子或趋化因子将活化的巨噬细胞和其他免疫细胞募集到肿瘤部位，这允许识别和靶向破坏肿瘤部位[67,68]。IFN-α和IFN-β已被证明通过诱导细胞分化和凋亡来抑制肿瘤进展[69]。另外，IFN治疗具有抗增殖作用，并且可增加细胞周期中的S期时间[70,71]。Zhang和同事使用靶向人前列腺癌细胞的IFN-β基因治疗对裸鼠进行研究。他们的结果表明，腺病毒递送的IFN-β基因治疗涉及巨噬细胞，并有助于抑制其生长和转移[72]。
巨噬细胞抑制因子(MIF)是可用于癌症免疫治疗的另一种细胞因子。MIF通常发现于实体瘤中，并表明预后不良。MIF抑制攻击性巨噬细胞功能，并使巨噬细胞趋向于M2表型，从而有助于肿瘤的生长和发展。Simpson、Templeton和Cross(2012)发现，MIF诱导髓样细胞(巨噬细胞前体)分化为表达M2表型的髓样细胞[73]。通过靶向MIF，它们能够减少这种抑制性巨噬细胞群，抑制其生长，从而控制肿瘤的生长和转移[73]。
2型趋化因子受体CCR2对于将单核细胞募集至炎性部位至关重要，并且已被证明是防止巨噬细胞募集至肿瘤部位、血管生成和转移的靶标。Sanford和同事(2013)在胰腺小鼠模型中研究了新型CCR2抑制剂(PF-04136309)，表明CCR2抑制剂减少了肿瘤部位的单核细胞/巨噬细胞募集，降低了肿瘤生长和转移，并提高了抗肿瘤免疫性[74]。Schmall等人最近的另一项研究结果显示，与10种不同的人类肺癌细胞共培养的巨噬细胞上调了CCR2表达。此外，他们表明在用CCR2拮抗剂治疗的肺部小鼠模型中，肿瘤生长和转移减少[75]。
其他研究已使用脂质体来递送药物，以从肿瘤中耗尽M2巨噬细胞并阻止血管生成。表达高水平IL-1β的癌细胞在体内生长更快并诱导更多的血管生成。Kimura和同事发现，暴露于表达IL-1β的肿瘤细胞的巨噬细胞产生更高水平的血管生成因子和趋化因子，诸如血管内皮生长因子A(VEG-A)、IL-8、单核细胞趋化蛋白1等，从而促进肿瘤生长和血管生成[76]。当他们使用氯膦酸盐脂质体耗尽巨噬细胞时，他们发现产生IL-1β的肿瘤细胞更少。他们还发现，通过抑制癌细胞中的NF-κB和AP-1转录因子，减少了肿瘤生长和血管生成。这些发现可能表明，肿瘤部位周围的巨噬细胞可能参与促进肿瘤生长和血管生成[76]。
化合物诸如甲硫氨酸脑啡肽(MENK)在体内和体外均具有抗肿瘤特性。MENK能够通过下调CD206和精氨酸酶-1(M2标记物)，同时上调CD64、MHC-II和一氧化氮的产生(M1标记物)，将M2巨噬细胞极化为M1巨噬细胞。MENK也可上调TNF-α并下调IL-10[77]。
最近的研究集中在作为M2巨噬细胞的潜在抑制剂的双膦酸盐。双膦酸盐通常用于治疗转移性乳腺癌患者，以预防骨骼并发症诸如骨吸收[78]。尽管双膦酸盐在体内停留的时间很短，但由于它们对羟基磷灰石的亲和力很高，因此它们可靶向破骨细胞，即与巨噬细胞属于同一家族的细胞。一旦双膦酸盐与骨结合，骨基质就通过内吞作用使双膦酸盐内化。一旦进入细胞质，双膦酸盐就可抑制蛋白质异戊二烯化，这是一个阻止整联蛋白信号传导和内体运输的事件，从而迫使细胞凋亡[69]。直到最近，尽管尚不清楚双膦酸盐是否可靶向肿瘤相关巨噬细胞，但是Junankar等人最近的一项研究表明，巨噬细胞通过胞饮作用和吞噬作用摄取含氮双膦酸盐化合物，这是在肿瘤周围的上皮细胞中不会发生的事件[79]。使用双膦酸盐迫使TAM凋亡可减少血管生成和转移。
癌症免疫治疗的另外的方法包括使用可引起免疫反应的生物材料。阳离子聚合物一旦溶解于水中，便具有反应性，因此可用于免疫治疗。Chen等人使用包括PEI、聚赖氨酸、阳离子葡聚糖和阳离子明胶的阳离子聚合物来产生强Th1免疫反应[77]。它们还能够诱导CD4+细胞增殖和M1巨噬细胞典型的IL-12分泌[77]。Huang和同事还使用生物材料通过靶向TLR4触发TAM产生抗肿瘤反应[80]。该研究发现，TAM能够极化为M1表型并表达IL-12。他们发现这些阳离子分子具有直接的杀肿瘤活性，并证明在小鼠中肿瘤减少了[80]。
TLR4
Toll样受体4是人中由TLR4基因编码的蛋白质。TLR 4检测革兰氏阴性细菌上的脂多糖(LPS)，因此在识别危险和激活先天免疫系统中起着极其重要的作用(图7)。当巨噬细胞被LPS诱导时，它与LY96(MD-2)和CD14协作以介导信号转导。当M1巨噬细胞检测到LPS的存在时，TLR4的细胞质结构域负责活化M1巨噬细胞。这是受体的功能部分，它将与MOTO-CAR(即，嵌合受体)偶联，以在CAR结合其靶蛋白时诱导单核细胞/巨噬细胞的活化。
接头蛋白MyD88和TIRAP通过与TLR4的Toll/白介素-1受体(IL-1R)(TIR)结构域的直接相互作用，促成多种途径甚至可能是所有途径的活化。但是，可能存在活化特定途径子集所需的另外的接头，这可有助于靶基因的差异调控。
胸苷激酶
人胸苷激酶1(TK1)是熟知的核苷酸补救途径酶，在其在肿瘤中过表达的背景下已得到大量研究。由于TK1最初通过其在癌症患者血清(sTK)中的表达而普及，因此已对其诊断和预后潜力进行了广泛的研究。例如，若干研究表明，许多不同癌症患者中的sTK1以类似阶段的方式升高，而TK1的水平越高，则表明肿瘤越晚期[81]。
其他研究已调查了TK1的预后潜力。一项此类研究表明，原发性乳腺肿瘤中的TK1水平可用于预测复发。其他令人兴奋的TK1预后研究显示，当患者对治疗产生反应时，sTK1水平显著降低，而对于似乎未对其治疗产生反应的患者，sTK1水平继续升高。还已知sTK1水平在复发之前就开始升高，并且注意到在某些情况下，sTK1水平可在“临床症状发作前1-6个月”预测复发。其他几项研究证实了TK1作为癌症诊断和预后指标的巨大潜力[82]。
尽管已经很好地确定了TK1的诊断和预后潜力，但相比之下，TK1的治疗潜力仍然模糊不清。虽然确实已将HSV-TK用于基因治疗，而PET成像利用TK1来识别增殖性癌细胞，但很少(如果有的话)研究涉及TK1免疫治疗的可能性。可能这主要是因为TK1是已知的胞浆蛋白。最近发现，TK1不仅在癌细胞中表达，而且在多种肿瘤类型的表面膜上也表达，因此是肿瘤免疫治疗的非常可行的靶标。
发明内容
本文描述了嵌合受体。嵌合受体包含细胞质结构域、跨膜结构域和细胞外结构域。在实施方案中，细胞质结构域包含受体的细胞质部分，该细胞质部分在活化时使巨噬细胞极化。在另外的实施方案中，包含细胞质部分的野生型蛋白质不包含嵌合受体的细胞外结构域(参见，例如图21)。在实施方案中，配体与嵌合受体的细胞外结构域的结合活化嵌合受体的细胞内部分(参见，例如图22)。嵌合受体的细胞内部分的活化可使巨噬细胞极化成M1或M2巨噬细胞(参见，例如图23和图24(A)和图25)。
在某些实施方案中，细胞外结构域可以包含特异性地结合配体的抗体或其片段。在实施方案中，嵌合受体可包含接头。在实施方案中，嵌合受体可以包含铰链区。
另外的实施方案包括包含嵌合受体的细胞或编码嵌合受体的核酸。
实施方案包括通过将包含嵌合受体的巨噬细胞与嵌合受体的细胞外结构域的配体接触来使巨噬细胞极化的方法；将配体结合到嵌合受体的细胞外结构域。配体与嵌合受体的细胞外结构域的结合活化细胞质部分，并且细胞质部分的活化使巨噬细胞极化。
根据本文所包含的教导内容，本公开的这些和其他方面对于技术人员将变得显而易见。
附图说明
图1A示出了嵌合受体TK1-MOTO1中的元件顺序的框图。图1B示出了TK1-MOTO1(SEQID NO:35)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-313位氨基酸是TLR4跨膜结构域，第314-496位氨基酸是TLR4胞质结构域。
图2A示出了嵌合受体TK1-MOTO2中的元件顺序的框图。图2B示出了TK1-MOTO2(SEQID NO:36)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-295位氨基酸是LRR短铰链，第296-318位氨基酸是TLR4跨膜结构域，第319-500位氨基酸是TLR4胞质结构域。
图3A示出了嵌合受体TK1-MOTO3中的元件顺序的框图。图3B示出了TK1-MOTO3(SEQID NO:37)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-345位氨基酸是LRR长铰链，第346-368位氨基酸是TLR4跨膜结构域，第269-501位氨基酸是TLR4胞质结构域。
图4A示出了嵌合受体TK1-MOTO4中的元件顺序的框图。图4B示出了TK1-MOTO4(SEQID NO:38)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-302位氨基酸是IgG4短铰链，第303-325位氨基酸是TLR4跨膜结构域，第326-508位氨基酸是TLR4胞质结构域。
图5A示出了嵌合受体TK1-MOTO5中的元件顺序的框图。图5B示出了TK1-MOTO5(SEQID NO:39)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-409位氨基酸是IgG 119氨基酸中等铰链，第410-432位氨基酸是TLR4跨膜结构域，第433-615位氨基酸是TLR4胞质结构域。
图6A示出了嵌合受体TK1-MOTO6中的元件顺序的框图。图6B示出了TK1-MOTO6(SEQID NO:40)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-518位氨基酸是IgG4长铰链，第519-541位氨基酸是TLR4跨膜结构域，第542-724位氨基酸是TLR4胞质结构域。
图7A示出了嵌合受体TK1-MOTO7中的元件顺序的框图。图7B示出了TK1-MOTO7(SEQID NO:41)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-358位氨基酸是带有C339S和C356S的突变CD8铰链，第359-381位氨基酸是TLR4跨膜结构域，第382-564位氨基酸是TLR4胞质结构域。
图8A示出了嵌合受体TK1-MOTO8中的元件顺序的框图。图8B示出了TK1-MOTO8(SEQID NO:42)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-358位氨基酸是CD8铰链的一部分，第359-381位氨基酸是TLR4跨膜结构域，第382-564位氨基酸是TLR4胞质结构域。
图9A示出了嵌合受体TK1-MO-FCGRA-CAR-1中的元件顺序的框图。图9B示出了TK1-MO-FCGRA-CAR-1(SEQ ID NO:43)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-311位氨基酸是FCGR3A跨膜结构域，第312-336位氨基酸是FCGR3A胞质结构域，第337-378位氨基酸是FCER1G胞质结构域。
图10A示出了嵌合受体TK1-MO-FCGRA-CAR-2中的元件顺序的框图。图10B示出了TK1-MO-FCGRA-CAR-2(SEQ ID NO:44)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-358位氨基酸是带有C339S和C356S的突变CD8铰链，第359-379位氨基酸是FCGR3A跨膜结构域，第380-404位氨基酸是FCGR3A胞质结构域，第405-446位氨基酸是FCER1G胞质结构域。
图11A示出了嵌合受体TK1-MO-FCGRA-CAR-3中的元件顺序的框图。图11B示出了TK1-MO-FCGRA-CAR-3(SEQ ID NO:45)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-358位氨基酸是CD8铰链的一部分，第359-379位氨基酸是FCGR3A跨膜结构域，第380-404位氨基酸是FCGR3A胞质结构域，第405-446位氨基酸是FCER1G胞质结构域。
图12A示出了嵌合受体TK1-MO-FCGRA-CAR-4中的元件顺序的框图。图12B示出了TK1-MO-FCGRA-CAR-4(SEQ ID NO:46)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-303位氨基酸是IgG4短铰链，第304-324位氨基酸是FCGR3A跨膜结构域，第325-349位氨基酸是FCGR3A胞质结构域，第350-391位氨基酸是FCER1G胞质结构域。
图13A示出了嵌合受体TK1-MO-FCGRA-CAR-5中的元件顺序的框图。图13B示出了TK1-MO-FCGRA-CAR-5(SEQ ID NO:47)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-409位氨基酸是IgG4 119氨基酸铰链，第410-430位氨基酸是FCGR3A跨膜结构域，第431-455位氨基酸是FCGR3A胞质结构域，第456-497位氨基酸是FCER1G胞质结构域。
图14A示出了嵌合受体TK1-MO-FCGRA-CAR-6中的元件顺序的框图。图14B示出了TK1-MO-FCGRA-CAR-6(SEQ ID NO:48)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-519位氨基酸是IgG4长铰链，第520-540位氨基酸是FCGR3A跨膜结构域，第541-565位氨基酸是FCGR3A胞质结构域，第566-607位氨基酸是FCER1G胞质结构域。
图15A示出了嵌合受体TK1-MO-FCG2A-CAR-1中的元件顺序的框图。图15B示出了TK1-MO-FCG2A-CAR-1(SEQ ID NO:49)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-312位氨基酸是FCGR2A跨膜结构域，第313-390位氨基酸是FCGR2A胞质结构域。
图16A示出了嵌合受体TK1-MO-FCG2A-CAR-2中的元件顺序的框图。图16B示出了TK1-MO-FCG2A-CAR-2(SEQ ID NO:50)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-358位氨基酸是带有C339S和C356S的突变CD8铰链，第359-380位氨基酸是FCGR2A跨膜结构域，第381-458位氨基酸是FCGR2A胞质结构域。
图17A示出了嵌合受体TK1-MO-FCG2A-CAR-3中的元件顺序的框图。图17B示出了TK1-MO-FCG2A-CAR-3(SEQ ID NO:51)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-358位氨基酸是CD8铰链的一部分，第359-380位氨基酸是FCGR2A跨膜结构域，第381-458位氨基酸是FCGR2A胞质结构域。
图18A示出了嵌合受体TK1-MO-FCG2A-CAR-4中的元件顺序的框图。图18B示出了TK1-MO-FCG2A-CAR-4(SEQ ID NO:52)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-303位氨基酸是IgG4短铰链，第304-325位氨基酸是FCGR2A跨膜结构域，第326-403位氨基酸是FCGR2A胞质结构域。
图19A示出了嵌合受体TK1-MO-FCG2A-CAR-5中的元件顺序的框图。图19B示出了TK1-MO-FCG2A-CAR-5(SEQ ID NO:53)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-409位氨基酸是IgG4 119氨基酸铰链，第410-431位氨基酸是FCGR2A跨膜结构域，第432-509位氨基酸是FCGR2A胞质结构域。
图20A示出了嵌合受体TK1-MO-FCG2A-CAR-6中的元件顺序的框图。图20B示出了TK1-MO-FCG2A-CAR-6(SEQ ID NO:54)的序列。第1-18位氨基酸是信号肽(SP)，第19-275位氨基酸是抗TK1 ScFv，第276-290位氨基酸是GS接头，第291-519位氨基酸是IgG4长铰链，第520-541位氨基酸是FCGR2A跨膜结构域，第542-619位氨基酸是FCGR2A胞质结构域。
图21是示出嵌合受体的示意图。
图22是示出表达嵌合受体的巨噬细胞的示意图。如图所示，嵌合受体包含toll样受体的胞质结构域、跨膜结构域和特异于配体的ScFv。箭头描绘了在ScFv结合配体时使巨噬细胞极化的信号传导。
图23是示出可用于构建嵌合受体的不同巨噬细胞受体的示意图。
图24A至图24C。图24A是显示导致细胞活化的Fcγ受体III信号级联的示意图。图24B是显示导致钙通量和增殖抑制的Fcγ受体III信号级联的示意图。图24C是显示导致细胞凋亡的Fcγ受体III信号级联的示意图。
图25是示出Toll样受体信号级联的示意图。
图26呈现了图示流式细胞术的图表，证实表达的抗体片段与所关注的配体结合。
图27呈现了用嵌合受体转导后巨噬细胞表型变化的两幅图像。
图28呈现了证实嵌合受体在单核细胞中的表达的两幅图像。
图29呈现了荧光激活细胞分选的三个散点图，证明了dTomato的表达。最左边的图显示了对照，其中只有0.58％的细胞显示出荧光，这表明dTomato的表达。右边的两个图显示转导后的转导效率为27.1％。
图30呈现了荧光激活细胞分选的六个散点图，其展示了染料(Alexa 647)的保留，以及在用嵌合受体转导的巨噬细胞中CD80、CD163、CD206和CD14的表达。
图31呈现了直方图，展示了在用嵌合受体转导的巨噬细胞中CD80、CD163、CD206和CD14的相对表达水平。
图32呈现了表达嵌合受体的转导巨噬细胞在肺癌细胞系(NCI-H460)中检测、攻击和诱导细胞死亡的六幅图像。
具体实施方式
本文描述了嵌合受体。嵌合受体包含细胞质结构域、跨膜结构域和细胞外结构域。在实施方案中，细胞质结构域包含受体的细胞质部分，该细胞质部分在活化时使巨噬细胞极化。在另外的实施方案中，包含细胞质部分的野生型蛋白质不包含嵌合受体的细胞外结构域。在实施方案中，配体与嵌合受体的细胞外结构域的结合活化嵌合受体的细胞内部分。嵌合受体的细胞内部分的活化可使巨噬细胞极化成M1或M2巨噬细胞。
在某些实施方案中，嵌合受体的细胞质部分可以包括来自toll样受体、骨髓分化初反应蛋白(MYD88)(SEQ ID NO:19)、toll样受体3(TLR3)(SEQ ID NO:1)、toll样受体4(TLR4)(SEQ ID NO:3)、toll样受体7(TLR7)(SEQ ID NO:7)、toll样受体8(TLR8)(SEQ IDNO:9)、toll样受体9(TLR9)(SEQ ID NO:11)、髓磷脂和淋巴细胞蛋白(MAL)(SEQ ID NO:21)、白介素-1受体相关激酶1(IRAK1)(SEQ ID NO:23)、低亲和力免疫球蛋白γFc区受体III-A(FCGR3A)(SEQ ID NO:15)、低亲和力免疫球蛋白γFc区受体II-a(FCGR2A)(SEQ IDNO:13)、高亲和力免疫球蛋白ε受体亚基γ(FCER1G)(SEQ ID NO:19)或与前述任一项所述的细胞质结构域具有至少90％序列同一性的序列的细胞质结构域。在某些实施方案中，细胞质部分不是来自toll样受体、FCGR3A、IL-1受体或IFN-γ受体的细胞质结构域。在实施方案中，胞质部分可以是在被活化时将导致巨噬细胞极化的任何多肽。
在另外的实施方案中，结合至细胞外结构域的配体的示例可以是但不限于胸苷激酶(TK1)、次黄嘌呤-鸟嘌呤磷酸核苷转移酶(HPRT)、受体酪氨酸激酶样孤儿受体1(ROR1)、黏蛋白-16(MUC-16)、表皮生长因子受体vIII(EGFRvIII)、间皮素、人表皮生长因子受体2(HER2)、癌胚抗原(CEA)、B-细胞成熟抗原(BCMA)、磷脂酰肌醇蛋白聚糖3(GPC3)、成纤维细胞活化蛋白(FAP)、促红细胞生成素产生肝细胞受体A2(EphA2)、自然杀伤细胞家族2D(NKG2D)配体、双唾液酸神经节苷脂2(GD2)、CD19、CD20、CD30、CD33、CD123、CD133、CD138和CD171。在某些实施方案中，配体不是TK1或HPRT。
可适于生成嵌合受体的细胞外结构域的抗体是本领域众所周知的，并且是可商购获得的。可商购获得的抗体的示例包括但不限于：HGPRT抗体、克隆13H11.1(EMDMillipore)、ROR1抗体(ab135669)(Abcam)、MUC1抗体[EP1024Y](ab45167)(Abcam)、MUC16抗体[X75](ab1107)(Abcam)、EGFRvIII抗体[L8A4](Absolute antibody)、间皮素抗体[EPR2685(2)](ab134109)(Abcam)、HER2[3B5](ab16901)(Abcam)、CEA抗体(LS-C84299-1000)(LifeSpan BioSciences)、BCMA抗体(ab5972)(Abcam)、磷脂酰肌醇蛋白聚糖-3抗体[9C2](ab129381)(Abcam)、FAP抗体(ab53066)(Abcam)、EphA2抗体[RM-0051-8F21](ab73254)(Abcam)、GD2抗体(LS-C546315)(LifeSpan BioSciences)、CD19抗体[2E2B6B10](ab31947)(Abcam)、CD20抗体[EP459Y](ab78237)(Abcam)、CD30抗体[EPR4102](ab134080)(Abcam)、CD33抗体[SP266](ab199432)(Abcam)、CD123抗体(ab53698)(Abcam)、CD133抗体(BioLegend)、CD123抗体(1A3H4)ab181789(Abcam)和CD171抗体(L1.1)(Invitrogenantibodies)。通过已知抗体创建抗体片段诸如ScFv的技术是本领域的常规技术。此外，生成编码此类片段的序列并重组性地将它们包含为编码嵌合蛋白的多核苷酸的一部分也是本领域中的常规技术。
在某些实施方案中，细胞外结构域可以包含特异性地结合配体的抗体或其片段。抗体及其片段的示例包括但不限于IgA、IgD、IgE、IgG、IgM、Fab片段、F(ab′)2片段、一价抗体、ScFv片段、scRv-Fc片段、IgNAR、hcIgG、VhH抗体、纳米抗体和alpha抗体。在另外的实施方案中，细胞外结构域可包括允许特异性结合配体的任何氨基酸序列，包括但不限于二聚体化结构域、受体、结合口袋等。
在实施方案中，嵌合受体可包含接头。非限制性地，接头可以位于嵌合受体的细胞外结构域和跨膜结构域之间。非限制性地，接头可以是G接头、GS接头、G4S接头、EAAAK接头、PAPAP接头或(Ala-Pro)n接头。接头的其他示例是本领域众所周知的。
在实施方案中，嵌合受体可以包含铰链区。非限制性地，铰链区可以位于嵌合受体的细胞外结构域和跨膜结构域之间。在另外的实施方案中，铰链区可以位于接头和跨膜结构域之间。非限制性地，接头可以是富亮氨酸重复序列(LRR)，或来自toll样受体、IgG、IgG4、CD8m或FcγIIIa-hing的铰链区。在实施方案中，可以用丝氨酸置换铰链区中的半胱氨酸。铰链区的其他示例是本领域众所周知的。
本文所述的嵌合受体可以包括SEQ ID NO:1、3、4、5、7、9、11、13、15、17、19、21、23及25-34中的一者或多者、它们中任一者的片段、和/或与SEQ ID NO:1、3、4、5、7、9、11、13、15、17、19、21、23及25-34中的至少一者或它们的片段具有至少90％序列同一性的多肽。嵌合受体的示例包括但不限于SEQ ID NO:35-54，或者其同源物或片段。在另一实施方案中，多肽含有选自与SEQ ID NO:35-54中的至少一者具有至少90％序列同一性的多肽的氨基酸序列。
实施方案包括含有编码上文所述嵌合受体的核酸序列的核酸序列。此类核酸的示例可以包括SEQ ID NO:2、6、8、10、12、14、16、18、20、22和24中的一者或多者、它们中任一者的片段、和/或与SEQ ID NO:2、6、8、10、12、14、16、18、20、22和24中的至少一者或它们的片段具有至少90％序列同一性的核酸。另外的示例包括编码SEQ ID NO:24-54中的一者或多者以及它们中任一者的片段的核酸。
在实施方案中，嵌合受体可以是糖基化的、聚乙二醇化的和/或以其他方式翻译后修饰的。此外，核酸序列可以是载体的一部分。以举例的方式，载体可以是质粒、噬菌体、黏质粒、人工染色体、病毒载体、AAV载体、腺病毒载体或慢病毒载体。在某些实施方案中，可将编码嵌合受体的核酸可操作地连接到启动子和/或其他调节序列(例如，增强子、沉默子、绝缘子、基因座控制区、顺式作用元件等)。
另外的实施方案包括包含嵌合受体的细胞或编码嵌合受体的核酸。此类细胞的非限制性示例包括骨髓细胞、骨髓祖细胞、单核细胞、中性粒细胞、嗜碱性粒细胞、嗜酸性粒细胞、巨核细胞、T细胞、B细胞、自然杀伤细胞、白细胞、淋巴细胞、树突状细胞和巨噬细胞。
实施方案包括通过使包含嵌合受体的巨噬细胞与用于嵌合受体的细胞外结构域的配体接触并使配体与嵌合受体的细胞外结构域结合来使巨噬细胞极化的方法。配体与嵌合受体的细胞外结构域的结合活化细胞质部分，并且细胞质部分的活化使巨噬细胞极化。
根据本公开，核苷酸、多核苷酸或核酸序列将被理解为是指单体和二聚体(所谓的串联)形式的双链或单链DNA或RNA以及DNA或RNA的转录产物。
本公开的各方面涉及能够以分离方法(诸如，通过基于分子体积的排除或通过亲和力进行的离子交换层析；或另选的基于在不同溶剂中的可溶性的分级分离技术)开始、或以基因工程方法(诸如，扩增、克隆和亚克隆)开始进行分离、纯化或部分纯化的核苷酸序列，能够由载体携带这些核苷酸序列。
核苷酸序列片段将被理解为指任何核苷酸片段，并且作为非限制性示例，可以包含该核苷酸序列片段所源自的序列的至少8、12、20、25、50、75、100、200、300、400、500、1000个或更多个连续核苷酸。
核苷酸序列的特定片段将被理解为是指经与对应的野生型序列进行比对和比较得到的具有至少一个核苷酸或碱基的任何核苷酸片段。
如本文所用的同源核苷酸序列应理解为是指至少与核苷酸序列的碱基具有至少约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.6％或99.7％的同一性的核苷酸序列，该百分比仅仅是统计上的，并且能够在两个核苷酸序列的整个长度上随机分布这两个核苷酸序列之间的差异。
在本公开的意义上，特定同源核苷酸序列应理解为是指具有特定片段的至少一个序列的同源序列，如上文所定义的。“特定”同源序列可以包括例如与基因序列或其表示基因序列变体的片段的序列对应的序列。这些特定同源序列因此可以对应于与序列内的突变相关的变异，并且尤其对应于至少一个核苷酸的截短、置换、缺失和/或添加。同源序列同样可以对应于与遗传密码的简并性相关的变异。
如本申请中所定义的，术语“序列同源性的程度或百分比”是指“经最佳比对得到的两个序列之间的序列同一性的程度或百分比”。
在如下文所述的那样比对两个核苷酸序列的最大对应性时得到这两个序列中的氨基酸或核苷酸残基的序列相同的结果，则称这两个核苷酸序列是“相同的”。通常通过在片段或“比较窗口”上比较两个最佳比对序列的序列以识别和比较序列相似性的局部区域来执行两个(或更多个)肽或多核苷酸之间的序列比较。可以通过Smith和Waterman,Ad.App.Math 2:482(1981)的局部同源性算法、通过Neddleman和Wunsch,J.Mol.Biol.48:443(1970)的同源比对算法、通过Pearson和Lipman,Proc.Natl.Acad.Sci.(U.S.A.)85:2444(1988)的研究相似性方法、通过这些算法的计算机化实现(威斯康星遗传学软件包中的GAP、BESTFIT、FASTA和TFASTA，遗传学计算机组(GCG)，575Science Dr.,Madison,Wis.)或通过目视检查来进行用于比较的序列的最佳比对。
通过在比较窗口上比较两个最佳比对序列来确定“序列同一性百分比”(或同一性程度)，其中比较窗口中的肽或多核苷酸序列的部分与参考序列(其不包括添加或缺失)相比可以包括用于两个序列的最佳比对的添加或缺失(即，缺口)。通过以下方式来计算该百分比：确定两个序列中出现相同氨基酸残基或核酸碱基的位置的数量以得到匹配位置数，将匹配位置数除以比较窗口中的位置总数，并将所得结果乘以100以得到序列同一性百分比。
上文给出的序列同一性的定义是本领域技术人员将使用的定义。该定义本身不需要任何算法的帮助，算法仅有助于实现序列的最佳比对，而不是序列同一性的计算。
根据上文给出的定义，可以看出，两个比较序列之间的序列同一性仅具有良好限定的一个值，该值与获得最佳或最优比对的值对应。
在BLAST N或BLAST P“BLAST 2序列”中，本发明人和一般技术人员习惯使用可在网站worldwideweb.ncbi.nlm.nih.gov/gorf/bl2.html获得的软件来比较和确定两个序列之间的同一性，通过该软件直接选择取决于要比较的序列长度的空位罚分(即，对于长度大于85的取代矩阵BLOSUM-62，为11.2)。
如本文所用的序列的互补核苷酸序列被理解为是指其核苷酸与该序列的那些核苷酸互补且其取向相反的任何DNA(反义序列)。
如本文所用的在严格条件下与核苷酸序列进行的杂交应该被理解为是指在以使得允许保持互补DNA的两个片段之间的杂交的方式选择的温度和离子强度条件下进行的杂交。
以例示的方式，以限定上述核苷酸片段为目的的杂交步骤的高度严格条件有利地如下。
在SSC缓冲液，1×SSC(对应于0.15M NaCl和0.05M柠檬酸钠)的存在下，在65℃的优先温度下进行杂交。洗涤步骤例如可如下：在环境温度下，2×SSC，随后在65℃下，用2×SSC、0.5％SDS洗涤两次；2×0.5×SSC、0.5％SDS，在65℃下各洗涤10分钟。
中等严格条件，例如，在2×SSC缓冲液的存在下，使用42℃的温度；或低严格条件，例如，在2×SSC缓冲液的存在下，使用37℃的温度，这两者分别需要用于两个序列之间的杂交的全局重要性较低的互补性。
根据Sambrook等人，1989的教导内容，本领域技术人员将针对具有更大或更小的大小的寡核苷酸调整上文所述的用于大小为具有约350个碱基的多核苷酸的严格杂交条件。
本文所述的核苷酸序列中存在可以在允许获得同源序列的方法中用作引物或探针的那些序列，这些方法(诸如，聚合酶链反应(PCR)、核酸克隆和基因测序)是本领域技术人员熟知的。
在这些核苷酸序列中存在可以在允许确定如下文所定义的特定核酸、特定核酸的片段之一、或特定核酸的变体之一的存在的方法中用作引物或探针的那些序列。在实施方案中，核苷酸序列可包含SEQ ID NO:2、6、8、10、12、14、16、18、20、22和24的片段，这些片段编码跨膜结构域、胞质结构域、或它们的一部分。其他片段可以包括编码接头、铰链或它们的片段的核苷酸序列，诸如编码SEQ ID NO:26-34中的一者或多者的核苷酸。其他片段可以包括编码SEQ ID NO:35-54中的一者或多者的核苷酸序列的片段。
核苷酸序列片段可以例如通过特异性扩增诸如PCR，或通过用合适的核苷酸序列限制性内切酶消化获得，这些方法特别描述于Sambrook等人，1989的著作中。同样，可以使用可从公司诸如获得的基因合成标准技术来获得此类片段。同样可以根据本领域普通技术人员熟知的方法通过化学合成获得此类代表性片段。
经修饰的核苷酸序列将被理解为是指根据本领域技术人员熟知的技术通过诱变获得的任何核苷酸序列，该核苷酸序列包含相对于野生型序列进行的修饰，例如，多肽表达的调节和/或启动子序列中的突变，特别是会导致多肽表达速率改变或复制周期得到调节。
经修饰的核苷酸序列同样将被理解为是指如下文所定义的编码经修饰的多肽的任何核苷酸序列。
本发明公开了编码嵌合受体的核苷酸序列，该核苷酸序列包括选自SEQ ID NO:2、6、8、10、12、14、16、18、20、22和24的核苷酸序列或它们的片段中的一者。此类片段可以编码特定结构域，诸如，跨膜结构域或胞质结构域，或它们的一部分。编码嵌合受体的其他核苷酸序列可以包括编码接头、铰链或它们的片段的核苷酸序列，诸如编码SEQ ID NO:26-34中的一者或多者的核苷酸。编码嵌合受体的核苷酸序列还可以是编码SEQ ID NO:35-54中的一者或多者或它们的片段的核苷酸序列。
实施方案同样涉及核苷酸序列，其特征在于，这些核苷酸序列包含选自以下各项的核苷酸序列：a)SEQ ID NO:2、6、8、10、12、14、16、18、20、22和24的核苷酸序列中的至少一者，编码SEQ ID NO:25-54中的至少一者或它们的片段之一的核苷酸序列；b)如a)中所定义的序列的特定片段的核苷酸序列；c)与如a)或b)中所定义的序列具有至少80％同一性的同源核苷酸序列；d)与如a)、b)或c)中所定义的序列对应的互补核苷酸序列或RNA序列；以及e)被如a)、b)、c)或d)中所定义的序列修饰的核苷酸序列。
这些核苷酸序列中存在SEQ ID NO:2、6、8、10、12、14、16、18、20、22和24的核苷酸序列、编码SEQ ID NO:25-54中的至少一者或它们的片段的核苷酸序列、以及与SEQ ID NO:2、6、8、10、12、14、16、18、20、22和24的序列中的至少一者具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.6％或99.7％同一性的同源性的任何核苷酸序列，编码SEQ ID NO:25-54中的至少一者或它们的片段的核苷酸序列。同源序列可以包括例如与野生型序列对应的序列。同样，这些特定同源序列可以对应于与野生型序列内的突变相关的变异，并且尤其对应于至少一个核苷酸的截短、置换、缺失和/或添加。如对于本领域普通技术人员将显而易见的，使用标准技术和公开可用的计算机程序(诸如，BLAST)，可以容易地创建和识别此类同源物。因此，以上所提及的每个同源物都应被认为如本文所述并且被充分描述。
实施方案包括由本文所述的核苷酸序列编码的嵌合受体或其片段，其序列由片段表示。对应于可以根据SEQ ID No:35-54的至少一种序列的三个可能的阅读框之一编码的多肽的氨基酸序列。
实施方案同样涉及嵌合受体，其特征在于它们包含选自SEQ ID NO:1、3、5、7、9、11、13、15、17、19、21、23和25至54的至少一种氨基酸序列或其片段之一的多肽。
根据实施方案，在这些多肽中有氨基酸序列SEQ ID NO:3、5、7、9、11、13、15、17、19、21、23和25至54或其片段的多肽，或与SEQ ID NO:1、3、5、7、9、11、13、15、17、19、21、23和25至54中的至少一种序列或其片段具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.6％或99.7％同一性的同源性的任何其他多肽。如对于本领域普通技术人员将显而易见的，使用标准技术和公开可用的计算机程序(诸如，BLAST)，可以容易地创建和识别此类同源物。因此，以上所提及的每个同源物都应被认为如本文所述并且被充分描述。
实施方案还涉及多肽，其特征在于它们包含选自以下的多肽：a)氨基酸序列的多肽的至少5个氨基酸的特异片段；b)与诸如a)中定义的多肽同源的多肽；c)诸如a)或b)中定义的多肽的特异生物活性片段；以及d)被例如a)、b)或c)中定义的多肽修饰的多肽。
在本说明书中，术语多肽、肽和蛋白质是可互换的。
在实施方案中，嵌合受体可以被糖基化、聚乙二醇化和/或以其他方式翻译后修饰。在另一个实施方案中，糖基化、聚乙二醇化和/或其他翻译后修饰可以在体内或体外发生和/或可以使用化学技术执行。在另外的实施方案中，任何糖基化、聚乙二醇化和/或其他翻译后修饰可以是N-连接或O-连接的。
在实施方案中，嵌合受体中的任一种可以是酶促或官能活性的，使得当细胞外结构域被配体结合时，信号被转导以使巨噬细胞极化。
如本文所用，“极化的巨噬细胞”是与M1或M2巨噬细胞表型相关的巨噬细胞。M1极化的巨噬细胞分泌IL-12和IL-23。可以通过使用标准细胞因子测定来测量IL-12和/或IL-23的表达并且将该表达与新分化的非极化巨噬细胞的表达进行比较来确定极化至M1的巨噬细胞。另选地，可以通过确定细胞是否为CD14+、CD80+、CD206+和CDCD163-来进行确定。M2极化的巨噬细胞分泌IL-10。可以通过使用标准细胞因子测定来测量IL-10的表达并且将该表达与新分化的非极化巨噬细胞的表达进行比较来确定极化至M2的巨噬细胞。另选地，可以通过确定细胞是否为CD14+、CD80-、CD206+和CDCD163+来进行确定。
本公开的方面涉及通过遗传重组或另选地通过化学合成获得的嵌合受体，因此它们可以包含非天然氨基酸，如下文所述。
根据实施方案的“多肽片段”应当理解为是指含有至少5个连续氨基酸、优选10个连续氨基酸或15个连续氨基酸的多肽。
在本文中，特异多肽片段应当理解为是指由核苷酸序列的特异片段编码的连续多肽片段。
“同源多肽”应当理解为是指相对于天然多肽具有某些修饰的多肽，诸如尤其是至少一个氨基酸的缺失、添加或置换、截短、延长、嵌合融合和/或突变。在同源多肽中，优选那些氨基酸序列与本文所述多肽的氨基酸序列具有至少80％或90％同源性的多肽。
“特异同源多肽”应当理解为是指如上所定义的同源多肽，并且具有本文所述的多肽的特异片段。
就置换而言，一个或多个连续或不连续的氨基酸被“等效”氨基酸替换。此处表达“等效”氨基酸在这里是指能够被基本结构的一种氨基酸置换而又不实质上改变对应肽的生物活性的任何氨基酸，因此将定义如下。对于本领域的普通技术人员将显而易见的是，使用标准分子生物学技术和可公开获得的计算机程序诸如BLAST，可以容易地创建和识别此类置换。因此，以上所提及的每个置换都应被认为如本文所述并且被充分描述。
这些等效氨基酸可以根据它们与它们所置换的氨基酸的结构同源性来确定，也可以根据能够进行的不同多肽之间的生物活性的对比测试结果来确定。
作为非限制性示例，将提及在不导致对应修饰的多肽的生物活性的广泛修饰的情况下进行置换的可能性，例如，用缬氨酸或异亮氨酸替换亮氨酸，用谷氨酸替换天冬氨酸，用天冬酰胺替换谷氨酰胺，用赖氨酸替换精氨酸等，在相同条件下自然可以想到逆向置换。
在另一个实施方案中，置换限于在具有相似识别的酶活性的其他蛋白质中不保守的氨基酸中的置换。例如，本领域的普通技术人员可以比对相似生物中具有相同功能的蛋白质，并且确定在具有该功能的蛋白质中通常保守的氨基酸。可用于生成此类比对的程序的一个示例是wordlwideweb.charite.de/bioinf/strap/连同由NCBI提供的数据库。
因此，根据一个实施方案，可以在具有该功能的蛋白质中通常保守的位置上进行置换或突变。在另一个实施方案中，核酸序列可以突变或置换，使得它们编码的氨基酸是不变的(简并置换和/突变)和/或突变或置换的，使得在具有该功能的蛋白质中通常保守的位置处进行任何所得的氨基酸置换或突变。
特异同源多肽同样对应于由如上文所定义的特异同源核苷酸序列编码的多肽，因此在本定义中包含突变的多肽或对应于可以存在于野生型序列中的变体的多肽，并且尤其对应于至少一个氨基酸残基的截短、置换、缺失和/或添加的多肽。
如本文所用，“多肽的特异生物活性片段”将尤其应当理解为是指具有如本文所述多肽的特征中的至少一个特征的特异多肽片段，诸如上文所定义的。在某些实施方案中，该肽能够表现为嵌合抗原受体，其在被活化时使巨噬细胞极化。
如本文所用，多肽的“修饰的多肽”应当理解为是指通过遗传重组或通过如下所述的化学合成获得的多肽，其相对于野生型序列具有至少一种修饰。这些修饰可能能够或可能不能够在如本文所述的多肽的特异性和/或活性的起点、或在结构构象、定位和膜插入能力的起点上携带氨基酸。因此，有可能创建具有等效、增加或降低的活性以及等效、更窄或更宽的特异性的多肽。在经修饰的多肽中，有必要提及其中可以修饰多达5个或更多个氨基酸、在N-或C-末端被截短甚至缺失或添加的多肽。
允许证实对真核或原核细胞的调节的方法是本领域普通技术人员众所周知的。同样应当理解，可以使用编码经修饰的多肽的核苷酸序列，例如通过载体并且如下文所述来进行调节。
前述经修饰的多肽可通过使用组合化学获得，其中可以在例如模型、细胞培养物或微生物上测试多肽之前，系统地改变多肽的部分，以选择活性最高或具有所寻求特性的化合物。
化学合成同样具有能够使用非天然氨基酸或非肽键的优点。
因此，为了延长多肽的寿命，可能感兴趣的是使用非天然氨基酸，例如为D形式，或者使用氨基酸类似物，特别是例如含硫形式。
最后，有可能将多肽、其特异性或经修饰的同源形式的结构整合到多肽类型或其他类型的化学结构中。因此，可能感兴趣的是在N-末端和C-末端提供不被蛋白酶识别的化合物。
本文同样公开了编码多肽的核苷酸序列。
实施方案同样涉及可用作引物或探针的核苷酸序列，其特征在于序列选自本文所述的核苷酸序列。
应当理解，各种实施方案同样涉及特异多肽，包括由核苷酸序列编码的嵌合受体，能够通过从天然多肽中纯化、通过遗传重组或通过本领域的技术人员熟知的程序并且诸如在下文中特别描述的化学合成来获得。以相同的方式，本公开内容还包括针对由核苷酸序列所编码的特异多肽的标记或未标记的单克隆或多克隆抗体。
实施方案另外涉及核苷酸序列作为用于检测和/或扩增核酸序列的引物或探针的用途。
因此，根据实施方案的核苷酸序列可以用于扩增核苷酸序列，特别是通过PCR技术(聚合酶链反应)(Erlich，1989年；Innis等人，1990年；Rolfs等人，1991年；以及White等人，1997年)来进行。
这些寡脱氧核糖核苷酸或寡核糖核苷酸引物有利地具有至少8个核苷酸、优选地至少12个核苷酸、甚至更优选地至少20个核苷酸的长度。
靶核酸的其他扩增技术可以有利地用作PCR的另选方案。
本文所述的核苷酸序列，尤其是引物，同样可以用于靶核酸扩增的其他程序中，诸如：Kwoh等人于1989年描述的TAS技术(基于转录的扩增系统)；Guatelli等人在1990年描述的3SR技术(自动维持序列扩增)；Kievitis等人在1991年描述的NASBA技术(基于核酸序列的扩增)；SDA技术(链置换扩增)(Walker等人，1992年)；TMA技术(转录介导扩增)。
包括嵌合受体的多核苷酸也可用于扩增或修饰用作探针的核酸的技术，诸如：Landegren等人在1988年描述的LCR技术(连接酶链反应)，并且Barany等人在1991年进行了改进，它采用了热稳定的连接酶；Segev在1992年描述的RCR技术(修复链反应)；Duck等人在1990年描述的CPR技术(循环探针反应)；Miele等人在1983年描述的Q-beta复制酶扩增技术，特别是由Chu等人在1986年，Lizardi等人在1988年，然后Burg等人以及Stone等人在1996年进行了改进。
在待检测的靶多核苷酸可能是RNA例如mRNA的情况下，在借助于至少一种引物进行扩增反应之前或借助于至少一种探针进行检测程序之前，可以使用逆转录酶类型的酶，以便从生物样品中包含的RNA中获得cDNA。因此，获得的cDNA将用作扩增或检测程序中使用的引物或探针的靶标。
检测探针将以此类方式选择，使其与靶序列或从靶序列产生的扩增子杂交。作为序列，此类探针将有利地具有至少12个核苷酸、尤其是至少20个核苷酸、优选至少100个核苷酸的序列。
实施方案还包含可用作探针或引物的核苷酸序列，其特征在于它们用放射性化合物或非放射性化合物标记。
未标记的核苷酸序列可以直接用作探针或引物，尽管通常用放射性同位素(32P、35S、3H、125I)或非放射性分子(生物素、乙酰氨基芴、洋地黄毒苷、5-溴脱氧尿苷、荧光素)标记该序列，以获得可用于多种应用的探针。
核苷酸序列的非放射性标记的示例描述于例如法国专利No.78.10975或由Urdea等人或Sanchez-Pescador等人于1988年描述。
在后一种情况下，也可以使用专利FR-2 422 956和FR-2 518 755中描述的一种标记方法。
杂交技术可以以各种方式进行(Matthews等人，1988年)。最通用的方法包括将细胞的核酸提取物固定在支持物(诸如硝化纤维、尼龙、聚苯乙烯)上，并且在明确的条件下用探针孵育固定的靶核酸。在杂交后，消除了过量的探针，并且通过适当的方法(与探针连接的放射性、荧光或酶活性的测量)检测了形成的杂合分子。
各种实施方案同样包含本文所述的核苷酸序列或多肽序列，其特征在于它们共价或非共价地固定在支持物上。
根据采用核苷酸序列的另一种有利模式，后者可以固定在支持物上使用，并且因此可以通过特异杂交用于捕获从待测生物样品中获得的靶核酸。如果需要，将固体支持物与样品分离，然后在捕获探针与靶核酸之间形成的杂交复合物借助第二探针(所谓的检测探针)进行检测，该探针用易于检测的元素标记。
另一个方面是用于克隆和/或表达序列的载体，其特征在于它包含本文所述的核苷酸序列。
同样提供了载体，其特征在于它们包含允许核苷酸序列在确定的宿主细胞中整合、表达和/或分泌的元件。
载体然后可以包含启动子、翻译的起始和终止信号以及转录调节的适当区域。它可能能够稳定地维持在宿主细胞中，并且可以任选地具有指定翻译蛋白质的分泌的特定信号。可以根据所使用的宿主细胞选择这些不同的元件。为此，可以将本文所述的核苷酸序列插入所选宿主内的自主复制载体或所选宿主的整合载体中。
此类载体将根据本领域的技术人员当前使用的方法来制备，并且可以通过标准方法，诸如磷酸钙沉淀、脂质转染、电穿孔和热冲击，将由此产生的克隆引入合适的宿主中。
依据的载体是例如质粒或病毒来源的载体。用于本文描述的多肽表达的载体的示例是质粒、噬菌体、粘粒、人工染色体、病毒载体、AAV载体、杆状病毒载体、腺病毒载体、慢病毒载体、逆转录病毒载体、嵌合病毒载体和嵌合腺病毒诸如AD5/F35。
这些载体可用于转化宿主细胞以克隆或表达本文所述的核苷酸序列。
实施方案同样包含被载体转化的宿主细胞。
这些细胞可通过将插入如上述定义的载体中的核苷酸序列引入宿主细胞中，然后在允许复制和/或表达转染的核苷酸序列的条件下培养细胞而获得。
宿主细胞可以选自原核或真核系统，例如细菌细胞(Olins和Lee，1993年)，但同样地酵母细胞(Buckholz，1993年)，以及植物细胞(诸如拟南芥属)和动物细胞，尤其是哺乳动物细胞的培养物(Edwards和Aruffo，1993年)，例如HEK 293细胞、HEK 293T细胞、中国仓鼠卵巢(CHO)细胞、骨髓细胞、骨髓祖细胞、单核细胞、嗜中性粒细胞、嗜碱性粒细胞、嗜酸性粒细胞、巨核细胞、T细胞、B细胞、自然杀伤细胞、白细胞、淋巴细胞、树突状细胞和巨噬细胞，但是同样，可以使用采用杆状病毒的程序的昆虫细胞，例如sf9昆虫细胞(Luckow，1993年)。
实施方案同样涉及包含转化细胞中的一个的生物。
表达一种或多种核酸或部分核酸的转基因生物的获得可根据本领域的技术人员熟知的方法，诸如通过病毒或非病毒转染，在例如大鼠、小鼠或兔子中进行。通过在普遍存在的强启动子的控制下转染基因的多个拷贝或者对一种类型的组织具有选择性，将有可能获得表达一种或多种基因的转基因生物。同样可能通过在胚胎细胞株中进行同源重组，将这些细胞株转移至胚胎，在生殖系水平上选择受影响的嵌合体以及嵌合体的生长来获得转基因生物。
转化细胞以及转基因生物可用于制备重组多肽的程序中。
如今，有可能通过使用由表达载体转化的细胞或使用转基因生物进行的基因工程来产生相对大量的重组多肽。
在重组形式的多肽诸如嵌合受体的制备程序中，其特征在于它们采用载体和/或由载体转化的细胞和/或包含转化细胞中的一个的转基因生物本身包括在本公开中。
如本文所用，“转化”和“转化的”涉及将核酸引入细胞，无论是原核还是真核。此外，本文所用的“转化”和“转化的”不必涉及生长控制或生长失调。
在重组形式的多肽诸如嵌合受体的制备程序中，采用载体和/或由该载体转化的细胞和/或包含转化细胞中的一个的转基因生物的制备程序包含核苷酸序列，诸如编码嵌合受体的核苷酸序列。
如本文所用，依据的变体可包括产生与“载体”蛋白质(嵌合蛋白质)融合的重组多肽。该系统的优点在于当融合配偶体对特异配体具有亲和力时，它可以允许重组产物的蛋白水解的稳定化和/或降低、在体外复性过程中溶解度的增加和/或纯化的简化。
更具体地，实施方案涉及包括以下步骤的多肽的制备程序：a)在允许表达核苷酸序列的重组多肽的条件下培养转化细胞；b)如果需要，回收重组多肽。
当用于制备多肽诸如嵌合受体的程序采用转基因生物时，然后可将重组多肽从该生物中提取或将其留在原地。
实施方案还涉及能够通过程序诸如前述而获得的多肽。
实施方案还包括用于制备合成多肽的程序，其特征在于其使用多肽的氨基酸序列。
本公开同样涉及通过程序获得的合成多肽，诸如嵌合受体。
多肽，诸如嵌合受体，同样可以通过肽合成领域中常规的技术来制备。该合成可以在均相溶液或固相中进行。
例如，可以求助于Houben-Weyl在1974年描述的均质溶液中的合成技术。
这种合成方法包括按要求的顺序两个接两个依次缩合的连续氨基酸，或者缩合先前形成的并且已经包含适当顺序的几个氨基酸的氨基酸和片段，或以前用这种方法制备的另选的几个片段，应当理解，有必要事先保护这些氨基酸或片段所携带的所有反应性官能团，不同的是一个是胺官能团而另一个是羧基，或者反之亦然，根据肽合成中众所周知的方法，这些官能团通常必须参与肽键的形成，特别是在羧基官能团活化之后。
也可以求助于Merrifield描述的技术。
为了根据Merrifield程序制备肽链，可以求助于非常多孔的聚合物树脂，该树脂上固定了该链的第一个C-末端氨基酸。该氨基酸通过其羧基固定在树脂上，并且其胺官能团得到了保护。因此，将要形成肽链的氨基酸一个接一个地固定在氨基上，该氨基每次已经预先脱保护，已经形成了一部分肽链，并且与树脂连接。当已经形成了全部所需的肽链时，形成肽链的不同氨基酸的保护基团被消除，并且借助于酸将肽与树脂分离。
这些杂合分子可以部分由与可能的免疫原性部分相关联的多肽载体分子或其片段形成，尤其是白喉毒素的表位、破伤风毒素、乙型肝炎病毒的表面抗原(专利FR 7921811)、脊髓灰质炎病毒的VP1抗原或任何其他病毒或细菌毒素或抗原。
包括嵌合受体的多肽、下文所述的抗体和编码前述任一种的核苷酸序列可有利地用于巨噬细胞极化的程序中。
在实施方案中，将编码嵌合受体的核酸序列提供给细胞。然后细胞可以表达编码的嵌合受体。表达的嵌合受体可以存在于细胞表面或细胞质中。在特定的实施方案中，表达嵌合受体的细胞是巨噬细胞。如前所述，巨噬细胞表达的嵌合受体可以结合配体，并且配体的结合可以活化嵌合受体，从而诱导巨噬细胞的极化。
在实施方案中，可以从受试者分离提供有编码嵌合受体的核酸序列的细胞。在向细胞提供核酸之后，可以例如通过注射或输血将细胞返回到从其获得的受试者。在其他实施方案中，提供有核酸的细胞可以由供体提供。在给供体细胞提供核酸后，可随后将该细胞提供给供体以外的个体。供体细胞的示例包括但不限于来自受试者的原代细胞和来自细胞系的细胞。
在其他实施方案中，可以将嵌合受体直接引入细胞中。可以使用将蛋白质引入细胞的任何方法，包括但不限于显微注射、电穿孔、膜融合和蛋白质转导域的使用。在向细胞提供嵌合受体之后，可以例如通过注射或输血将细胞返回到从其获得的受试者。在其他实施方案中，提供有嵌合受体的细胞可以由供体提供。在给供体细胞提供核酸后，可随后将该细胞提供给供体以外的个体。供体细胞的示例包括但不限于来自受试者的原代细胞和来自细胞系的细胞。
实施方案同样涉及借助适当的标签诸如酶、荧光或放射性类型标记的多肽，诸如嵌合受体。
这些多肽允许制备单克隆或多克隆抗体，其特征在于它们特异性识别该多肽。根据Kohler和Milstein在1975年描述的技术，从杂交瘤中制备单克隆抗体将是有利的。例如通过用与免疫反应的佐剂相关的多肽或DNA免疫动物，特别是小鼠，然后在亲和柱上纯化免疫动物血清中所含的特异抗体，将可能制备多克隆抗体，该亲和柱上已先前固定有用作抗原的多肽。多克隆抗体还可以通过在已先前固定有多肽的亲和柱上纯化由嵌合受体免疫攻击的动物血清中所含的抗体或其多肽或片段来制备。
此外，抗体可用于制备其他形式的结合分子，包括但不限于IgA、IgD、IgE、IgG、IgM、Fab片段、F(ab′)2片段、单价抗体、scFv片段、scRv-Fc片段、IgNAR、hcIgG、VhH抗体、纳米抗体和alpha抗体。
实施方案同样涉及单克隆或多克隆抗体或其片段，或嵌合抗体或其片段，其特征在于它们能够特异性识别本文所述的多肽或多肽和/或嵌合受体的配体。
同样有可能以与先前对核酸探针所述相同的方式标记抗体，诸如酶、荧光或放射性类型的标记。也可能包括此类抗体和/或其片段作为嵌合受体的一部分。作为非限制性示例，此类抗体或其片段可以构成嵌合受体的细胞外结构域的一部分。
另外，实施方案针对用于检测和/或识别样品中的嵌合受体的程序，其特征在于它包括以下步骤：a)使样品与单克隆或多克隆接触(在允许抗体与生物样品中可能存在的嵌合受体之间发生免疫反应的条件下)；b)表明可能形成的抗原-抗体复合物。
在以下例示性示例中进一步详细地描述了实施方案。尽管这些示例可以仅代表选定的实施方案，但是应当理解，以下示例是例示性的而不是限制性的。
实施例
实施例1：分离特异配体的ScFv片段
从表达对人TK1特异的抗体的单克隆抗体杂交瘤细胞(CB1)中纯化cDNA。分离的cDNA用于经由聚合酶链反应(PCR)扩增CB1可变区的重链和轻链，使用NCBI Blast确认了来自重链和轻链的序列。经由位点重叠延伸(SOE)PCR将CB1重链和轻链融合在一起，从而使用G4S接头形成单链片段变量(scFv)。G4S接头使用由IDT(https://www.idtdna.com/CodonOpt)提供的密码子优化工具对酵母和人进行了密码子优化，以使蛋白质表达最大化。使用限制性内切酶切出CB1 scFv，并且插入到pMP71 CAR载体中。
从酵母抗体文库中分离出TK-1和HPRT特异的人scFv片段。分离TK-1和HPRT蛋白质，用His标记并纯化。用抗His生物素化抗体标记TK-1和HPRT蛋白质，然后将其添加到文库中以选择TK-1和HPRT特异抗体克隆。TK-1和HPRT抗体克隆用链霉亲和素或抗生物素微珠交替染色，并使用磁柱富集。执行了另外两轮分选和选择，以分离TK-1和HPRT特异抗体。对于最终选择，通过另选地用荧光偶联的抗HA或抗c-myc抗体进行标记，以分离TK-1和HPRT特异抗体，可能的TK-1和HPRT抗体克隆及其各自的蛋白质通过荧光激活细胞分选(FACS)进行分选。选择高亲和力克隆用于嵌合受体构造。通过使用噬菌体显示或其他重组方法，可以选择其他人抗体或来自其他动物的人源化抗体或将其更改为TK-1或HPRT特异性的。
然后将选定的scFv克隆与人IgG1恒定域结合，以创建用于应用诸如蛋白质印记或ELISA的抗体，从而确认scFv的结合特异性。将抗体构建体插入pPNL9酵母分泌载体，并且用该构建体转化YVH10酵母并且诱导产生抗体。其他表达系统，诸如大肠杆菌或哺乳动物系统也可以用于分泌抗体。
蛋白质特异抗体片段的分离和表征。
参考图26，将105个酵母与2.5μg用荧光标签APC标记的目标蛋白质一起温育。较高的左(红色)峰表示未与目标蛋白质结合的酵母种群(阴性对照)。左侧的左下(蓝色)峰表明酵母菌不表达其表面蛋白，而右侧的高(蓝色)峰表明表达的抗体片段与目标蛋白质结合。
“Structural Consensus among Antibodies Defines the Antigen 5BindingSite”，PLoS Comput.Biol.第8卷第2期：e1002388，doi：10.1371/journal.pcbi.1002388，Kunik V，Ashkenazi S，Ofran Y(2012年)。“Paratome：An online tool for systematicidentification of antigen binding regions in antibodies based on sequence orstructure”，Nucleic Acids Res.2012年7月；40(Web Server issue)：W521-4.doi：10.1093/nar/gks480.Epub2012年6月6日。
实施例2：嵌合受体的创建
嵌合受体载体的构造：
该过程的第一步是设计合成嵌合受体基因的核苷酸序列，并且选择合适的慢病毒载体。所有的矢量设计均在版本为9.1.6的通用软件中进行。该序列可以从Uniprot和人蛋白参考数据库以及NCBI中检索。
载体是结合重组DNA技术和基因合成来合成的。
单链可变片段的序列是用人源化抗体酵母显示文库或噬菌体显示文库产生的。编码特异于TK1、HPRT、ROR1、MUC-16、EGFRvIII、间皮素、HER2、CEA、BCMA、GPC3、FAP、EphA2、NKG2D配体、GD2、CD19、CD20、CD30、CD33、CD123、CD133、CD138和CD171中的每一个的ScFv的核酸。编码具有a)、b)、c)、d)和e)中的至少一个的嵌合受体的核酸的所有可能组合，其中a)、b)、c)、d)和e)为：
a)特异于TK1、HPRT、ROR1、MUC-16、EGFRvIII、间皮素、HER2、CEA、BCMA、GPC3、FAP、EphA2、NKG2D配体、GD2、CD19、CD20、CD30、CD33、CD123、CD133、CD138和CD171的ScFv；
b)GS接头或没有GS接头；
c)选自LRR 5氨基酸短铰链、LRR长铰链、IgG4短铰链、IgG 119氨基酸中等铰链和IgG4长铰链、CD8铰链、半胱氨酸转换为丝氨酸的CD8铰链、无铰链的铰链区；
d)选自MYD88、TLR3、TLR4、TLR7、TLR8、TLR9、MAL、IRAK1、FCGR2A、FCGR3A和FCER1G跨膜结构域的跨膜结构域；以及
e)选自MYD88、TLR3、TLR4、TLR7、TLR8、TLR9、MAL、IRAK1、FCGR2A、FCGR3A和FCER1G胞质结构域的胞质结构域。
通过重组DNA技术和基因合成的组合来合成上述编码嵌合受体的核酸。
巨噬细胞经由慢病毒介导的基因转移以整合的基因递送方法进行遗传修饰，以提供编码嵌合受体的核酸。addgene的第三代慢病毒系统用于包装我们的慢病毒载体。pHIV-dTomato(#21374)和pUltra-chilli(#48687)是基因转移质粒。pCMV-VSV-G(#8454)、pMDLg/pRRE(#12251)、pRSV-Rev(#12253)、pHCMV-AmphoEnv(#15799)是包装质粒。慢病毒介导的人类淋巴细胞基因转移已经标准化，先前获得高达50％的转导效率。HEK293T细胞用磷酸钙法(SIGMA CAPHOS)转染。每次转染使用约10μg每种包装质粒和20μg编码嵌合受体的载体。在48至36小时后，收获病毒颗粒并且无菌过滤。确定感染HT1080和U937细胞的病毒滴定。
通过流式细胞仪检测红色荧光蛋白来执行分析。在病毒滴定后，使用Retronectin板(Clonetech，T100B)和旋转感染方法转导人单核细胞。
在慢病毒转导之前，使用人单核细胞分离试剂盒II(MACS130-091-153)，通过阴性选择和磁性分选从整个PBMNC中分离单核细胞。在单核细胞分离后，将细胞分成2个nunclon6孔板(Thermo，145380)，每个载体在每个孔中接种1.5×106个细胞。立即转导一个板，而第二个板用于单核细胞向M1巨噬细胞的离体分化。使用培养基M1-巨噬细胞产生培养基DFX(Promocell，C-28055)产生M1巨噬细胞。在7天后，在第9天用LPS(500X)(Affimetryx，00-4976-03)和IFN-γ(Promokine，C-60724)转导并活化巨噬细胞。通过流式细胞仪分析转导效率。通过使用FACS Aria细胞分选仪进行细胞分选来分离转导的细胞。在细胞分选后，将转导的单核细胞离体培养几天，然后分化，而分化的巨噬细胞可以持续一个月。
实施例3：通过嵌合受体极化巨噬细胞
将实施例2中制备的转导巨噬细胞分别暴露于TK1、HPRT、ROR1、MUC-16、EGFRvIII、间皮素、HER2、CEA、BCMA、GPC3、FAP、EphA2、NKG2D缀合的配体、GD2、CD19、CD20、CD30、CD33、CD123、CD133、CD138和CD171，然后使用标准的细胞因子测定或通过测量RNA的产生来监测IL-12和IL-23的分泌，从而检测向M1表型的极化。当暴露于特异于特定嵌合受体的配体时，带有嵌合受体的巨噬细胞被极化为M1表型，并由IL-12和/或IL-23的分泌增加来确定。除了特异嵌合受体的特异配体以外的配体在IL-12和/或IL-21中没有显示增加。
实施例4：单核细胞衍生的巨噬细胞的产生和转导
在分化7天后，单核细胞衍生的巨噬细胞经历了表型改变。在转导的细胞与非转导的细胞之间比较这些变化。如在图27中可以观察到的，转导的细胞具有与M1或经典活化的巨噬细胞相似的更具攻击性的表型。图27示出了分化第8天的非转导和转导的单核细胞衍生的巨噬细胞的图像。此时未添加干扰素γ和LPS。可以观察到，用嵌合受体转导的巨噬细胞的表型与未转导的巨噬细胞不同。转导的细胞显示出经典活化或M1样表型，表明巨噬细胞活化。改变的表型可能是转导过程和新合成受体表达的综合效应。
图28提供了编码嵌合受体的构建体的插入和表达的证实，如通过转导后48至72小时的dTomato的表达所证实的。这证明了人单核细胞衍生的巨噬细胞的成功转导。
实施例5：转导效率
在分化的第10天后，评估转导效率，并且对表达嵌合受体的巨噬细胞进行细胞分选。慢病毒转导在巨噬细胞中具有挑战性。然而，使用带有EF1-α启动子的HIV-1基系统，可实现近30％的巨噬细胞转导。在巨噬细胞分化的早期阶段，细胞的转导表现出不同的转导效率。分化早期的单核细胞或巨噬细胞更易于转导。带有嵌合腺病毒AD5/F35的腺病毒转导已成为巨噬细胞转导的另一种选择。图29示出了使用FACSAria系统对转导的巨噬细胞进行细胞分选的结果。使用慢病毒方法实现了约30％的巨噬细胞转导。最左边的图显示了对照，其中只有0.58％的细胞显示出荧光，这表明dTomato的表达。右边的两个图显示转导后的转导效率为27.1％。
实施例6：转导的巨噬细胞的免疫表型
进行了用载体转导的用于表达嵌合受体的巨噬细胞的免疫表型，以鉴定所转导细胞的活化状态。据报道，对TLR-4细胞外结构域的修饰可能诱导其信号域的持续活化(Gay等人，2014年)。TLR-4信号的持续活化可能导致巨噬细胞活化或M1表型。不知道所使用的基于TLR-4的构建体是否能够通过从TLR-4获得的TIR域触发信号的持续活化。然而，在转导过程之后，观察到表型的改变和巨噬细胞中细胞表面标记物的表达的改变。这可能是由于慢病毒转导和嵌合受体蛋白的表达相结合。CD14、CD80、D206的表达和CD163的低表达是巨噬细胞向M1表型极化的指示。在转导的细胞中观察到这些细胞表面标记物的表达。图30呈现了荧光激活细胞分选的六个散点图，其展示了染料(Alexa 647)的保留，以及在用嵌合受体转导的巨噬细胞中CD80、CD163、CD206和CD14的表达。
图31呈现了用载体转导以表达嵌合受体的巨噬细胞中M1细胞表面标记物的相对表达水平的直方图。
实施例7：靶向TK1的嵌合受体所转导的巨噬细胞对NCI-H460细胞的体外毒性。
测试了靶向TK1的嵌合受体所转导的巨噬细胞对NCI-H460-GFP细胞的杀肿瘤活性。使用的E:T比率为1:10。用共聚焦显微镜执行分析。在12小时期间每5分钟执行一次荧光检测。在时间流逝期间，观察到靶向TK1的嵌合受体所转导的巨噬细胞向H460-GFP细胞迁移并且攻击它们。在突触后，在靶细胞中诱导特异细胞死亡。如图32中的图像所示，靶向TK1的嵌合受体所转导的巨噬细胞可以检测、攻击和诱导表达TK1的肺癌细胞系中的细胞死亡。修饰NCI-H460细胞以表达GFP。用共聚焦显微镜将靶向TK1的嵌合受体所转导的巨噬细胞的杀肿瘤活性作为靶细胞中荧光损失来检测。
本公开可以在本公开的实质和范围内进一步修改。因此，本申请旨在涵盖使用其一般原理的本公开的任何变化、使用或改编。此外，本申请旨在涵盖与本公开有关的，在本公开所属领域的已知或习惯实践内的，并且落入所附权利要求及其合法等同物的范围内的此类偏离。
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序列表
<110> 吉姆•奥尼尔
<120> 转基因巨噬细胞、嵌合抗原受体和相关方法
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Met Arg Gln Thr Leu Pro Cys Ile Tyr Phe Trp Gly Gly Leu Leu Pro
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Phe Gly Met Leu Cys Ala Ser Ser Thr Thr Lys Cys Thr Val Ser His
20 25 30
Glu Val Ala Asp Cys Ser His Leu Lys Leu Thr Gln Val Pro Asp Asp
35 40 45
Leu Pro Thr Asn Ile Thr Val Leu Asn Leu Thr His Asn Gln Leu Arg
50 55 60
Arg Leu Pro Ala Ala Asn Phe Thr Arg Tyr Ser Gln Leu Thr Ser Leu
65 70 75 80
Asp Val Gly Phe Asn Thr Ile Ser Lys Leu Glu Pro Glu Leu Cys Gln
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Lys Leu Pro Met Leu Lys Val Leu Asn Leu Gln His Asn Glu Leu Ser
100 105 110
Gln Leu Ser Asp Lys Thr Phe Ala Phe Cys Thr Asn Leu Thr Glu Leu
115 120 125
His Leu Met Ser Asn Ser Ile Gln Lys Ile Lys Asn Asn Pro Phe Val
130 135 140
Lys Gln Lys Asn Leu Ile Thr Leu Asp Leu Ser His Asn Gly Leu Ser
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Ser Thr Lys Leu Gly Thr Gln Val Gln Leu Glu Asn Leu Gln Glu Leu
165 170 175
Leu Leu Ser Asn Asn Lys Ile Gln Ala Leu Lys Ser Glu Glu Leu Asp
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Ile Phe Ala Asn Ser Ser Leu Lys Lys Leu Glu Leu Ser Ser Asn Gln
195 200 205
Ile Lys Glu Phe Ser Pro Gly Cys Phe His Ala Ile Gly Arg Leu Phe
210 215 220
Gly Leu Phe Leu Asn Asn Val Gln Leu Gly Pro Ser Leu Thr Glu Lys
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Leu Cys Leu Glu Leu Ala Asn Thr Ser Ile Arg Asn Leu Ser Leu Ser
245 250 255
Asn Ser Gln Leu Ser Thr Thr Ser Asn Thr Thr Phe Leu Gly Leu Lys
260 265 270
Trp Thr Asn Leu Thr Met Leu Asp Leu Ser Tyr Asn Asn Leu Asn Val
275 280 285
Val Gly Asn Asp Ser Phe Ala Trp Leu Pro Gln Leu Glu Tyr Phe Phe
290 295 300
Leu Glu Tyr Asn Asn Ile Gln His Leu Phe Ser His Ser Leu His Gly
305 310 315 320
Leu Phe Asn Val Arg Tyr Leu Asn Leu Lys Arg Ser Phe Thr Lys Gln
325 330 335
Ser Ile Ser Leu Ala Ser Leu Pro Lys Ile Asp Asp Phe Ser Phe Gln
340 345 350
Trp Leu Lys Cys Leu Glu His Leu Asn Met Glu Asp Asn Asp Ile Pro
355 360 365
Gly Ile Lys Ser Asn Met Phe Thr Gly Leu Ile Asn Leu Lys Tyr Leu
370 375 380
Ser Leu Ser Asn Ser Phe Thr Ser Leu Arg Thr Leu Thr Asn Glu Thr
385 390 395 400
Phe Val Ser Leu Ala His Ser Pro Leu His Ile Leu Asn Leu Thr Lys
405 410 415
Asn Lys Ile Ser Lys Ile Glu Ser Asp Ala Phe Ser Trp Leu Gly His
420 425 430
Leu Glu Val Leu Asp Leu Gly Leu Asn Glu Ile Gly Gln Glu Leu Thr
435 440 445
Gly Gln Glu Trp Arg Gly Leu Glu Asn Ile Phe Glu Ile Tyr Leu Ser
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Tyr Asn Lys Tyr Leu Gln Leu Thr Arg Asn Ser Phe Ala Leu Val Pro
465 470 475 480
Ser Leu Gln Arg Leu Met Leu Arg Arg Val Ala Leu Lys Asn Val Asp
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Ser Ser Pro Ser Pro Phe Gln Pro Leu Arg Asn Leu Thr Ile Leu Asp
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Leu Glu Lys Leu Glu Ile Leu Asp Leu Gln His Asn Asn Leu Ala Arg
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Leu Trp Lys His Ala Asn Pro Gly Gly Pro Ile Tyr Phe Leu Lys Gly
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Leu Ser His Leu His Ile Leu Asn Leu Glu Ser Asn Gly Phe Asp Glu
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Ile Pro Val Glu Val Phe Lys Asp Leu Phe Glu Leu Lys Ile Ile Asp
580 585 590
Leu Gly Leu Asn Asn Leu Asn Thr Leu Pro Ala Ser Val Phe Asn Asn
595 600 605
Gln Val Ser Leu Lys Ser Leu Asn Leu Gln Lys Asn Leu Ile Thr Ser
610 615 620
Val Glu Lys Lys Val Phe Gly Pro Ala Phe Arg Asn Leu Thr Glu Leu
625 630 635 640
Asp Met Arg Phe Asn Pro Phe Asp Cys Thr Cys Glu Ser Ile Ala Trp
645 650 655
Phe Val Asn Trp Ile Asn Glu Thr His Thr Asn Ile Pro Glu Leu Ser
660 665 670
Ser His Tyr Leu Cys Asn Thr Pro Pro His Tyr His Gly Phe Pro Val
675 680 685
Arg Leu Phe Asp Thr Ser Ser Cys Lys Asp Ser Ala Pro Phe Glu Leu
690 695 700
Phe Phe Met Ile Asn Thr Ser Ile Leu Leu Ile Phe Ile Phe Ile Val
705 710 715 720
Leu Leu Ile His Phe Glu Gly Trp Arg Ile Ser Phe Tyr Trp Asn Val
725 730 735
Ser Val His Arg Val Leu Gly Phe Lys Glu Ile Asp Arg Gln Thr Glu
740 745 750
Gln Phe Glu Tyr Ala Ala Tyr Ile Ile His Ala Tyr Lys Asp Lys Asp
755 760 765
Trp Val Trp Glu His Phe Ser Ser Met Glu Lys Glu Asp Gln Ser Leu
770 775 780
Lys Phe Cys Leu Glu Glu Arg Asp Phe Glu Ala Gly Val Phe Glu Leu
785 790 795 800
Glu Ala Ile Val Asn Ser Ile Lys Arg Ser Arg Lys Ile Ile Phe Val
805 810 815
Ile Thr His His Leu Leu Lys Asp Pro Leu Cys Lys Arg Phe Lys Val
820 825 830
His His Ala Val Gln Gln Ala Ile Glu Gln Asn Leu Asp Ser Ile Ile
835 840 845
Leu Val Phe Leu Glu Glu Ile Pro Asp Tyr Lys Leu Asn His Ala Leu
850 855 860
Cys Leu Arg Arg Gly Met Phe Lys Ser His Cys Ile Leu Asn Trp Pro
865 870 875 880
Val Gln Lys Glu Arg Ile Gly Ala Phe Arg His Lys Leu Gln Val Ala
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Leu Gly Ser Lys Asn Ser Val His
900
<210> 2
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<212> DNA
<213> 人类（Homo sapiens）
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atgagacaga ctttgccttg tatctacttt tgggggggcc ttttgccctt tgggatgctg 60
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aagttgactc aggtacccga tgatctaccc acaaacataa cagtgttgaa ccttacccat 180
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ggaagattat ttggcctctt tctgaacaat gtccagctgg gtcccagcct tacagagaag 720
ctatgtttgg aattagcaaa cacaagcatt cggaatctgt ctctgagtaa cagccagctg 780
tccaccacca gcaatacaac tttcttggga ctaaagtgga caaatctcac tatgctcgat 840
ctttcctaca acaacttaaa tgtggttggt aacgattcct ttgcttggct tccacaacta 900
gaatatttct tcctagagta taataatata cagcatttgt tttctcactc tttgcacggg 960
cttttcaatg tgaggtacct gaatttgaaa cggtctttta ctaaacaaag tatttccctt 1020
gcctcactcc ccaagattga tgatttttct tttcagtggc taaaatgttt ggagcacctt 1080
aacatggaag ataatgatat tccaggcata aaaagcaata tgttcacagg attgataaac 1140
ctgaaatact taagtctatc caactccttt acaagtttgc gaactttgac aaatgaaaca 1200
tttgtatcac ttgctcattc tcccttacac atactcaacc taaccaagaa taaaatctca 1260
aaaatagaga gtgatgcttt ctcttggttg ggccacctag aagtacttga cctgggcctt 1320
aatgaaattg ggcaagaact cacaggccag gaatggagag gtctagaaaa tattttcgaa 1380
atctatcttt cctacaacaa gtacctgcag ctgactagga actcctttgc cttggtccca 1440
agccttcaac gactgatgct ccgaagggtg gcccttaaaa atgtggatag ctctccttca 1500
ccattccagc ctcttcgtaa cttgaccatt ctggatctaa gcaacaacaa catagccaac 1560
ataaatgatg acatgttgga gggtcttgag aaactagaaa ttctcgattt gcagcataac 1620
aacttagcac ggctctggaa acacgcaaac cctggtggtc ccatttattt cctaaagggt 1680
ctgtctcacc tccacatcct taacttggag tccaacggct ttgacgagat cccagttgag 1740
gtcttcaagg atttatttga actaaagatc atcgatttag gattgaataa tttaaacaca 1800
cttccagcat ctgtctttaa taatcaggtg tctctaaagt cattgaacct tcagaagaat 1860
ctcataacat ccgttgagaa gaaggttttc gggccagctt tcaggaacct gactgagtta 1920
gatatgcgct ttaatccctt tgattgcacg tgtgaaagta ttgcctggtt tgttaattgg 1980
attaacgaga cccataccaa catccctgag ctgtcaagcc actacctttg caacactcca 2040
cctcactatc atgggttccc agtgagactt tttgatacat catcttgcaa agacagtgcc 2100
ccctttgaac tctttttcat gatcaatacc agtatcctgt tgatttttat ctttattgta 2160
cttctcatcc actttgaggg ctggaggata tctttttatt ggaatgtttc agtacatcga 2220
gttcttggtt tcaaagaaat agacagacag acagaacagt ttgaatatgc agcatatata 2280
attcatgcct ataaagataa ggattgggtc tgggaacatt tctcttcaat ggaaaaggaa 2340
gaccaatctc tcaaattttg tctggaagaa agggactttg aggcgggtgt ttttgaacta 2400
gaagcaattg ttaacagcat caaaagaagc agaaaaatta tttttgttat aacacaccat 2460
ctattaaaag acccattatg caaaagattc aaggtacatc atgcagttca acaagctatt 2520
gaacaaaatc tggattccat tatattggtt ttccttgagg agattccaga ttataaactg 2580
aaccatgcac tctgtttgcg aagaggaatg tttaaatctc actgcatctt gaactggcca 2640
gttcagaaag aacggatagg tgcctttcgt cataaattgc aagtagcact tggatccaaa 2700
aactctgtac attaa 2715
<210> 3
<211> 839
<212> PRT
<213> 人类（Homo sapiens）
<400> 3
Met Met Ser Ala Ser Arg Leu Ala Gly Thr Leu Ile Pro Ala Met Ala
1 5 10 15
Phe Leu Ser Cys Val Arg Pro Glu Ser Trp Glu Pro Cys Val Glu Val
20 25 30
Val Pro Asn Ile Thr Tyr Gln Cys Met Glu Leu Asn Phe Tyr Lys Ile
35 40 45
Pro Asp Asn Leu Pro Phe Ser Thr Lys Asn Leu Asp Leu Ser Phe Asn
50 55 60
Pro Leu Arg His Leu Gly Ser Tyr Ser Phe Phe Ser Phe Pro Glu Leu
65 70 75 80
Gln Val Leu Asp Leu Ser Arg Cys Glu Ile Gln Thr Ile Glu Asp Gly
85 90 95
Ala Tyr Gln Ser Leu Ser His Leu Ser Thr Leu Ile Leu Thr Gly Asn
100 105 110
Pro Ile Gln Ser Leu Ala Leu Gly Ala Phe Ser Gly Leu Ser Ser Leu
115 120 125
Gln Lys Leu Val Ala Val Glu Thr Asn Leu Ala Ser Leu Glu Asn Phe
130 135 140
Pro Ile Gly His Leu Lys Thr Leu Lys Glu Leu Asn Val Ala His Asn
145 150 155 160
Leu Ile Gln Ser Phe Lys Leu Pro Glu Tyr Phe Ser Asn Leu Thr Asn
165 170 175
Leu Glu His Leu Asp Leu Ser Ser Asn Lys Ile Gln Ser Ile Tyr Cys
180 185 190
Thr Asp Leu Arg Val Leu His Gln Met Pro Leu Leu Asn Leu Ser Leu
195 200 205
Asp Leu Ser Leu Asn Pro Met Asn Phe Ile Gln Pro Gly Ala Phe Lys
210 215 220
Glu Ile Arg Leu His Lys Leu Thr Leu Arg Asn Asn Phe Asp Ser Leu
225 230 235 240
Asn Val Met Lys Thr Cys Ile Gln Gly Leu Ala Gly Leu Glu Val His
245 250 255
Arg Leu Val Leu Gly Glu Phe Arg Asn Glu Gly Asn Leu Glu Lys Phe
260 265 270
Asp Lys Ser Ala Leu Glu Gly Leu Cys Asn Leu Thr Ile Glu Glu Phe
275 280 285
Arg Leu Ala Tyr Leu Asp Tyr Tyr Leu Asp Asp Ile Ile Asp Leu Phe
290 295 300
Asn Cys Leu Thr Asn Val Ser Ser Phe Ser Leu Val Ser Val Thr Ile
305 310 315 320
Glu Arg Val Lys Asp Phe Ser Tyr Asn Phe Gly Trp Gln His Leu Glu
325 330 335
Leu Val Asn Cys Lys Phe Gly Gln Phe Pro Thr Leu Lys Leu Lys Ser
340 345 350
Leu Lys Arg Leu Thr Phe Thr Ser Asn Lys Gly Gly Asn Ala Phe Ser
355 360 365
Glu Val Asp Leu Pro Ser Leu Glu Phe Leu Asp Leu Ser Arg Asn Gly
370 375 380
Leu Ser Phe Lys Gly Cys Cys Ser Gln Ser Asp Phe Gly Thr Thr Ser
385 390 395 400
Leu Lys Tyr Leu Asp Leu Ser Phe Asn Gly Val Ile Thr Met Ser Ser
405 410 415
Asn Phe Leu Gly Leu Glu Gln Leu Glu His Leu Asp Phe Gln His Ser
420 425 430
Asn Leu Lys Gln Met Ser Glu Phe Ser Val Phe Leu Ser Leu Arg Asn
435 440 445
Leu Ile Tyr Leu Asp Ile Ser His Thr His Thr Arg Val Ala Phe Asn
450 455 460
Gly Ile Phe Asn Gly Leu Ser Ser Leu Glu Val Leu Lys Met Ala Gly
465 470 475 480
Asn Ser Phe Gln Glu Asn Phe Leu Pro Asp Ile Phe Thr Glu Leu Arg
485 490 495
Asn Leu Thr Phe Leu Asp Leu Ser Gln Cys Gln Leu Glu Gln Leu Ser
500 505 510
Pro Thr Ala Phe Asn Ser Leu Ser Ser Leu Gln Val Leu Asn Met Ser
515 520 525
His Asn Asn Phe Phe Ser Leu Asp Thr Phe Pro Tyr Lys Cys Leu Asn
530 535 540
Ser Leu Gln Val Leu Asp Tyr Ser Leu Asn His Ile Met Thr Ser Lys
545 550 555 560
Lys Gln Glu Leu Gln His Phe Pro Ser Ser Leu Ala Phe Leu Asn Leu
565 570 575
Thr Gln Asn Asp Phe Ala Cys Thr Cys Glu His Gln Ser Phe Leu Gln
580 585 590
Trp Ile Lys Asp Gln Arg Gln Leu Leu Val Glu Val Glu Arg Met Glu
595 600 605
Cys Ala Thr Pro Ser Asp Lys Gln Gly Met Pro Val Leu Ser Leu Asn
610 615 620
Ile Thr Cys Gln Met Asn Lys Thr Ile Ile Gly Val Ser Val Leu Ser
625 630 635 640
Val Leu Val Val Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe
645 650 655
His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn
660 665 670
Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val
675 680 685
Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln
690 695 700
Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala
705 710 715 720
Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val
725 730 735
Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu
740 745 750
Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe
755 760 765
Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu
770 775 780
Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser
785 790 795 800
Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu
805 810 815
Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn
820 825 830
Trp Gln Glu Ala Thr Ser Ile
835
<210> 4
<211> 23
<212> PRT
<213> 人类（Homo sapiens）
<400> 4
Ile Gly Val Ser Val Leu Ser Val Leu Val Val Ser Val Val Ala Val
1 5 10 15
Leu Val Tyr Lys Phe Tyr Phe
20
<210> 5
<211> 183
<212> PRT
<213> 人类（Homo sapiens）
<400> 5
His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn
1 5 10 15
Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val
20 25 30
Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln
35 40 45
Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala
50 55 60
Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val
65 70 75 80
Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu
85 90 95
Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe
100 105 110
Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu
115 120 125
Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser
130 135 140
Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu
145 150 155 160
Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn
165 170 175
Trp Gln Glu Ala Thr Ser Ile
180
<210> 6
<211> 2520
<212> DNA
<213> 人类（Homo sapiens）
<400> 6
atgatgtctg cctcgcgcct ggctgggact ctgatcccag ccatggcctt cctctcctgc 60
gtgagaccag aaagctggga gccctgcgtg gaggtggttc ctaatattac ttatcaatgc 120
atggagctga atttctacaa aatccccgac aacctcccct tctcaaccaa gaacctggac 180
ctgagcttta atcccctgag gcatttaggc agctatagct tcttcagttt cccagaactg 240
caggtgctgg atttatccag gtgtgaaatc cagacaattg aagatggggc atatcagagc 300
ctaagccacc tctctacctt aatattgaca ggaaacccca tccagagttt agccctggga 360
gccttttctg gactatcaag tttacagaag ctggtggctg tggagacaaa tctagcatct 420
ctagagaact tccccattgg acatctcaaa actttgaaag aacttaatgt ggctcacaat 480
cttatccaat ctttcaaatt acctgagtat ttttctaatc tgaccaatct agagcacttg 540
gacctttcca gcaacaagat tcaaagtatt tattgcacag acttgcgggt tctacatcaa 600
atgcccctac tcaatctctc tttagacctg tccctgaacc ctatgaactt tatccaacca 660
ggtgcattta aagaaattag gcttcataag ctgactttaa gaaataattt tgatagttta 720
aatgtaatga aaacttgtat tcaaggtctg gctggtttag aagtccatcg tttggttctg 780
ggagaattta gaaatgaagg aaacttggaa aagtttgaca aatctgctct agagggcctg 840
tgcaatttga ccattgaaga attccgatta gcatacttag actactacct cgatgatatt 900
attgacttat ttaattgttt gacaaatgtt tcttcatttt ccctggtgag tgtgactatt 960
gaaagggtaa aagacttttc ttataatttc ggatggcaac atttagaatt agttaactgt 1020
aaatttggac agtttcccac attgaaactc aaatctctca aaaggcttac tttcacttcc 1080
aacaaaggtg ggaatgcttt ttcagaagtt gatctaccaa gccttgagtt tctagatctc 1140
agtagaaatg gcttgagttt caaaggttgc tgttctcaaa gtgattttgg gacaaccagc 1200
ctaaagtatt tagatctgag cttcaatggt gttattacca tgagttcaaa cttcttgggc 1260
ttagaacaac tagaacatct ggatttccag cattccaatt tgaaacaaat gagtgagttt 1320
tcagtattcc tatcactcag aaacctcatt taccttgaca tttctcatac tcacaccaga 1380
gttgctttca atggcatctt caatggcttg tccagtctcg aagtcttgaa aatggctggc 1440
aattctttcc aggaaaactt ccttccagat atcttcacag agctgagaaa cttgaccttc 1500
ctggacctct ctcagtgtca actggagcag ttgtctccaa cagcatttaa ctcactctcc 1560
agtcttcagg tactaaatat gagccacaac aacttctttt cattggatac gtttccttat 1620
aagtgtctga actccctcca ggttcttgat tacagtctca atcacataat gacttccaaa 1680
aaacaggaac tacagcattt tccaagtagt ctagctttct taaatcttac tcagaatgac 1740
tttgcttgta cttgtgaaca ccagagtttc ctgcaatgga tcaaggacca gaggcagctc 1800
ttggtggaag ttgaacgaat ggaatgtgca acaccttcag ataagcaggg catgcctgtg 1860
ctgagtttga atatcacctg tcagatgaat aagaccatca ttggtgtgtc ggtcctcagt 1920
gtgcttgtag tatctgttgt agcagttctg gtctataagt tctattttca cctgatgctt 1980
cttgctggct gcataaagta tggtagaggt gaaaacatct atgatgcctt tgttatctac 2040
tcaagccagg atgaggactg ggtaaggaat gagctagtaa agaatttaga agaaggggtg 2100
cctccatttc agctctgcct tcactacaga gactttattc ccggtgtggc cattgctgcc 2160
aacatcatcc atgaaggttt ccataaaagc cgaaaggtga ttgttgtggt gtcccagcac 2220
ttcatccaga gccgctggtg tatctttgaa tatgagattg ctcagacctg gcagtttctg 2280
agcagtcgtg ctggtatcat cttcattgtc ctgcagaagg tggagaagac cctgctcagg 2340
cagcaggtgg agctgtaccg ccttctcagc aggaacactt acctggagtg ggaggacagt 2400
gtcctggggc ggcacatctt ctggagacga ctcagaaaag ccctgctgga tggtaaatca 2460
tggaatccag aaggaacagt gggtacagga tgcaattggc aggaagcaac atctatctga 2520
<210> 7
<211> 1049
<212> PRT
<213> 人类（Homo sapiens）
<400> 7
Met Val Phe Pro Met Trp Thr Leu Lys Arg Gln Ile Leu Ile Leu Phe
1 5 10 15
Asn Ile Ile Leu Ile Ser Lys Leu Leu Gly Ala Arg Trp Phe Pro Lys
20 25 30
Thr Leu Pro Cys Asp Val Thr Leu Asp Val Pro Lys Asn His Val Ile
35 40 45
Val Asp Cys Thr Asp Lys His Leu Thr Glu Ile Pro Gly Gly Ile Pro
50 55 60
Thr Asn Thr Thr Asn Leu Thr Leu Thr Ile Asn His Ile Pro Asp Ile
65 70 75 80
Ser Pro Ala Ser Phe His Arg Leu Asp His Leu Val Glu Ile Asp Phe
85 90 95
Arg Cys Asn Cys Val Pro Ile Pro Leu Gly Ser Lys Asn Asn Met Cys
100 105 110
Ile Lys Arg Leu Gln Ile Lys Pro Arg Ser Phe Ser Gly Leu Thr Tyr
115 120 125
Leu Lys Ser Leu Tyr Leu Asp Gly Asn Gln Leu Leu Glu Ile Pro Gln
130 135 140
Gly Leu Pro Pro Ser Leu Gln Leu Leu Ser Leu Glu Ala Asn Asn Ile
145 150 155 160
Phe Ser Ile Arg Lys Glu Asn Leu Thr Glu Leu Ala Asn Ile Glu Ile
165 170 175
Leu Tyr Leu Gly Gln Asn Cys Tyr Tyr Arg Asn Pro Cys Tyr Val Ser
180 185 190
Tyr Ser Ile Glu Lys Asp Ala Phe Leu Asn Leu Thr Lys Leu Lys Val
195 200 205
Leu Ser Leu Lys Asp Asn Asn Val Thr Ala Val Pro Thr Val Leu Pro
210 215 220
Ser Thr Leu Thr Glu Leu Tyr Leu Tyr Asn Asn Met Ile Ala Lys Ile
225 230 235 240
Gln Glu Asp Asp Phe Asn Asn Leu Asn Gln Leu Gln Ile Leu Asp Leu
245 250 255
Ser Gly Asn Cys Pro Arg Cys Tyr Asn Ala Pro Phe Pro Cys Ala Pro
260 265 270
Cys Lys Asn Asn Ser Pro Leu Gln Ile Pro Val Asn Ala Phe Asp Ala
275 280 285
Leu Thr Glu Leu Lys Val Leu Arg Leu His Ser Asn Ser Leu Gln His
290 295 300
Val Pro Pro Arg Trp Phe Lys Asn Ile Asn Lys Leu Gln Glu Leu Asp
305 310 315 320
Leu Ser Gln Asn Phe Leu Ala Lys Glu Ile Gly Asp Ala Lys Phe Leu
325 330 335
His Phe Leu Pro Ser Leu Ile Gln Leu Asp Leu Ser Phe Asn Phe Glu
340 345 350
Leu Gln Val Tyr Arg Ala Ser Met Asn Leu Ser Gln Ala Phe Ser Ser
355 360 365
Leu Lys Ser Leu Lys Ile Leu Arg Ile Arg Gly Tyr Val Phe Lys Glu
370 375 380
Leu Lys Ser Phe Asn Leu Ser Pro Leu His Asn Leu Gln Asn Leu Glu
385 390 395 400
Val Leu Asp Leu Gly Thr Asn Phe Ile Lys Ile Ala Asn Leu Ser Met
405 410 415
Phe Lys Gln Phe Lys Arg Leu Lys Val Ile Asp Leu Ser Val Asn Lys
420 425 430
Ile Ser Pro Ser Gly Asp Ser Ser Glu Val Gly Phe Cys Ser Asn Ala
435 440 445
Arg Thr Ser Val Glu Ser Tyr Glu Pro Gln Val Leu Glu Gln Leu His
450 455 460
Tyr Phe Arg Tyr Asp Lys Tyr Ala Arg Ser Cys Arg Phe Lys Asn Lys
465 470 475 480
Glu Ala Ser Phe Met Ser Val Asn Glu Ser Cys Tyr Lys Tyr Gly Gln
485 490 495
Thr Leu Asp Leu Ser Lys Asn Ser Ile Phe Phe Val Lys Ser Ser Asp
500 505 510
Phe Gln His Leu Ser Phe Leu Lys Cys Leu Asn Leu Ser Gly Asn Leu
515 520 525
Ile Ser Gln Thr Leu Asn Gly Ser Glu Phe Gln Pro Leu Ala Glu Leu
530 535 540
Arg Tyr Leu Asp Phe Ser Asn Asn Arg Leu Asp Leu Leu His Ser Thr
545 550 555 560
Ala Phe Glu Glu Leu His Lys Leu Glu Val Leu Asp Ile Ser Ser Asn
565 570 575
Ser His Tyr Phe Gln Ser Glu Gly Ile Thr His Met Leu Asn Phe Thr
580 585 590
Lys Asn Leu Lys Val Leu Gln Lys Leu Met Met Asn Asp Asn Asp Ile
595 600 605
Ser Ser Ser Thr Ser Arg Thr Met Glu Ser Glu Ser Leu Arg Thr Leu
610 615 620
Glu Phe Arg Gly Asn His Leu Asp Val Leu Trp Arg Glu Gly Asp Asn
625 630 635 640
Arg Tyr Leu Gln Leu Phe Lys Asn Leu Leu Lys Leu Glu Glu Leu Asp
645 650 655
Ile Ser Lys Asn Ser Leu Ser Phe Leu Pro Ser Gly Val Phe Asp Gly
660 665 670
Met Pro Pro Asn Leu Lys Asn Leu Ser Leu Ala Lys Asn Gly Leu Lys
675 680 685
Ser Phe Ser Trp Lys Lys Leu Gln Cys Leu Lys Asn Leu Glu Thr Leu
690 695 700
Asp Leu Ser His Asn Gln Leu Thr Thr Val Pro Glu Arg Leu Ser Asn
705 710 715 720
Cys Ser Arg Ser Leu Lys Asn Leu Ile Leu Lys Asn Asn Gln Ile Arg
725 730 735
Ser Leu Thr Lys Tyr Phe Leu Gln Asp Ala Phe Gln Leu Arg Tyr Leu
740 745 750
Asp Leu Ser Ser Asn Lys Ile Gln Met Ile Gln Lys Thr Ser Phe Pro
755 760 765
Glu Asn Val Leu Asn Asn Leu Lys Met Leu Leu Leu His His Asn Arg
770 775 780
Phe Leu Cys Thr Cys Asp Ala Val Trp Phe Val Trp Trp Val Asn His
785 790 795 800
Thr Glu Val Thr Ile Pro Tyr Leu Ala Thr Asp Val Thr Cys Val Gly
805 810 815
Pro Gly Ala His Lys Gly Gln Ser Val Ile Ser Leu Asp Leu Tyr Thr
820 825 830
Cys Glu Leu Asp Leu Thr Asn Leu Ile Leu Phe Ser Leu Ser Ile Ser
835 840 845
Val Ser Leu Phe Leu Met Val Met Met Thr Ala Ser His Leu Tyr Phe
850 855 860
Trp Asp Val Trp Tyr Ile Tyr His Phe Cys Lys Ala Lys Ile Lys Gly
865 870 875 880
Tyr Gln Arg Leu Ile Ser Pro Asp Cys Cys Tyr Asp Ala Phe Ile Val
885 890 895
Tyr Asp Thr Lys Asp Pro Ala Val Thr Glu Trp Val Leu Ala Glu Leu
900 905 910
Val Ala Lys Leu Glu Asp Pro Arg Glu Lys His Phe Asn Leu Cys Leu
915 920 925
Glu Glu Arg Asp Trp Leu Pro Gly Gln Pro Val Leu Glu Asn Leu Ser
930 935 940
Gln Ser Ile Gln Leu Ser Lys Lys Thr Val Phe Val Met Thr Asp Lys
945 950 955 960
Tyr Ala Lys Thr Glu Asn Phe Lys Ile Ala Phe Tyr Leu Ser His Gln
965 970 975
Arg Leu Met Asp Glu Lys Val Asp Val Ile Ile Leu Ile Phe Leu Glu
980 985 990
Lys Pro Phe Gln Lys Ser Lys Phe Leu Gln Leu Arg Lys Arg Leu Cys
995 1000 1005
Gly Ser Ser Val Leu Glu Trp Pro Thr Asn Pro Gln Ala His Pro
1010 1015 1020
Tyr Phe Trp Gln Cys Leu Lys Asn Ala Leu Ala Thr Asp Asn His
1025 1030 1035
Val Ala Tyr Ser Gln Val Phe Lys Glu Thr Val
1040 1045
<210> 8
<211> 3150
<212> DNA
<213> 人类（Homo sapiens）
<400> 8
atggtgtttc caatgtggac actgaagaga caaattctta tcctttttaa cataatccta 60
atttccaaac tccttggggc tagatggttt cctaaaactc tgccctgtga tgtcactctg 120
gatgttccaa agaaccatgt gatcgtggac tgcacagaca agcatttgac agaaattcct 180
ggaggtattc ccacgaacac cacgaacctc accctcacca ttaaccacat accagacatc 240
tccccagcgt cctttcacag actggaccat ctggtagaga tcgatttcag atgcaactgt 300
gtacctattc cactggggtc aaaaaacaac atgtgcatca agaggctgca gattaaaccc 360
agaagcttta gtggactcac ttatttaaaa tccctttacc tggatggaaa ccagctacta 420
gagataccgc agggcctccc gcctagctta cagcttctca gccttgaggc caacaacatc 480
ttttccatca gaaaagagaa tctaacagaa ctggccaaca tagaaatact ctacctgggc 540
caaaactgtt attatcgaaa tccttgttat gtttcatatt caatagagaa agatgccttc 600
ctaaacttga caaagttaaa agtgctctcc ctgaaagata acaatgtcac agccgtccct 660
actgttttgc catctacttt aacagaacta tatctctaca acaacatgat tgcaaaaatc 720
caagaagatg attttaataa cctcaaccaa ttacaaattc ttgacctaag tggaaattgc 780
cctcgttgtt ataatgcccc atttccttgt gcgccgtgta aaaataattc tcccctacag 840
atccctgtaa atgcttttga tgcgctgaca gaattaaaag ttttacgtct acacagtaac 900
tctcttcagc atgtgccccc aagatggttt aagaacatca acaaactcca ggaactggat 960
ctgtcccaaa acttcttggc caaagaaatt ggggatgcta aatttctgca ttttctcccc 1020
agcctcatcc aattggatct gtctttcaat tttgaacttc aggtctatcg tgcatctatg 1080
aatctatcac aagcattttc ttcactgaaa agcctgaaaa ttctgcggat cagaggatat 1140
gtctttaaag agttgaaaag ctttaacctc tcgccattac ataatcttca aaatcttgaa 1200
gttcttgatc ttggcactaa ctttataaaa attgctaacc tcagcatgtt taaacaattt 1260
aaaagactga aagtcataga tctttcagtg aataaaatat caccttcagg agattcaagt 1320
gaagttggct tctgctcaaa tgccagaact tctgtagaaa gttatgaacc ccaggtcctg 1380
gaacaattac attatttcag atatgataag tatgcaagga gttgcagatt caaaaacaaa 1440
gaggcttctt tcatgtctgt taatgaaagc tgctacaagt atgggcagac cttggatcta 1500
agtaaaaata gtatattttt tgtcaagtcc tctgattttc agcatctttc tttcctcaaa 1560
tgcctgaatc tgtcaggaaa tctcattagc caaactctta atggcagtga attccaacct 1620
ttagcagagc tgagatattt ggacttctcc aacaaccggc ttgatttact ccattcaaca 1680
gcatttgaag agcttcacaa actggaagtt ctggatataa gcagtaatag ccattatttt 1740
caatcagaag gaattactca tatgctaaac tttaccaaga acctaaaggt tctgcagaaa 1800
ctgatgatga acgacaatga catctcttcc tccaccagca ggaccatgga gagtgagtct 1860
cttagaactc tggaattcag aggaaatcac ttagatgttt tatggagaga aggtgataac 1920
agatacttac aattattcaa gaatctgcta aaattagagg aattagacat ctctaaaaat 1980
tccctaagtt tcttgccttc tggagttttt gatggtatgc ctccaaatct aaagaatctc 2040
tctttggcca aaaatgggct caaatctttc agttggaaga aactccagtg tctaaagaac 2100
ctggaaactt tggacctcag ccacaaccaa ctgaccactg tccctgagag attatccaac 2160
tgttccagaa gcctcaagaa tctgattctt aagaataatc aaatcaggag tctgacgaag 2220
tattttctac aagatgcctt ccagttgcga tatctggatc tcagctcaaa taaaatccag 2280
atgatccaaa agaccagctt cccagaaaat gtcctcaaca atctgaagat gttgcttttg 2340
catcataatc ggtttctgtg cacctgtgat gctgtgtggt ttgtctggtg ggttaaccat 2400
acggaggtga ctattcctta cctggccaca gatgtgactt gtgtggggcc aggagcacac 2460
aagggccaaa gtgtgatctc cctggatctg tacacctgtg agttagatct gactaacctg 2520
attctgttct cactttccat atctgtatct ctctttctca tggtgatgat gacagcaagt 2580
cacctctatt tctgggatgt gtggtatatt taccatttct gtaaggccaa gataaagggg 2640
tatcagcgtc taatatcacc agactgttgc tatgatgctt ttattgtgta tgacactaaa 2700
gacccagctg tgaccgagtg ggttttggct gagctggtgg ccaaactgga agacccaaga 2760
gagaaacatt ttaatttatg tctcgaggaa agggactggt taccagggca gccagttctg 2820
gaaaaccttt cccagagcat acagcttagc aaaaagacag tgtttgtgat gacagacaag 2880
tatgcaaaga ctgaaaattt taagatagca ttttacttgt cccatcagag gctcatggat 2940
gaaaaagttg atgtgattat cttgatattt cttgagaagc cctttcagaa gtccaagttc 3000
ctccagctcc ggaaaaggct ctgtgggagt tctgtccttg agtggccaac aaacccgcaa 3060
gctcacccat acttctggca gtgtctaaag aacgccctgg ccacagacaa tcatgtggcc 3120
tatagtcagg tgttcaagga aacggtctag 3150
<210> 9
<211> 1041
<212> PRT
<213> 人类（Homo sapiens）
<400> 9
Met Glu Asn Met Phe Leu Gln Ser Ser Met Leu Thr Cys Ile Phe Leu
1 5 10 15
Leu Ile Ser Gly Ser Cys Glu Leu Cys Ala Glu Glu Asn Phe Ser Arg
20 25 30
Ser Tyr Pro Cys Asp Glu Lys Lys Gln Asn Asp Ser Val Ile Ala Glu
35 40 45
Cys Ser Asn Arg Arg Leu Gln Glu Val Pro Gln Thr Val Gly Lys Tyr
50 55 60
Val Thr Glu Leu Asp Leu Ser Asp Asn Phe Ile Thr His Ile Thr Asn
65 70 75 80
Glu Ser Phe Gln Gly Leu Gln Asn Leu Thr Lys Ile Asn Leu Asn His
85 90 95
Asn Pro Asn Val Gln His Gln Asn Gly Asn Pro Gly Ile Gln Ser Asn
100 105 110
Gly Leu Asn Ile Thr Asp Gly Ala Phe Leu Asn Leu Lys Asn Leu Arg
115 120 125
Glu Leu Leu Leu Glu Asp Asn Gln Leu Pro Gln Ile Pro Ser Gly Leu
130 135 140
Pro Glu Ser Leu Thr Glu Leu Ser Leu Ile Gln Asn Asn Ile Tyr Asn
145 150 155 160
Ile Thr Lys Glu Gly Ile Ser Arg Leu Ile Asn Leu Lys Asn Leu Tyr
165 170 175
Leu Ala Trp Asn Cys Tyr Phe Asn Lys Val Cys Glu Lys Thr Asn Ile
180 185 190
Glu Asp Gly Val Phe Glu Thr Leu Thr Asn Leu Glu Leu Leu Ser Leu
195 200 205
Ser Phe Asn Ser Leu Ser His Val Pro Pro Lys Leu Pro Ser Ser Leu
210 215 220
Arg Lys Leu Phe Leu Ser Asn Thr Gln Ile Lys Tyr Ile Ser Glu Glu
225 230 235 240
Asp Phe Lys Gly Leu Ile Asn Leu Thr Leu Leu Asp Leu Ser Gly Asn
245 250 255
Cys Pro Arg Cys Phe Asn Ala Pro Phe Pro Cys Val Pro Cys Asp Gly
260 265 270
Gly Ala Ser Ile Asn Ile Asp Arg Phe Ala Phe Gln Asn Leu Thr Gln
275 280 285
Leu Arg Tyr Leu Asn Leu Ser Ser Thr Ser Leu Arg Lys Ile Asn Ala
290 295 300
Ala Trp Phe Lys Asn Met Pro His Leu Lys Val Leu Asp Leu Glu Phe
305 310 315 320
Asn Tyr Leu Val Gly Glu Ile Ala Ser Gly Ala Phe Leu Thr Met Leu
325 330 335
Pro Arg Leu Glu Ile Leu Asp Leu Ser Phe Asn Tyr Ile Lys Gly Ser
340 345 350
Tyr Pro Gln His Ile Asn Ile Ser Arg Asn Phe Ser Lys Leu Leu Ser
355 360 365
Leu Arg Ala Leu His Leu Arg Gly Tyr Val Phe Gln Glu Leu Arg Glu
370 375 380
Asp Asp Phe Gln Pro Leu Met Gln Leu Pro Asn Leu Ser Thr Ile Asn
385 390 395 400
Leu Gly Ile Asn Phe Ile Lys Gln Ile Asp Phe Lys Leu Phe Gln Asn
405 410 415
Phe Ser Asn Leu Glu Ile Ile Tyr Leu Ser Glu Asn Arg Ile Ser Pro
420 425 430
Leu Val Lys Asp Thr Arg Gln Ser Tyr Ala Asn Ser Ser Ser Phe Gln
435 440 445
Arg His Ile Arg Lys Arg Arg Ser Thr Asp Phe Glu Phe Asp Pro His
450 455 460
Ser Asn Phe Tyr His Phe Thr Arg Pro Leu Ile Lys Pro Gln Cys Ala
465 470 475 480
Ala Tyr Gly Lys Ala Leu Asp Leu Ser Leu Asn Ser Ile Phe Phe Ile
485 490 495
Gly Pro Asn Gln Phe Glu Asn Leu Pro Asp Ile Ala Cys Leu Asn Leu
500 505 510
Ser Ala Asn Ser Asn Ala Gln Val Leu Ser Gly Thr Glu Phe Ser Ala
515 520 525
Ile Pro His Val Lys Tyr Leu Asp Leu Thr Asn Asn Arg Leu Asp Phe
530 535 540
Asp Asn Ala Ser Ala Leu Thr Glu Leu Ser Asp Leu Glu Val Leu Asp
545 550 555 560
Leu Ser Tyr Asn Ser His Tyr Phe Arg Ile Ala Gly Val Thr His His
565 570 575
Leu Glu Phe Ile Gln Asn Phe Thr Asn Leu Lys Val Leu Asn Leu Ser
580 585 590
His Asn Asn Ile Tyr Thr Leu Thr Asp Lys Tyr Asn Leu Glu Ser Lys
595 600 605
Ser Leu Val Glu Leu Val Phe Ser Gly Asn Arg Leu Asp Ile Leu Trp
610 615 620
Asn Asp Asp Asp Asn Arg Tyr Ile Ser Ile Phe Lys Gly Leu Lys Asn
625 630 635 640
Leu Thr Arg Leu Asp Leu Ser Leu Asn Arg Leu Lys His Ile Pro Asn
645 650 655
Glu Ala Phe Leu Asn Leu Pro Ala Ser Leu Thr Glu Leu His Ile Asn
660 665 670
Asp Asn Met Leu Lys Phe Phe Asn Trp Thr Leu Leu Gln Gln Phe Pro
675 680 685
Arg Leu Glu Leu Leu Asp Leu Arg Gly Asn Lys Leu Leu Phe Leu Thr
690 695 700
Asp Ser Leu Ser Asp Phe Thr Ser Ser Leu Arg Thr Leu Leu Leu Ser
705 710 715 720
His Asn Arg Ile Ser His Leu Pro Ser Gly Phe Leu Ser Glu Val Ser
725 730 735
Ser Leu Lys His Leu Asp Leu Ser Ser Asn Leu Leu Lys Thr Ile Asn
740 745 750
Lys Ser Ala Leu Glu Thr Lys Thr Thr Thr Lys Leu Ser Met Leu Glu
755 760 765
Leu His Gly Asn Pro Phe Glu Cys Thr Cys Asp Ile Gly Asp Phe Arg
770 775 780
Arg Trp Met Asp Glu His Leu Asn Val Lys Ile Pro Arg Leu Val Asp
785 790 795 800
Val Ile Cys Ala Ser Pro Gly Asp Gln Arg Gly Lys Ser Ile Val Ser
805 810 815
Leu Glu Leu Thr Thr Cys Val Ser Asp Val Thr Ala Val Ile Leu Phe
820 825 830
Phe Phe Thr Phe Phe Ile Thr Thr Met Val Met Leu Ala Ala Leu Ala
835 840 845
His His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu
850 855 860
Ala Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr
865 870 875 880
Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp
885 890 895
Val Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn
900 905 910
Val Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile
915 920 925
Ile Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe
930 935 940
Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe
945 950 955 960
Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile
965 970 975
Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu
980 985 990
Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro
995 1000 1005
Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu
1010 1015 1020
Thr Glu Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile
1025 1030 1035
Lys Gln Tyr
1040
<210> 10
<211> 3126
<212> DNA
<213> 人类（Homo sapiens）
<400> 10
atggaaaaca tgttccttca gtcgtcaatg ctgacctgca ttttcctgct aatatctggt 60
tcctgtgagt tatgcgccga agaaaatttt tctagaagct atccttgtga tgagaaaaag 120
caaaatgact cagttattgc agagtgcagc aatcgtcgac tacaggaagt tccccaaacg 180
gtgggcaaat atgtgacaga actagacctg tctgataatt tcatcacaca cataacgaat 240
gaatcatttc aagggctgca aaatctcact aaaataaatc taaaccacaa ccccaatgta 300
cagcaccaga acggaaatcc cggtatacaa tcaaatggct tgaatatcac agacggggca 360
ttcctcaacc taaaaaacct aagggagtta ctgcttgaag acaaccagtt accccaaata 420
ccctctggtt tgccagagtc tttgacagaa cttagtctaa ttcaaaacaa tatatacaac 480
ataactaaag agggcatttc aagacttata aacttgaaaa atctctattt ggcctggaac 540
tgctatttta acaaagtttg cgagaaaact aacatagaag atggagtatt tgaaacgctg 600
acaaatttgg agttgctatc actatctttc aattctcttt cacacgtgcc acccaaactg 660
ccaagctccc tacgcaaact ttttctgagc aacacccaga tcaaatacat tagtgaagaa 720
gatttcaagg gattgataaa tttaacatta ctagatttaa gcgggaactg tccgaggtgc 780
ttcaatgccc catttccatg cgtgccttgt gatggtggtg cttcaattaa tatagatcgt 840
tttgcttttc aaaacttgac ccaacttcga tacctaaacc tctctagcac ttccctcagg 900
aagattaatg ctgcctggtt taaaaatatg cctcatctga aggtgctgga tcttgaattc 960
aactatttag tgggagaaat agcctctggg gcatttttaa cgatgctgcc ccgcttagaa 1020
atacttgact tgtcttttaa ctatataaag gggagttatc cacagcatat taatatttcc 1080
agaaacttct ctaaactttt gtctctacgg gcattgcatt taagaggtta tgtgttccag 1140
gaactcagag aagatgattt ccagcccctg atgcagcttc caaacttatc gactatcaac 1200
ttgggtatta attttattaa gcaaatcgat ttcaaacttt tccaaaattt ctccaatctg 1260
gaaattattt acttgtcaga aaacagaata tcaccgttgg taaaagatac ccggcagagt 1320
tatgcaaata gttcctcttt tcaacgtcat atccggaaac gacgctcaac agattttgag 1380
tttgacccac attcgaactt ttatcatttc acccgtcctt taataaagcc acaatgtgct 1440
gcttatggaa aagccttaga tttaagcctc aacagtattt tcttcattgg gccaaaccaa 1500
tttgaaaatc ttcctgacat tgcctgttta aatctgtctg caaatagcaa tgctcaagtg 1560
ttaagtggaa ctgaattttc agccattcct catgtcaaat atttggattt gacaaacaat 1620
agactagact ttgataatgc tagtgctctt actgaattgt ccgacttgga agttctagat 1680
ctcagctata attcacacta tttcagaata gcaggcgtaa cacatcatct agaatttatt 1740
caaaatttca caaatctaaa agttttaaac ttgagccaca acaacattta tactttaaca 1800
gataagtata acctggaaag caagtccctg gtagaattag ttttcagtgg caatcgcctt 1860
gacattttgt ggaatgatga tgacaacagg tatatctcca ttttcaaagg tctcaagaat 1920
ctgacacgtc tggatttatc ccttaatagg ctgaagcaca tcccaaatga agcattcctt 1980
aatttgccag cgagtctcac tgaactacat ataaatgata atatgttaaa gttttttaac 2040
tggacattac tccagcagtt tcctcgtctc gagttgcttg acttacgtgg aaacaaacta 2100
ctctttttaa ctgatagcct atctgacttt acatcttccc ttcggacact gctgctgagt 2160
cataacagga tttcccacct accctctggc tttctttctg aagtcagtag tctgaagcac 2220
ctcgatttaa gttccaatct gctaaaaaca atcaacaaat ccgcacttga aactaagacc 2280
accaccaaat tatctatgtt ggaactacac ggaaacccct ttgaatgcac ctgtgacatt 2340
ggagatttcc gaagatggat ggatgaacat ctgaatgtca aaattcccag actggtagat 2400
gtcatttgtg ccagtcctgg ggatcaaaga gggaagagta ttgtgagtct ggagctaaca 2460
acttgtgttt cagatgtcac tgcagtgata ttatttttct tcacgttctt tatcaccacc 2520
atggttatgt tggctgccct ggctcaccat ttgttttact gggatgtttg gtttatatat 2580
aatgtgtgtt tagctaaggt aaaaggctac aggtctcttt ccacatccca aactttctat 2640
gatgcttaca tttcttatga caccaaagat gcctctgtta ctgactgggt gataaatgag 2700
ctgcgctacc accttgaaga gagccgagac aaaaacgttc tcctttgtct agaggagagg 2760
gattgggacc cgggattggc catcatcgac aacctcatgc agagcatcaa ccaaagcaag 2820
aaaacagtat ttgttttaac caaaaaatat gcaaaaagct ggaactttaa aacagctttt 2880
tacttggctt tgcagaggct aatggatgag aacatggatg tgattatatt tatcctgctg 2940
gagccagtgt tacagcattc tcagtatttg aggctacggc agcggatctg taagagctcc 3000
atcctccagt ggcctgacaa cccgaaggca gaaggcttgt tttggcaaac tctgagaaat 3060
gtggtcttga ctgaaaatga ttcacggtat aacaatatgt atgtcgattc cattaagcaa 3120
tactaa 3126
<210> 11
<211> 1032
<212> PRT
<213> 人类（Homo sapiens）
<400> 11
Met Gly Phe Cys Arg Ser Ala Leu His Pro Leu Ser Leu Leu Val Gln
1 5 10 15
Ala Ile Met Leu Ala Met Thr Leu Ala Leu Gly Thr Leu Pro Ala Phe
20 25 30
Leu Pro Cys Glu Leu Gln Pro His Gly Leu Val Asn Cys Asn Trp Leu
35 40 45
Phe Leu Lys Ser Val Pro His Phe Ser Met Ala Ala Pro Arg Gly Asn
50 55 60
Val Thr Ser Leu Ser Leu Ser Ser Asn Arg Ile His His Leu His Asp
65 70 75 80
Ser Asp Phe Ala His Leu Pro Ser Leu Arg His Leu Asn Leu Lys Trp
85 90 95
Asn Cys Pro Pro Val Gly Leu Ser Pro Met His Phe Pro Cys His Met
100 105 110
Thr Ile Glu Pro Ser Thr Phe Leu Ala Val Pro Thr Leu Glu Glu Leu
115 120 125
Asn Leu Ser Tyr Asn Asn Ile Met Thr Val Pro Ala Leu Pro Lys Ser
130 135 140
Leu Ile Ser Leu Ser Leu Ser His Thr Asn Ile Leu Met Leu Asp Ser
145 150 155 160
Ala Ser Leu Ala Gly Leu His Ala Leu Arg Phe Leu Phe Met Asp Gly
165 170 175
Asn Cys Tyr Tyr Lys Asn Pro Cys Arg Gln Ala Leu Glu Val Ala Pro
180 185 190
Gly Ala Leu Leu Gly Leu Gly Asn Leu Thr His Leu Ser Leu Lys Tyr
195 200 205
Asn Asn Leu Thr Val Val Pro Arg Asn Leu Pro Ser Ser Leu Glu Tyr
210 215 220
Leu Leu Leu Ser Tyr Asn Arg Ile Val Lys Leu Ala Pro Glu Asp Leu
225 230 235 240
Ala Asn Leu Thr Ala Leu Arg Val Leu Asp Val Gly Gly Asn Cys Arg
245 250 255
Arg Cys Asp His Ala Pro Asn Pro Cys Met Glu Cys Pro Arg His Phe
260 265 270
Pro Gln Leu His Pro Asp Thr Phe Ser His Leu Ser Arg Leu Glu Gly
275 280 285
Leu Val Leu Lys Asp Ser Ser Leu Ser Trp Leu Asn Ala Ser Trp Phe
290 295 300
Arg Gly Leu Gly Asn Leu Arg Val Leu Asp Leu Ser Glu Asn Phe Leu
305 310 315 320
Tyr Lys Cys Ile Thr Lys Thr Lys Ala Phe Gln Gly Leu Thr Gln Leu
325 330 335
Arg Lys Leu Asn Leu Ser Phe Asn Tyr Gln Lys Arg Val Ser Phe Ala
340 345 350
His Leu Ser Leu Ala Pro Ser Phe Gly Ser Leu Val Ala Leu Lys Glu
355 360 365
Leu Asp Met His Gly Ile Phe Phe Arg Ser Leu Asp Glu Thr Thr Leu
370 375 380
Arg Pro Leu Ala Arg Leu Pro Met Leu Gln Thr Leu Arg Leu Gln Met
385 390 395 400
Asn Phe Ile Asn Gln Ala Gln Leu Gly Ile Phe Arg Ala Phe Pro Gly
405 410 415
Leu Arg Tyr Val Asp Leu Ser Asp Asn Arg Ile Ser Gly Ala Ser Glu
420 425 430
Leu Thr Ala Thr Met Gly Glu Ala Asp Gly Gly Glu Lys Val Trp Leu
435 440 445
Gln Pro Gly Asp Leu Ala Pro Ala Pro Val Asp Thr Pro Ser Ser Glu
450 455 460
Asp Phe Arg Pro Asn Cys Ser Thr Leu Asn Phe Thr Leu Asp Leu Ser
465 470 475 480
Arg Asn Asn Leu Val Thr Val Gln Pro Glu Met Phe Ala Gln Leu Ser
485 490 495
His Leu Gln Cys Leu Arg Leu Ser His Asn Cys Ile Ser Gln Ala Val
500 505 510
Asn Gly Ser Gln Phe Leu Pro Leu Thr Gly Leu Gln Val Leu Asp Leu
515 520 525
Ser His Asn Lys Leu Asp Leu Tyr His Glu His Ser Phe Thr Glu Leu
530 535 540
Pro Arg Leu Glu Ala Leu Asp Leu Ser Tyr Asn Ser Gln Pro Phe Gly
545 550 555 560
Met Gln Gly Val Gly His Asn Phe Ser Phe Val Ala His Leu Arg Thr
565 570 575
Leu Arg His Leu Ser Leu Ala His Asn Asn Ile His Ser Gln Val Ser
580 585 590
Gln Gln Leu Cys Ser Thr Ser Leu Arg Ala Leu Asp Phe Ser Gly Asn
595 600 605
Ala Leu Gly His Met Trp Ala Glu Gly Asp Leu Tyr Leu His Phe Phe
610 615 620
Gln Gly Leu Ser Gly Leu Ile Trp Leu Asp Leu Ser Gln Asn Arg Leu
625 630 635 640
His Thr Leu Leu Pro Gln Thr Leu Arg Asn Leu Pro Lys Ser Leu Gln
645 650 655
Val Leu Arg Leu Arg Asp Asn Tyr Leu Ala Phe Phe Lys Trp Trp Ser
660 665 670
Leu His Phe Leu Pro Lys Leu Glu Val Leu Asp Leu Ala Gly Asn Gln
675 680 685
Leu Lys Ala Leu Thr Asn Gly Ser Leu Pro Ala Gly Thr Arg Leu Arg
690 695 700
Arg Leu Asp Val Ser Cys Asn Ser Ile Ser Phe Val Ala Pro Gly Phe
705 710 715 720
Phe Ser Lys Ala Lys Glu Leu Arg Glu Leu Asn Leu Ser Ala Asn Ala
725 730 735
Leu Lys Thr Val Asp His Ser Trp Phe Gly Pro Leu Ala Ser Ala Leu
740 745 750
Gln Ile Leu Asp Val Ser Ala Asn Pro Leu His Cys Ala Cys Gly Ala
755 760 765
Ala Phe Met Asp Phe Leu Leu Glu Val Gln Ala Ala Val Pro Gly Leu
770 775 780
Pro Ser Arg Val Lys Cys Gly Ser Pro Gly Gln Leu Gln Gly Leu Ser
785 790 795 800
Ile Phe Ala Gln Asp Leu Arg Leu Cys Leu Asp Glu Ala Leu Ser Trp
805 810 815
Asp Cys Phe Ala Leu Ser Leu Leu Ala Val Ala Leu Gly Leu Gly Val
820 825 830
Pro Met Leu His His Leu Cys Gly Trp Asp Leu Trp Tyr Cys Phe His
835 840 845
Leu Cys Leu Ala Trp Leu Pro Trp Arg Gly Arg Gln Ser Gly Arg Asp
850 855 860
Glu Asp Ala Leu Pro Tyr Asp Ala Phe Val Val Phe Asp Lys Thr Gln
865 870 875 880
Ser Ala Val Ala Asp Trp Val Tyr Asn Glu Leu Arg Gly Gln Leu Glu
885 890 895
Glu Cys Arg Gly Arg Trp Ala Leu Arg Leu Cys Leu Glu Glu Arg Asp
900 905 910
Trp Leu Pro Gly Lys Thr Leu Phe Glu Asn Leu Trp Ala Ser Val Tyr
915 920 925
Gly Ser Arg Lys Thr Leu Phe Val Leu Ala His Thr Asp Arg Val Ser
930 935 940
Gly Leu Leu Arg Ala Ser Phe Leu Leu Ala Gln Gln Arg Leu Leu Glu
945 950 955 960
Asp Arg Lys Asp Val Val Val Leu Val Ile Leu Ser Pro Asp Gly Arg
965 970 975
Arg Ser Arg Tyr Val Arg Leu Arg Gln Arg Leu Cys Arg Gln Ser Val
980 985 990
Leu Leu Trp Pro His Gln Pro Ser Gly Gln Arg Ser Phe Trp Ala Gln
995 1000 1005
Leu Gly Met Ala Leu Thr Arg Asp Asn His His Phe Tyr Asn Arg
1010 1015 1020
Asn Phe Cys Gln Gly Pro Thr Ala Glu
1025 1030
<210> 12
<211> 3099
<212> DNA
<213> 人类（Homo sapiens）
<400> 12
atgggtttct gccgcagcgc cctgcacccg ctgtctctcc tggtgcaggc catcatgctg 60
gccatgaccc tggccctggg taccttgcct gccttcctac cctgtgagct ccagccccac 120
ggcctggtga actgcaactg gctgttcctg aagtctgtgc cccacttctc catggcagca 180
ccccgtggca atgtcaccag cctttccttg tcctccaacc gcatccacca cctccatgat 240
tctgactttg cccacctgcc cagcctgcgg catctcaacc tcaagtggaa ctgcccgccg 300
gttggcctca gccccatgca cttcccctgc cacatgacca tcgagcccag caccttcttg 360
gctgtgccca ccctggaaga gctaaacctg agctacaaca acatcatgac tgtgcctgcg 420
ctgcccaaat ccctcatatc cctgtccctc agccatacca acatcctgat gctagactct 480
gccagcctcg ccggcctgca tgccctgcgc ttcctattca tggacggcaa ctgttattac 540
aagaacccct gcaggcaggc actggaggtg gccccgggtg ccctccttgg cctgggcaac 600
ctcacccacc tgtcactcaa gtacaacaac ctcactgtgg tgccccgcaa cctgccttcc 660
agcctggagt atctgctgtt gtcctacaac cgcatcgtca aactggcgcc tgaggacctg 720
gccaatctga ccgccctgcg tgtgctcgat gtgggcggaa attgccgccg ctgcgaccac 780
gctcccaacc cctgcatgga gtgccctcgt cacttccccc agctacatcc cgataccttc 840
agccacctga gccgtcttga aggcctggtg ttgaaggaca gttctctctc ctggctgaat 900
gccagttggt tccgtgggct gggaaacctc cgagtgctgg acctgagtga gaacttcctc 960
tacaaatgca tcactaaaac caaggccttc cagggcctaa cacagctgcg caagcttaac 1020
ctgtccttca attaccaaaa gagggtgtcc tttgcccacc tgtctctggc cccttccttc 1080
gggagcctgg tcgccctgaa ggagctggac atgcacggca tcttcttccg ctcactcgat 1140
gagaccacgc tccggccact ggcccgcctg cccatgctcc agactctgcg tctgcagatg 1200
aacttcatca accaggccca gctcggcatc ttcagggcct tccctggcct gcgctacgtg 1260
gacctgtcgg acaaccgcat cagcggagct tcggagctga cagccaccat gggggaggca 1320
gatggagggg agaaggtctg gctgcagcct ggggaccttg ctccggcccc agtggacact 1380
cccagctctg aagacttcag gcccaactgc agcaccctca acttcacctt ggatctgtca 1440
cggaacaacc tggtgaccgt gcagccggag atgtttgccc agctctcgca cctgcagtgc 1500
ctgcgcctga gccacaactg catctcgcag gcagtcaatg gctcccagtt cctgccgctg 1560
accggtctgc aggtgctaga cctgtcccac aataagctgg acctctacca cgagcactca 1620
ttcacggagc taccgcgact ggaggccctg gacctcagct acaacagcca gccctttggc 1680
atgcagggcg tgggccacaa cttcagcttc gtggctcacc tgcgcaccct gcgccacctc 1740
agcctggccc acaacaacat ccacagccaa gtgtcccagc agctctgcag tacgtcgctg 1800
cgggccctgg acttcagcgg caatgcactg ggccatatgt gggccgaggg agacctctat 1860
ctgcacttct tccaaggcct gagcggtttg atctggctgg acttgtccca gaaccgcctg 1920
cacaccctcc tgccccaaac cctgcgcaac ctccccaaga gcctacaggt gctgcgtctc 1980
cgtgacaatt acctggcctt ctttaagtgg tggagcctcc acttcctgcc caaactggaa 2040
gtcctcgacc tggcaggaaa ccagctgaag gccctgacca atggcagcct gcctgctggc 2100
acccggctcc ggaggctgga tgtcagctgc aacagcatca gcttcgtggc ccccggcttc 2160
ttttccaagg ccaaggagct gcgagagctc aaccttagcg ccaacgccct caagacagtg 2220
gaccactcct ggtttgggcc cctggcgagt gccctgcaaa tactagatgt aagcgccaac 2280
cctctgcact gcgcctgtgg ggcggccttt atggacttcc tgctggaggt gcaggctgcc 2340
gtgcccggtc tgcccagccg ggtgaagtgt ggcagtccgg gccagctcca gggcctcagc 2400
atctttgcac aggacctgcg cctctgcctg gatgaggccc tctcctggga ctgtttcgcc 2460
ctctcgctgc tggctgtggc tctgggcctg ggtgtgccca tgctgcatca cctctgtggc 2520
tgggacctct ggtactgctt ccacctgtgc ctggcctggc ttccctggcg ggggcggcaa 2580
agtgggcgag atgaggatgc cctgccctac gatgccttcg tggtcttcga caaaacgcag 2640
agcgcagtgg cagactgggt gtacaacgag cttcgggggc agctggagga gtgccgtggg 2700
cgctgggcac tccgcctgtg cctggaggaa cgcgactggc tgcctggcaa aaccctcttt 2760
gagaacctgt gggcctcggt ctatggcagc cgcaagacgc tgtttgtgct ggcccacacg 2820
gaccgggtca gtggtctctt gcgcgccagc ttcctgctgg cccagcagcg cctgctggag 2880
gaccgcaagg acgtcgtggt gctggtgatc ctgagccctg acggccgccg ctcccgctac 2940
gtgcggctgc gccagcgcct ctgccgccag agtgtcctcc tctggcccca ccagcccagt 3000
ggtcagcgca gcttctgggc ccagctgggc atggccctga ccagggacaa ccaccacttc 3060
tataaccgga acttctgcca gggacccacg gccgaatag 3099
<210> 13
<211> 317
<212> PRT
<213> 人类（Homo sapiens）
<400> 13
Met Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu
1 5 10 15
Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp
20 25 30
Ser Gln Ala Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro
35 40 45
Trp Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly
50 55 60
Ala Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn
65 70 75 80
Leu Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn
85 90 95
Asn Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser
100 105 110
Asp Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr
115 120 125
Pro His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His
130 135 140
Ser Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly
145 150 155 160
Lys Ser Gln Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile Pro Gln
165 170 175
Ala Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly
180 185 190
Tyr Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro
195 200 205
Ser Met Gly Ser Ser Ser Pro Met Gly Ile Ile Val Ala Val Val Ile
210 215 220
Ala Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr
225 230 235 240
Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala
245 250 255
Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg
260 265 270
Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr
275 280 285
Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr
290 295 300
Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn
305 310 315
<210> 14
<211> 954
<212> DNA
<213> 人类（Homo sapiens）
<400> 14
atgactatgg agacccaaat gtctcagaat gtatgtccca gaaacctgtg gctgcttcaa 60
ccattgacag ttttgctgct gctggcttct gcagacagtc aagctgcagc tcccccaaag 120
gctgtgctga aacttgagcc cccgtggatc aacgtgctcc aggaggactc tgtgactctg 180
acatgccagg gggctcgcag ccctgagagc gactccattc agtggttcca caatgggaat 240
ctcattccca cccacacgca gcccagctac aggttcaagg ccaacaacaa tgacagcggg 300
gagtacacgt gccagactgg ccagaccagc ctcagcgacc ctgtgcatct gactgtgctt 360
tccgaatggc tggtgctcca gacccctcac ctggagttcc aggagggaga aaccatcatg 420
ctgaggtgcc acagctggaa ggacaagcct ctggtcaagg tcacattctt ccagaatgga 480
aaatcccaga aattctccca tttggatccc accttctcca tcccacaagc aaaccacagt 540
cacagtggtg attaccactg cacaggaaac ataggctaca cgctgttctc atccaagcct 600
gtgaccatca ctgtccaagt gcccagcatg ggcagctctt caccaatggg gatcattgtg 660
gctgtggtca ttgcgactgc tgtagcagcc attgttgctg ctgtagtggc cttgatctac 720
tgcaggaaaa agcggatttc agccaattcc actgatcctg tgaaggctgc ccaatttgag 780
ccacctggac gtcaaatgat tgccatcaga aagagacaac ttgaagaaac caacaatgac 840
tatgaaacag ctgacggcgg ctacatgact ctgaacccca gggcacctac tgacgatgat 900
aaaaacatct acctgactct tcctcccaac gaccatgtca acagtaataa ctaa 954
<210> 15
<211> 254
<212> PRT
<213> 人类（Homo sapiens）
<400> 15
Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala
1 5 10 15
Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro
20 25 30
Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln
35 40 45
Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu
50 55 60
Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr
65 70 75 80
Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu
85 90 95
Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln
100 105 110
Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys
115 120 125
His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
130 135 140
Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro
145 150 155 160
Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe
165 170 175
Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
180 185 190
Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln
195 200 205
Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly
210 215 220
Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp
225 230 235 240
Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys
245 250
<210> 16
<211> 765
<212> DNA
<213> 人类（Homo sapiens）
<400> 16
atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60
gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120
gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180
tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240
gtcgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300
cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360
gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420
tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480
aaagccacac tcaaagacag cggctcctac ttctgcaggg ggctttttgg gagtaaaaat 540
gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600
tcattctttc cacctgggta ccaagtctct ttctgcttgg tgatggtact cctttttgca 660
gtggacacag gactatattt ctctgtgaag acaaacattc gaagctcaac aagagactgg 720
aaggaccata aatttaaatg gagaaaggac cctcaagaca aatga 765
<210> 17
<211> 86
<212> PRT
<213> 人类（Homo sapiens）
<400> 17
Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val Glu Gln Ala
1 5 10 15
Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala Ile Leu
20 25 30
Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile
35 40 45
Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val
50 55 60
Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys
65 70 75 80
His Glu Lys Pro Pro Gln
85
<210> 18
<211> 261
<212> DNA
<213> 人类（Homo sapiens）
<400> 18
atgattccag cagtggtctt gctcttactc cttttggttg aacaagcagc ggccctggga 60
gagcctcagc tctgctatat cctggatgcc atcctgtttc tgtatggaat tgtcctcacc 120
ctcctctact gtcgactgaa gatccaagtg cgaaaggcag ctataaccag ctatgagaaa 180
tcagatggtg tttacacggg cctgagcacc aggaaccagg agacttacga gactctgaag 240
catgagaaac caccacagta g 261
<210> 19
<211> 296
<212> PRT
<213> 人类（Homo sapiens）
<400> 19
Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser
1 5 10 15
Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg
20 25 30
Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr
35 40 45
Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu
50 55 60
Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly
65 70 75 80
Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu
85 90 95
Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Glu Glu Asp
100 105 110
Cys Gln Lys Tyr Ile Leu Lys Gln Gln Gln Glu Glu Ala Glu Lys Pro
115 120 125
Leu Gln Val Ala Ala Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu
130 135 140
Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly His Met Pro Glu Arg
145 150 155 160
Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile Gln Phe Val Gln
165 170 175
Glu Met Ile Arg Gln Leu Glu Gln Thr Asn Tyr Arg Leu Lys Leu Cys
180 185 190
Val Ser Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp Ser Ile Ala
195 200 205
Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val Val Val Ser
210 215 220
Asp Asp Tyr Leu Gln Ser Lys Glu Cys Asp Phe Gln Thr Lys Phe Ala
225 230 235 240
Leu Ser Leu Ser Pro Gly Ala His Gln Lys Arg Leu Ile Pro Ile Lys
245 250 255
Tyr Lys Ala Met Lys Lys Glu Phe Pro Ser Ile Leu Arg Phe Ile Thr
260 265 270
Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Ser Trp Phe Trp Thr Arg
275 280 285
Leu Ala Lys Ala Leu Ser Leu Pro
290 295
<210> 20
<211> 954
<212> DNA
<213> 人类（Homo sapiens）
<400> 20
atgcgacccg accgcgctga ggctccagga ccgcccgcca tggctgcagg aggtcccggc 60
gcggggtctg cggccccggt ctcctccaca tcctcccttc ccctggctgc tctcaacatg 120
cgagtgcggc gccgcctgtc tctgttcttg aacgtgcgga cacaggtggc ggccgactgg 180
accgcgctgg cggaggagat ggactttgag tacttggaga tccggcaact ggagacacaa 240
gcggacccca ctggcaggct gctggacgcc tggcagggac gccctggcgc ctctgtaggc 300
cgactgctcg agctgcttac caagctgggc cgcgacgacg tgctgctgga gctgggaccc 360
agcattgagg aggattgcca aaagtatatc ttgaagcagc agcaggagga ggctgagaag 420
cctttacagg tggccgctgt agacagcagt gtcccacgga cagcagagct ggcgggcatc 480
accacacttg atgaccccct ggggcatatg cctgagcgtt tcgatgcctt catctgctat 540
tgccccagcg acatccagtt tgtgcaggag atgatccggc aactggaaca gacaaactat 600
cgactgaagt tgtgtgtgtc tgaccgcgat gtcctgcctg gcacctgtgt ctggtctatt 660
gctagtgagc tcatcgaaaa gaggttggct agaaggccac ggggtgggtg ccgccggatg 720
gtggtggttg tctctgatga ttacctgcag agcaaggaat gtgacttcca gaccaaattt 780
gcactcagcc tctctccagg tgcccatcag aagcgactga tccccatcaa gtacaaggca 840
atgaagaaag agttccccag catcctgagg ttcatcactg tctgcgacta caccaacccc 900
tgcaccaaat cttggttctg gactcgcctt gccaaggcct tgtccctgcc ctga 954
<210> 21
<211> 153
<212> PRT
<213> 人类（Homo sapiens）
<400> 21
Met Ala Pro Ala Ala Ala Thr Gly Gly Ser Thr Leu Pro Ser Gly Phe
1 5 10 15
Ser Val Phe Thr Thr Leu Pro Asp Leu Leu Phe Ile Phe Glu Phe Ile
20 25 30
Phe Gly Gly Leu Val Trp Ile Leu Val Ala Ser Ser Leu Val Pro Trp
35 40 45
Pro Leu Val Gln Gly Trp Val Met Phe Val Ser Val Phe Cys Phe Val
50 55 60
Ala Thr Thr Thr Leu Ile Ile Leu Tyr Ile Ile Gly Ala His Gly Gly
65 70 75 80
Glu Thr Ser Trp Val Thr Leu Asp Ala Ala Tyr His Cys Thr Ala Ala
85 90 95
Leu Phe Tyr Leu Ser Ala Ser Val Leu Glu Ala Leu Ala Thr Ile Thr
100 105 110
Met Gln Asp Gly Phe Thr Tyr Arg His Tyr His Glu Asn Ile Ala Ala
115 120 125
Val Val Phe Ser Tyr Ile Ala Thr Leu Leu Tyr Val Val His Ala Val
130 135 140
Phe Ser Leu Ile Arg Trp Lys Ser Ser
145 150
<210> 22
<211> 462
<212> DNA
<213> 人类（Homo sapiens）
<400> 22
atggcccccg cagcggcgac ggggggcagc accctgccca gtggcttctc ggtcttcacc 60
accttgcccg acttgctctt catctttgag tttatcttcg ggggcctggt gtggatcctg 120
gtggcctcct ccctggtgcc ctggcccctg gtccagggct gggtgatgtt cgtgtctgtg 180
ttctgcttcg tggccaccac caccttgatc atcctgtaca taattggagc ccacggtgga 240
gagacttcct gggtcacctt ggacgcagcc taccactgca ccgctgccct cttttacctc 300
agcgcctcag tcctggaggc cctggccacc atcacgatgc aagacggctt cacctacagg 360
cactaccatg aaaacattgc tgccgtggtg ttctcctaca tagccactct gctctacgtg 420
gtccatgcgg tgttctcttt aatcagatgg aagtcttcat aa 462
<210> 23
<211> 712
<212> PRT
<213> 人类（Homo sapiens）
<400> 23
Met Ala Gly Gly Pro Gly Pro Gly Glu Pro Ala Ala Pro Gly Ala Gln
1 5 10 15
His Phe Leu Tyr Glu Val Pro Pro Trp Val Met Cys Arg Phe Tyr Lys
20 25 30
Val Met Asp Ala Leu Glu Pro Ala Asp Trp Cys Gln Phe Ala Ala Leu
35 40 45
Ile Val Arg Asp Gln Thr Glu Leu Arg Leu Cys Glu Arg Ser Gly Gln
50 55 60
Arg Thr Ala Ser Val Leu Trp Pro Trp Ile Asn Arg Asn Ala Arg Val
65 70 75 80
Ala Asp Leu Val His Ile Leu Thr His Leu Gln Leu Leu Arg Ala Arg
85 90 95
Asp Ile Ile Thr Ala Trp His Pro Pro Ala Pro Leu Pro Ser Pro Gly
100 105 110
Thr Thr Ala Pro Arg Pro Ser Ser Ile Pro Ala Pro Ala Glu Ala Glu
115 120 125
Ala Trp Ser Pro Arg Lys Leu Pro Ser Ser Ala Ser Thr Phe Leu Ser
130 135 140
Pro Ala Phe Pro Gly Ser Gln Thr His Ser Gly Pro Glu Leu Gly Leu
145 150 155 160
Val Pro Ser Pro Ala Ser Leu Trp Pro Pro Pro Pro Ser Pro Ala Pro
165 170 175
Ser Ser Thr Lys Pro Gly Pro Glu Ser Ser Val Ser Leu Leu Gln Gly
180 185 190
Ala Arg Pro Phe Pro Phe Cys Trp Pro Leu Cys Glu Ile Ser Arg Gly
195 200 205
Thr His Asn Phe Ser Glu Glu Leu Lys Ile Gly Glu Gly Gly Phe Gly
210 215 220
Cys Val Tyr Arg Ala Val Met Arg Asn Thr Val Tyr Ala Val Lys Arg
225 230 235 240
Leu Lys Glu Asn Ala Asp Leu Glu Trp Thr Ala Val Lys Gln Ser Phe
245 250 255
Leu Thr Glu Val Glu Gln Leu Ser Arg Phe Arg His Pro Asn Ile Val
260 265 270
Asp Phe Ala Gly Tyr Cys Ala Gln Asn Gly Phe Tyr Cys Leu Val Tyr
275 280 285
Gly Phe Leu Pro Asn Gly Ser Leu Glu Asp Arg Leu His Cys Gln Thr
290 295 300
Gln Ala Cys Pro Pro Leu Ser Trp Pro Gln Arg Leu Asp Ile Leu Leu
305 310 315 320
Gly Thr Ala Arg Ala Ile Gln Phe Leu His Gln Asp Ser Pro Ser Leu
325 330 335
Ile His Gly Asp Ile Lys Ser Ser Asn Val Leu Leu Asp Glu Arg Leu
340 345 350
Thr Pro Lys Leu Gly Asp Phe Gly Leu Ala Arg Phe Ser Arg Phe Ala
355 360 365
Gly Ser Ser Pro Ser Gln Ser Ser Met Val Ala Arg Thr Gln Thr Val
370 375 380
Arg Gly Thr Leu Ala Tyr Leu Pro Glu Glu Tyr Ile Lys Thr Gly Arg
385 390 395 400
Leu Ala Val Asp Thr Asp Thr Phe Ser Phe Gly Val Val Val Leu Glu
405 410 415
Thr Leu Ala Gly Gln Arg Ala Val Lys Thr His Gly Ala Arg Thr Lys
420 425 430
Tyr Leu Lys Asp Leu Val Glu Glu Glu Ala Glu Glu Ala Gly Val Ala
435 440 445
Leu Arg Ser Thr Gln Ser Thr Leu Gln Ala Gly Leu Ala Ala Asp Ala
450 455 460
Trp Ala Ala Pro Ile Ala Met Gln Ile Tyr Lys Lys His Leu Asp Pro
465 470 475 480
Arg Pro Gly Pro Cys Pro Pro Glu Leu Gly Leu Gly Leu Gly Gln Leu
485 490 495
Ala Cys Cys Cys Leu His Arg Arg Ala Lys Arg Arg Pro Pro Met Thr
500 505 510
Gln Val Tyr Glu Arg Leu Glu Lys Leu Gln Ala Val Val Ala Gly Val
515 520 525
Pro Gly His Ser Glu Ala Ala Ser Cys Ile Pro Pro Ser Pro Gln Glu
530 535 540
Asn Ser Tyr Val Ser Ser Thr Gly Arg Ala His Ser Gly Ala Ala Pro
545 550 555 560
Trp Gln Pro Leu Ala Ala Pro Ser Gly Ala Ser Ala Gln Ala Ala Glu
565 570 575
Gln Leu Gln Arg Gly Pro Asn Gln Pro Val Glu Ser Asp Glu Ser Leu
580 585 590
Gly Gly Leu Ser Ala Ala Leu Arg Ser Trp His Leu Thr Pro Ser Cys
595 600 605
Pro Leu Asp Pro Ala Pro Leu Arg Glu Ala Gly Cys Pro Gln Gly Asp
610 615 620
Thr Ala Gly Glu Ser Ser Trp Gly Ser Gly Pro Gly Ser Arg Pro Thr
625 630 635 640
Ala Val Glu Gly Leu Ala Leu Gly Ser Ser Ala Ser Ser Ser Ser Glu
645 650 655
Pro Pro Gln Ile Ile Ile Asn Pro Ala Arg Gln Lys Met Val Gln Lys
660 665 670
Leu Ala Leu Tyr Glu Asp Gly Ala Leu Asp Ser Leu Gln Leu Leu Ser
675 680 685
Ser Ser Ser Leu Pro Gly Leu Gly Leu Glu Gln Asp Arg Gln Gly Pro
690 695 700
Glu Glu Ser Asp Glu Phe Gln Ser
705 710
<210> 24
<211> 2139
<212> DNA
<213> 人类（Homo sapiens）
<400> 24
atggccgggg ggccgggccc gggggagccc gcagcccccg gcgcccagca cttcttgtac 60
gaggtgccgc cctgggtcat gtgccgcttc tacaaagtga tggacgccct ggagcccgcc 120
gactggtgcc agttcgccgc cctgatcgtg cgcgaccaga ccgagctgcg gctgtgcgag 180
cgctccgggc agcgcacggc cagcgtcctg tggccctgga tcaaccgcaa cgcccgtgtg 240
gccgacctcg tgcacatcct cacgcacctg cagctgctcc gtgcgcggga catcatcaca 300
gcctggcacc ctcccgcccc gcttccgtcc ccaggcacca ctgccccgag gcccagcagc 360
atccctgcac ccgccgaggc cgaggcctgg agcccccgga agttgccatc ctcagcctcc 420
accttcctct ccccagcttt tccaggctcc cagacccatt cagggcctga gctcggcctg 480
gtcccaagcc ctgcttccct gtggcctcca ccgccatctc cagccccttc ttctaccaag 540
ccaggcccag agagctcagt gtccctcctg cagggagccc gcccctttcc gttttgctgg 600
cccctctgtg agatttcccg gggcacccac aacttctcgg aggagctcaa gatcggggag 660
ggtggctttg ggtgcgtgta ccgggcggtg atgaggaaca cggtgtatgc tgtgaagagg 720
ctgaaggaga acgctgacct ggagtggact gcagtgaagc agagcttcct gaccgaggtg 780
gagcagctgt ccaggtttcg tcacccaaac attgtggact ttgctggcta ctgtgctcag 840
aacggcttct actgcctggt gtacggcttc ctgcccaacg gctccctgga ggaccgtctc 900
cactgccaga cccaggcctg cccacctctc tcctggcctc agcgactgga catccttctg 960
ggtacagccc gggcaattca gtttctacat caggacagcc ccagcctcat ccatggagac 1020
atcaagagtt ccaacgtcct tctggatgag aggctgacac ccaagctggg agactttggc 1080
ctggcccggt tcagccgctt tgccgggtcc agccccagcc agagcagcat ggtggcccgg 1140
acacagacag tgcggggcac cctggcctac ctgcccgagg agtacatcaa gacgggaagg 1200
ctggctgtgg acacggacac cttcagcttt ggggtggtag tgctagagac cttggctggt 1260
cagagggctg tgaagacgca cggtgccagg accaagtatc tgaaagacct ggtggaagag 1320
gaggctgagg aggctggagt ggctttgaga agcacccaga gcacactgca agcaggtctg 1380
gctgcagatg cctgggctgc tcccatcgcc atgcagatct acaagaagca cctggacccc 1440
aggcccgggc cctgcccacc tgagctgggc ctgggcctgg gccagctggc ctgctgctgc 1500
ctgcaccgcc gggccaaaag gaggcctcct atgacccagg tgtacgagag gctagagaag 1560
ctgcaggcag tggtggcggg ggtgcccggg cattcggagg ccgccagctg catcccccct 1620
tccccgcagg agaactccta cgtgtccagc actggcagag cccacagtgg ggctgctcca 1680
tggcagcccc tggcagcgcc atcaggagcc agtgcccagg cagcagagca gctgcagaga 1740
ggccccaacc agcccgtgga gagtgacgag agcctaggcg gcctctctgc tgccctgcgc 1800
tcctggcact tgactccaag ctgccctctg gacccagcac ccctcaggga ggccggctgt 1860
cctcaggggg acacggcagg agaatcgagc tgggggagtg gcccaggatc ccggcccaca 1920
gccgtggaag gactggccct tggcagctct gcatcatcgt cgtcagagcc accgcagatt 1980
atcatcaacc ctgcccgaca gaagatggtc cagaagctgg ccctgtacga ggatggggcc 2040
ctggacagcc tgcagctgct gtcgtccagc tccctcccag gcttgggcct ggaacaggac 2100
aggcaggggc ccgaagaaag tgatgaattt cagagctga 2139
<210> 25
<211> 257
<212> PRT
<213> 人工序列
<220>
<223> ScFv 片段
<400> 25
Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser
1 5 10 15
Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser
20 25 30
Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys
35 40 45
Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg
50 55 60
Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile Ser Arg
65 70 75 80
Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser
85 90 95
Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Ser Gly
100 105 110
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ala
115 120 125
Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser Gly Ala
130 135 140
Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser
145 150 155 160
Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln Arg Pro
165 170 175
Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Asn
180 185 190
Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Thr Asp
195 200 205
Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu
210 215 220
Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His Trp Tyr
225 230 235 240
Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Val Asp
245 250 255
Leu
<210> 26
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 短铰链接头
<400> 26
Met Asn Lys Thr Ile
1 5
<210> 27
<211> 55
<212> PRT
<213> 人工序列
<220>
<223> 长铰链接头
<400> 27
Asn Asp Phe Ala Cys Thr Cys Glu His Gln Ser Phe Leu Gln Trp Ile
1 5 10 15
Lys Asp Gln Arg Gln Leu Leu Val Glu Val Glu Arg Met Glu Cys Ala
20 25 30
Thr Pro Ser Asp Lys Gln Gly Met Pro Val Leu Ser Leu Asn Ile Thr
35 40 45
Cys Gln Met Asn Lys Thr Ile
50 55
<210> 28
<211> 327
<212> PRT
<213> 人类（Homo sapiens）
<400> 28
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 29
<211> 12
<212> PRT
<213> 人类（Homo sapiens）
<400> 29
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
1 5 10
<210> 30
<211> 119
<212> PRT
<213> 人类（Homo sapiens）
<400> 30
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg
1 5 10 15
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
20 25 30
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
35 40 45
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
50 55 60
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
65 70 75 80
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
85 90 95
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
100 105 110
Leu Ser Leu Ser Leu Gly Lys
115
<210> 31
<211> 228
<212> PRT
<213> 人类（Homo sapiens）
<400> 31
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val
1 5 10 15
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
35 40 45
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
50 55 60
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr
65 70 75 80
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
85 90 95
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
100 105 110
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
130 135 140
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
145 150 155 160
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
180 185 190
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
195 200 205
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
210 215 220
Ser Leu Gly Lys
225
<210> 32
<211> 235
<212> PRT
<213> 人类（Homo sapiens）
<400> 32
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr
20 25 30
Trp Asn Leu Gly Glu Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser
35 40 45
Asn Pro Thr Ser Gly Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala
50 55 60
Ala Ser Pro Thr Phe Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala
65 70 75 80
Ala Glu Gly Leu Asp Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp
85 90 95
Thr Phe Val Leu Thr Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr
100 105 110
Tyr Phe Cys Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe
115 120 125
Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg
130 135 140
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
145 150 155 160
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
165 170 175
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
180 185 190
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His
195 200 205
Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser
210 215 220
Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val
225 230 235
<210> 33
<211> 68
<212> PRT
<213> 人工序列
<220>
<223> 突变的CD8铰链
<400> 33
Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val
1 5 10 15
Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
20 25 30
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
35 40 45
Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
50 55 60
Ala Ser Asp Ile
65
<210> 34
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> GS 接头
<400> 34
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 35
<211> 496
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1-ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS-接头
<220>
<221> MISC_FEATURE
<222> (291)..(313)
<223> TLR4-跨膜结构域
<220>
<221> MISC_FEATURE
<222> (314)..(496)
<223> TLR4-胞质结构域
<400> 35
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Ile Gly Val Ser Val Leu Ser Val Leu Val Val Ser Val Val
290 295 300
Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met Leu Leu Ala Gly
305 310 315 320
Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr Asp Ala Phe Val Ile
325 330 335
Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn Glu Leu Val Lys Asn
340 345 350
Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys Leu His Tyr Arg Asp
355 360 365
Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile Ile His Glu Gly Phe
370 375 380
His Lys Ser Arg Lys Val Ile Val Val Val Ser Gln His Phe Ile Gln
385 390 395 400
Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln Thr Trp Gln Phe
405 410 415
Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu Gln Lys Val Glu
420 425 430
Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg Leu Leu Ser Arg
435 440 445
Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly Arg His Ile Phe
450 455 460
Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly Lys Ser Trp Asn Pro
465 470 475 480
Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln Glu Ala Thr Ser Ile
485 490 495
<210> 36
<211> 500
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1-ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS-接头
<220>
<221> MISC_FEATURE
<222> (291)..(295)
<223> LRR 短铰链
<220>
<221> MISC_FEATURE
<222> (296)..(318)
<223> TLR4 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (319)..(500)
<223> TLR4 胞质结构域
<400> 36
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Met Asn Lys Thr Ile Ile Gly Val Ser Val Leu Ser Val Leu
290 295 300
Val Val Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu
305 310 315 320
Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr
325 330 335
Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn
340 345 350
Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys
355 360 365
Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile
370 375 380
Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val Val Ser
385 390 395 400
Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala
405 410 415
Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val
420 425 430
Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr
435 440 445
Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu
450 455 460
Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly
465 470 475 480
Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln
485 490 495
Glu Ala Thr Ser
500
<210> 37
<211> 551
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1-ScFv
<220>
<221> MISC_FEATURE
<222> (269)..(551)
<223> TLR4 胞质结构域
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS-接头
<220>
<221> MISC_FEATURE
<222> (291)..(345)
<223> LRR 长铰链
<220>
<221> MISC_FEATURE
<222> (346)..(368)
<223> TLR4 跨膜结构域
<400> 37
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Asn Asp Phe Ala Cys Thr Cys Glu His Gln Ser Phe Leu Gln
290 295 300
Trp Ile Lys Asp Gln Arg Gln Leu Leu Val Glu Val Glu Arg Met Glu
305 310 315 320
Cys Ala Thr Pro Ser Asp Lys Gln Gly Met Pro Val Leu Ser Leu Asn
325 330 335
Ile Thr Cys Gln Met Asn Lys Thr Ile Ile Gly Val Ser Val Leu Ser
340 345 350
Val Leu Val Val Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe
355 360 365
His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn
370 375 380
Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val
385 390 395 400
Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln
405 410 415
Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala
420 425 430
Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val
435 440 445
Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu
450 455 460
Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe
465 470 475 480
Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu
485 490 495
Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser
500 505 510
Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu
515 520 525
Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn
530 535 540
Trp Gln Glu Ala Thr Ser Ile
545 550
<210> 38
<211> 508
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(302)
<223> IgG4 短铰链
<220>
<221> MISC_FEATURE
<222> (303)..(325)
<223> TLR4 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (326)..(508)
<223> TLR4 胞质结构域
<400> 38
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ile Gly
290 295 300
Val Ser Val Leu Ser Val Leu Val Val Ser Val Val Ala Val Leu Val
305 310 315 320
Tyr Lys Phe Tyr Phe His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr
325 330 335
Gly Arg Gly Glu Asn Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln
340 345 350
Asp Glu Asp Trp Val Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly
355 360 365
Val Pro Pro Phe Gln Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly
370 375 380
Val Ala Ile Ala Ala Asn Ile Ile His Glu Gly Phe His Lys Ser Arg
385 390 395 400
Lys Val Ile Val Val Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys
405 410 415
Ile Phe Glu Tyr Glu Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg
420 425 430
Ala Gly Ile Ile Phe Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu
435 440 445
Arg Gln Gln Val Glu Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu
450 455 460
Glu Trp Glu Asp Ser Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu
465 470 475 480
Arg Lys Ala Leu Leu Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val
485 490 495
Gly Thr Gly Cys Asn Trp Gln Glu Ala Thr Ser Ile
500 505
<210> 39
<211> 615
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(409)
<223> IgG4 中等铰链
<220>
<221> MISC_FEATURE
<222> (410)..(432)
<223> TLR4 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (433)..(615)
<223> TLR4 胞质结构域
<400> 39
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln
290 295 300
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
305 310 315 320
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
325 330 335
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
340 345 350
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
355 360 365
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
370 375 380
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
385 390 395 400
Lys Ser Leu Ser Leu Ser Leu Gly Lys Ile Gly Val Ser Val Leu Ser
405 410 415
Val Leu Val Val Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe
420 425 430
His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn
435 440 445
Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val
450 455 460
Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln
465 470 475 480
Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala
485 490 495
Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val
500 505 510
Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu
515 520 525
Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe
530 535 540
Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu
545 550 555 560
Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser
565 570 575
Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu
580 585 590
Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn
595 600 605
Trp Gln Glu Ala Thr Ser Ile
610 615
<210> 40
<211> 724
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(518)
<223> IgG4 长铰链
<220>
<221> MISC_FEATURE
<222> (519)..(541)
<223> TLR4 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (542)..(724)
<223> TLR4 胞质结构域
<400> 40
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
290 295 300
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
305 310 315 320
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
325 330 335
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
340 345 350
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln
355 360 365
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
370 375 380
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
385 390 395 400
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
405 410 415
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
420 425 430
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
435 440 445
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
450 455 460
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
465 470 475 480
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
485 490 495
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
500 505 510
Ser Leu Ser Leu Gly Lys Ile Gly Val Ser Val Leu Ser Val Leu Val
515 520 525
Val Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met
530 535 540
Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr Asp
545 550 555 560
Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn Glu
565 570 575
Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys Leu
580 585 590
His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile Ile
595 600 605
His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val Val Ser Gln
610 615 620
His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln
625 630 635 640
Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu
645 650 655
Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg
660 665 670
Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly
675 680 685
Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly Lys
690 695 700
Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln Glu
705 710 715 720
Ala Thr Ser Ile
<210> 41
<211> 564
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(358)
<223> 突变的CD8铰链
<220>
<221> MISC_FEATURE
<222> (359)..(381)
<223> TLR4 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (382)..(564)
<223> TLR4 胞质结构域
<400> 41
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val
290 295 300
Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro
305 310 315 320
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
325 330 335
Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
340 345 350
Asp Phe Ala Ser Asp Ile Ile Gly Val Ser Val Leu Ser Val Leu Val
355 360 365
Val Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met
370 375 380
Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr Asp
385 390 395 400
Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn Glu
405 410 415
Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys Leu
420 425 430
His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile Ile
435 440 445
His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val Val Ser Gln
450 455 460
His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln
465 470 475 480
Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu
485 490 495
Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg
500 505 510
Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly
515 520 525
Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly Lys
530 535 540
Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln Glu
545 550 555 560
Ala Thr Ser Ile
<210> 42
<211> 564
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(358)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (359)..(381)
<223> TLR4 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (382)..(564)
<223> TLR4 胞质结构域
<400> 42
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val
290 295 300
Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro
305 310 315 320
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
325 330 335
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
340 345 350
Asp Phe Ala Cys Asp Ile Ile Gly Val Ser Val Leu Ser Val Leu Val
355 360 365
Val Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met
370 375 380
Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr Asp
385 390 395 400
Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn Glu
405 410 415
Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys Leu
420 425 430
His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile Ile
435 440 445
His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val Val Ser Gln
450 455 460
His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln
465 470 475 480
Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu
485 490 495
Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg
500 505 510
Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly
515 520 525
Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly Lys
530 535 540
Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln Glu
545 550 555 560
Ala Thr Ser Ile
<210> 43
<211> 378
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(311)
<223> FCGR3A 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (312)..(336)
<223> FCGR3A 胞质结构域
<220>
<221> MISC_FEATURE
<222> (337)..(378)
<223> FCER1G 胞质结构域
<400> 43
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp
290 295 300
Thr Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg
305 310 315 320
Asp Trp Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys
325 330 335
Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys
340 345 350
Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr
355 360 365
Glu Thr Leu Lys His Glu Lys Pro Pro Gln
370 375
<210> 44
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(358)
<223> 修饰的CD8a接头
<220>
<221> MISC_FEATURE
<222> (359)..(379)
<223> FCGR3A 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (380)..(404)
<223> FCGR3A 胞质结构域
<220>
<221> MISC_FEATURE
<222> (405)..(446)
<223> FCER1G 胞质结构域
<400> 44
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val
290 295 300
Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro
305 310 315 320
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
325 330 335
Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
340 345 350
Asp Phe Ala Ser Asp Ile Val Ser Phe Cys Leu Val Met Val Leu Leu
355 360 365
Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg
370 375 380
Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe Lys Trp Arg Lys Asp
385 390 395 400
Pro Gln Asp Lys Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile Thr
405 410 415
Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn
420 425 430
Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro Pro Gln
435 440 445
<210> 45
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(358)
<223> CD8铰链的部分
<220>
<221> MISC_FEATURE
<222> (359)..(379)
<223> FCGR3A 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (380)..(404)
<223> FCGR3A 胞质结构域
<220>
<221> MISC_FEATURE
<222> (405)..(446)
<223> FCER1G 胞质结构域
<400> 45
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val
290 295 300
Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro
305 310 315 320
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
325 330 335
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
340 345 350
Asp Phe Ala Cys Asp Ile Val Ser Phe Cys Leu Val Met Val Leu Leu
355 360 365
Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg
370 375 380
Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe Lys Trp Arg Lys Asp
385 390 395 400
Pro Gln Asp Lys Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile Thr
405 410 415
Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn
420 425 430
Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro Pro Gln
435 440 445
<210> 46
<211> 391
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(303)
<223> IgG4 短铰链
<220>
<221> MISC_FEATURE
<222> (304)..(324)
<223> FCGR3A 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (325)..(349)
<223> FCGR3A 胞质结构域
<220>
<221> MISC_FEATURE
<222> (350)..(391)
<223> FCER1G 胞质结构域
<400> 46
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ile Val
290 295 300
Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly Leu
305 310 315 320
Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys
325 330 335
Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys Arg Leu Lys
340 345 350
Ile Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly
355 360 365
Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu
370 375 380
Lys His Glu Lys Pro Pro Gln
385 390
<210> 47
<211> 497
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(409)
<223> IgG4 119 aa 铰链
<220>
<221> MISC_FEATURE
<222> (410)..(430)
<223> FCGR3A 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (431)..(455)
<223> FCGR3A 胞质结构域
<220>
<221> MISC_FEATURE
<222> (456)..(497)
<223> FCER1G 胞质结构域
<400> 47
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln
290 295 300
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
305 310 315 320
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
325 330 335
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
340 345 350
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
355 360 365
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
370 375 380
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
385 390 395 400
Lys Ser Leu Ser Leu Ser Leu Gly Lys Val Ser Phe Cys Leu Val Met
405 410 415
Val Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr
420 425 430
Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe Lys Trp
435 440 445
Arg Lys Asp Pro Gln Asp Lys Arg Leu Lys Ile Gln Val Arg Lys Ala
450 455 460
Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser
465 470 475 480
Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro Pro
485 490 495
Gln
<210> 48
<211> 607
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(519)
<223> IgG4 长铰链
<220>
<221> MISC_FEATURE
<222> (520)..(540)
<223> FCGR3A 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (541)..(565)
<223> FCGR3A 胞质结构域
<220>
<221> MISC_FEATURE
<222> (566)..(607)
<223> FCER1G 胞质结构域
<400> 48
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
290 295 300
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
305 310 315 320
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
325 330 335
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
340 345 350
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln
355 360 365
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
370 375 380
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
385 390 395 400
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
405 410 415
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
420 425 430
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
435 440 445
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
450 455 460
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
465 470 475 480
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
485 490 495
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
500 505 510
Ser Leu Ser Leu Gly Lys Ile Val Ser Phe Cys Leu Val Met Val Leu
515 520 525
Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile
530 535 540
Arg Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe Lys Trp Arg Lys
545 550 555 560
Asp Pro Gln Asp Lys Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile
565 570 575
Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg
580 585 590
Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro Pro Gln
595 600 605
<210> 49
<211> 390
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(312)
<223> FCGR2A 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (313)..(390)
<223> FCGR2A 胞质结构域
<400> 49
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Ile Ile Val Ala Val Val Ile Ala Thr Ala Val Ala Ala Ile
290 295 300
Val Ala Ala Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser
305 310 315 320
Ala Asn Ser Thr Asp Pro Val Lys Ala Ala Gln Phe Glu Pro Pro Gly
325 330 335
Arg Gln Met Ile Ala Ile Arg Lys Arg Gln Leu Glu Glu Thr Asn Asn
340 345 350
Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met Thr Leu Asn Pro Arg Ala
355 360 365
Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu Thr Leu Pro Pro Asn Asp
370 375 380
His Val Asn Ser Asn Asn
385 390
<210> 50
<211> 458
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(358)
<223> 修饰的CD8a铰链
<220>
<221> MISC_FEATURE
<222> (359)..(380)
<223> FCGR2A 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (381)..(458)
<223> FCGR2A 胞质结构域
<400> 50
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val
290 295 300
Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro
305 310 315 320
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
325 330 335
Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
340 345 350
Asp Phe Ala Ser Asp Ile Ile Ile Val Ala Val Val Ile Ala Thr Ala
355 360 365
Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys Arg Lys
370 375 380
Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala Gln Phe
385 390 395 400
Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln Leu Glu
405 410 415
Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met Thr Leu
420 425 430
Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu Thr Leu
435 440 445
Pro Pro Asn Asp His Val Asn Ser Asn Asn
450 455
<210> 51
<211> 458
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(358)
<223> CD8a接头的部分
<220>
<221> MISC_FEATURE
<222> (359)..(380)
<223> FCGR2A 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (381)..(458)
<223> FCGR2A 胞质结构域
<400> 51
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val
290 295 300
Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro
305 310 315 320
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
325 330 335
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
340 345 350
Asp Phe Ala Cys Asp Ile Ile Ile Val Ala Val Val Ile Ala Thr Ala
355 360 365
Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys Arg Lys
370 375 380
Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala Gln Phe
385 390 395 400
Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln Leu Glu
405 410 415
Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met Thr Leu
420 425 430
Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu Thr Leu
435 440 445
Pro Pro Asn Asp His Val Asn Ser Asn Asn
450 455
<210> 52
<211> 403
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(303)
<223> IgG4 短铰链
<220>
<221> MISC_FEATURE
<222> (304)..(325)
<223> FCGR2A 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (326)..(403)
<223> FCGR2A 胞质结构域
<400> 52
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ile Ile
290 295 300
Ile Val Ala Val Val Ile Ala Thr Ala Val Ala Ala Ile Val Ala Ala
305 310 315 320
Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser
325 330 335
Thr Asp Pro Val Lys Ala Ala Gln Phe Glu Pro Pro Gly Arg Gln Met
340 345 350
Ile Ala Ile Arg Lys Arg Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu
355 360 365
Thr Ala Asp Gly Gly Tyr Met Thr Leu Asn Pro Arg Ala Pro Thr Asp
370 375 380
Asp Asp Lys Asn Ile Tyr Leu Thr Leu Pro Pro Asn Asp His Val Asn
385 390 395 400
Ser Asn Asn
<210> 53
<211> 509
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK1 ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(409)
<223> IgG4 119 aa 铰链
<220>
<221> MISC_FEATURE
<222> (410)..(431)
<223> FCGR2A 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (432)..(509)
<223> FCGR2A 胞质结构域
<400> 53
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln
290 295 300
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
305 310 315 320
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
325 330 335
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
340 345 350
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
355 360 365
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
370 375 380
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
385 390 395 400
Lys Ser Leu Ser Leu Ser Leu Gly Lys Ile Ile Val Ala Val Val Ile
405 410 415
Ala Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr
420 425 430
Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala
435 440 445
Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg
450 455 460
Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr
465 470 475 480
Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr
485 490 495
Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn
500 505
<210> 54
<211> 619
<212> PRT
<213> 人工序列
<220>
<223> 嵌合抗原受体
<220>
<221> SIGNAL
<222> (1)..(18)
<223> 信号肽
<220>
<221> MISC_FEATURE
<222> (19)..(275)
<223> TK! ScFv
<220>
<221> MISC_FEATURE
<222> (276)..(290)
<223> GS 接头
<220>
<221> MISC_FEATURE
<222> (291)..(519)
<223> IgG4 长铰链
<220>
<221> MISC_FEATURE
<222> (520)..(541)
<223> FCGR2A 跨膜结构域
<220>
<221> MISC_FEATURE
<222> (542)..(619)
<223> FCGR2A 胞质结构域
<400> 54
Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr
165 170 175
Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln
180 185 190
Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
195 200 205
Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr
210 215 220
Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr
225 230 235 240
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His
245 250 255
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
260 265 270
Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
290 295 300
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
305 310 315 320
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
325 330 335
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
340 345 350
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln
355 360 365
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
370 375 380
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
385 390 395 400
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
405 410 415
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
420 425 430
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
435 440 445
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
450 455 460
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
465 470 475 480
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
485 490 495
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
500 505 510
Ser Leu Ser Leu Gly Lys Ile Ile Ile Val Ala Val Val Ile Ala Thr
515 520 525
Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys Arg
530 535 540
Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala Gln
545 550 555 560
Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln Leu
565 570 575
Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met Thr
580 585 590
Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu Thr
595 600 605
Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn
610 615
Claims (21)
1.一种嵌合受体，所述嵌合受体包括
细胞质结构域；
跨膜结构域；以及
细胞外结构域；
其中所述细胞质结构域包含受体的细胞质部分，所述细胞质部分在活化时使巨噬细胞极化；
其中包含所述细胞质部分的野生型蛋白质不包含所述细胞外结构域。
2.根据权利要求1所述的嵌合受体，其中配体与所述细胞外结构域的结合活化所述细胞质部分。
3.根据权利要求1所述的嵌合受体，其中所述细胞质部分被活化，所述部分将巨噬细胞极化为M1巨噬细胞。
4.根据权利要求1所述的嵌合受体，其中所述细胞质部分被活化，所述部分将巨噬细胞极化为M2巨噬细胞。
5.根据权利要求1所述的嵌合受体，其中所述细胞质部分包括来自toll样受体、骨髓分化初反应蛋白(MYD88)、toll样受体3 (TLR3)、toll样受体4 (TLR4)、toll样受体7 (TLR7)、toll样受体8 (TLR8)、toll样受体9 (TLR9)、髓磷脂和淋巴细胞蛋白(MAL)、白介素-1受体相关激酶1 (IRAK1)、低亲和力免疫球蛋白γ Fc区受体III-A (FCGR3A)、低亲和力免疫球蛋白γ Fc区受体II-a (FCGR2A)和高亲和力免疫球蛋白ε受体亚基γ (FCER1G)。
6.根据权利要求2所述的嵌合受体，其中所述配体选自胸苷激酶(TK1)、次黄嘌呤-鸟嘌呤磷酸核苷转移酶(HPRT)、受体酪氨酸激酶样孤儿受体1 (ROR1)、黏蛋白-16 (MUC-16)、表皮生长因子受体vIII (EGFRvIII)、间皮素、人表皮生长因子受体2 (HER2)、癌胚抗原(CEA)、B-细胞成熟抗原(BCMA)、磷脂酰肌醇蛋白聚糖3 (GPC3)、成纤维细胞活化蛋白(FAP)、促红细胞生成素产生肝细胞受体A2 (EphA2)、自然杀伤细胞家族2D (NKG2D)配体、双唾液酸神经节苷脂2 (GD2)、CD19、CD20、CD30、CD33、CD123、CD133、CD138和CD171。
7.根据权利要求1所述的嵌合受体，其中所述细胞外结构域是特异于选自如下的配体的抗体或片段：胸苷激酶(TK1)、次黄嘌呤-鸟嘌呤磷酸核苷转移酶(HPRT)、受体酪氨酸激酶样孤儿受体1 (ROR1)、黏蛋白-16 (MUC-16)、表皮生长因子受体vIII (EGFRvIII)、间皮素、人表皮生长因子受体2 (HER2)、癌胚抗原(CEA)、B-细胞成熟抗原(BCMA)、磷脂酰肌醇蛋白聚糖3 (GPC3)、成纤维细胞活化蛋白(FAP)、促红细胞生成素产生肝细胞受体A2 (EphA2)、自然杀伤细胞家族2D (NKG2D)配体、双唾液酸神经节苷脂2 (GD2)、CD19、CD20、CD30、CD33、CD123、CD133、CD138和CD171。
8.根据权利要求7所述的嵌合受体，其中所述抗体或其片段是ScFv片段。
9.根据权利要求1所述的嵌合受体，其中所述嵌合受体还包括所述跨膜结构域和所述细胞外结构域之间的接头。
10.根据权利要求9所述的嵌合受体，其中所述接头为GS接头。
11.根据权利要求1所述的嵌合受体，其中所述嵌合受体还包括所述跨膜结构域和所述细胞外结构域之间的铰链区。
12.根据权利要求9所述的嵌合受体，其中所述嵌合受体还包括所述跨膜结构域和所述接头之间的铰链区。
13.一种核酸，包括编码根据权利要求1所述的嵌合受体的多核苷酸。
14.根据权利要求13所述的核酸，还包括可操作地连接至所述多核苷酸的启动子。
15.一种载体，包括根据权利要求13所述的核酸。
16.根据权利要求15所述的载体，其中所述载体是慢病毒载体。
17.一种细胞，包括根据权利要求1所述的嵌合受体。
18.根据权利要求15所述的细胞，其中所述细胞是单核细胞或巨噬细胞。
19.一种细胞，包括根据权利要求13所述的核酸。
20.根据权利要求19所述的细胞，其中所述细胞是单核细胞或巨噬细胞。
21.一种使巨噬细胞极化的方法，所述方法包括：
使包括根据权利要求1所述的嵌合受体的巨噬细胞与所述嵌合受体的所述细胞外结构域的配体接触；以及
将所述配体结合到所述嵌合受体的所述细胞外结构域；
其中所述配体与所述嵌合受体的所述细胞外结构域的结合活化所述细胞质部分；并且
其中所述细胞质部分的活化使所述巨噬细胞极化。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109336980A (zh) * | 2017-07-27 | 2019-02-15 | 上海细胞治疗研究院 | 一种靶向Muc1的嵌合抗原受体修饰T细胞及其用途 |
| CN113980138A (zh) * | 2021-08-11 | 2022-01-28 | 卡瑞济(北京)生命科技有限公司 | EphA2嵌合抗原受体以及其用途 |
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| US10434153B1 (en) | 2015-05-20 | 2019-10-08 | Kim Leslie O'Neill | Use of car and bite technology coupled with an scFv from an antibody against human thymidine kinase 1 to specifically target tumors |
| US11352439B2 (en) | 2015-08-13 | 2022-06-07 | Kim Leslie O'Neill | Macrophage CAR (MOTO-CAR) in immunotherapy |
| RU2019112860A (ru) | 2016-09-27 | 2020-10-30 | Серо Терапьютикс, Инк. | Молекулы химерных интернализационных рецепторов |
| CA3073421A1 (en) | 2017-09-26 | 2019-04-04 | Daniel Mark COREY | Chimeric engulfment receptor molecules and methods of use |
| US20220332780A1 (en) | 2019-09-10 | 2022-10-20 | Obsidian Therapeutics, Inc. | Ca2-il15 fusion proteins for tunable regulation |
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| WO2017025944A2 (en) * | 2015-08-13 | 2017-02-16 | Brigham Young University | Macrophage car (moto-car) in imunotherapy |
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| FR2518755B1 (fr) | 1981-12-23 | 1986-04-11 | Pasteur Institut | Sonde contenant un acide nucleique modifie et reconnaissable par des anticorps specifiques et utilisation de cette sonde pour detecter et caracteriser une sequence d'adn homologue |
| ES2918501T3 (es) | 2013-12-19 | 2022-07-18 | Novartis Ag | Receptores de antígenos quiméricos de mesotelina humana y usos de los mismos |
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| WO2017019848A1 (en) * | 2015-07-28 | 2017-02-02 | The Trustees Of The University Of Pennsylvania | Modified monocytes/macrophage expressing chimeric antigen receptors and uses thereof |
| WO2017025944A2 (en) * | 2015-08-13 | 2017-02-16 | Brigham Young University | Macrophage car (moto-car) in imunotherapy |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109336980A (zh) * | 2017-07-27 | 2019-02-15 | 上海细胞治疗研究院 | 一种靶向Muc1的嵌合抗原受体修饰T细胞及其用途 |
| CN109336980B (zh) * | 2017-07-27 | 2022-04-12 | 上海细胞治疗研究院 | 一种靶向Muc1的嵌合抗原受体修饰T细胞及其用途 |
| CN113980138A (zh) * | 2021-08-11 | 2022-01-28 | 卡瑞济(北京)生命科技有限公司 | EphA2嵌合抗原受体以及其用途 |
| CN113980138B (zh) * | 2021-08-11 | 2023-08-11 | 卡瑞济(北京)生命科技有限公司 | EphA2嵌合抗原受体以及其用途 |
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| JP2020520252A (ja) | 2020-07-09 |
| EP3624834A1 (en) | 2020-03-25 |
| KR102508182B1 (ko) | 2023-03-09 |
| WO2018212770A1 (en) | 2018-11-22 |
| JP7164598B2 (ja) | 2022-11-01 |
| CA3062978A1 (en) | 2018-11-22 |
| AU2017414703A1 (en) | 2020-01-16 |
| JP2022189893A (ja) | 2022-12-22 |
| KR20200020708A (ko) | 2020-02-26 |
| ZA201908245B (en) | 2020-12-23 |
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