CN116419761A - 胞质溶胶递送 - Google Patents
胞质溶胶递送 Download PDFInfo
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- CN116419761A CN116419761A CN202180065007.XA CN202180065007A CN116419761A CN 116419761 A CN116419761 A CN 116419761A CN 202180065007 A CN202180065007 A CN 202180065007A CN 116419761 A CN116419761 A CN 116419761A
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Abstract
本发明涉及能够促进抗原的交叉呈递(XP)的嵌合受体及其实施方法。
Description
技术领域
本发明涉及用于将功能性大分子递送至胞质溶胶的方法。本发明提供了包含跨膜受体DNGR-1的部分的嵌合受体，以及表达所述嵌合受体的细胞，用于产生所述嵌合受体的方法，及其医学用途。还提供了功能性大分子，诸如生物聚合物，其结合并触发DNGR-1和/或本发明的嵌合受体，以及包含所述大分子的构建体。
背景技术
DNGR-1(也称为CLEC9A)是C型凝集素，其先前已被描述为1型常规树突细胞(cDC1)交叉呈递的关键介体(D Sancho等人Nature 200912；WO 2009/013484A120，两个文献通过引用明确并入本文)。交叉呈递(XP)是指由抗原呈递细胞(APC)进行的将MHC I类分子上的外源抗原呈递至细胞毒性T淋巴细胞(CTL)的过程。XP是诱导抗肿瘤和许多病毒的保护性CTL反应所必需的1-7。
巨噬细胞、单核细胞来源的树突细胞和其他髓样细胞类型以及非免疫细胞已广泛用于剖析XP中涉及的一些机制8、21、38。虽然这导致了认为XP在许多情况下涉及胞质溶胶途径(吞噬体至胞质溶胶转移；P2C)，但这些研究通常无法解释P2C如何发生，尤其是对于复杂底物(诸如死细胞)。实际上，据报道，由DNGR-1调节抗原自细胞尸体的交叉呈递的潜在实际机制仍然是个迷28。在体内，特别是在病毒感染和抗肿瘤免疫的情境下，XP在cDC1缺陷型Batf3-/-小鼠中被消除39，这意味着cDC1是非冗余交叉呈递APC。
相对少的论文集中在XP机制上，特别是在cDC1中7、30、36、40-42。典型地，认为濒死的病毒感染的细胞或肿瘤细胞是交叉呈递cDC1的外源抗原的来源。由这些细胞表达的DNGR-1检测暴露在死细胞碎片上的F-肌动蛋白/肌球蛋白复合物，并且促进尸体相关抗原的XP12 -16，从而将细胞死亡的检测与免疫偶联。
DNGR-1的胞质尾区中的含酪氨酸的hemITAM基序54允许在酪氨酸磷酸化后结合酪氨酸激酶Syk12。然而，到目前为止，对允许DNGR-1促进结合抗原的XP的后续细胞内信号传导事件(以及通常XP的潜在机制)仍然知之甚少。一些先前的公开内容通过产生嵌合蛋白检查了DNGR-1功能，所述嵌合蛋白包含融合至CD3ζ的细胞内结构域的DNGR-1的细胞外结构域12、20。在这种情境下，当二价配体结合DNGR-1细胞外结构域时，触发可检测的信号传导，导致由CD3ζ激活起始的可容易检测的信号传导级联。然而，对负责XP的细胞机制缺乏了解，抑制了对DNGR-1是否可以提供有用生物医学技术的基础的研究。
发明内容
诸位发明人揭示了DNGR-1介导交叉呈递(XP)的机制。这指引诸位发明人提供了本发明的嵌合受体，其提供了促进靶抗原(其不限于任何特定类型或类别的抗原)的XP的平台技术。出乎意料的是，这可以用于实现一系列细胞类型而不仅仅是专职抗原呈递细胞(APC)对靶抗原的XP。对XP途径的这种新认识还导致提供了一种将大分子(诸如生物聚合物)递送至胞质溶胶而不降解的方式。下面更详细地解释本发明及其潜在机制。
诸位发明人发现DNGR-1依赖型XP经由胞质溶胶途径进行。DNGR-1促进吞噬体破裂，这允许内化抗原释放到胞质溶胶中，在此它们经由常规MHC I类抗原加工途径进行加工和呈递。诸位发明人出乎意料地发现，内化抗原在被释放到胞质溶胶中之前基本上没有降解。使用嵌合受体，诸位发明人现已证明DNGR-1的胞质尾区是这个过程的关键介体，从而能够实现XP。诸位发明人表明这仅需要DNGR-1信号传导结构域，其募集和激活脾酪氨酸激酶(Syk)和NADPH氧化酶，导致脂质过氧化和吞噬体膜不稳定。值得注意的是，DNGR-1信号传导可以在异源细胞(包括非专职APC)中诱导吞噬体膜破裂和XP。这些结果表明吞噬体破裂与XP偶联，提供了外源抗原进入内源MHC I途径的简单机制。这种机制是非选择性的，并且不需要特定抗原转运蛋白。此外，吞噬体破裂的基本机构不限于DC，因此其他非APC具有交叉呈递外源抗原的潜力。然而，这种机构的有效参与需要专用的XP信号传导受体(诸如DNGR-1)。
因此，本发明提供了包含DNGR-1的胞质尾区的信号传导结构域的嵌合蛋白。本发明的嵌合蛋白可以促进XP。通过工程化细胞(不限于DC)表达嵌合蛋白，本发明提供了多种如下细胞，所述细胞可以识别和加工期望的靶标(不限于死细胞上的肌动蛋白/肌球蛋白II信号)，并将抗原从所述靶标呈递至CD8+T细胞，以引发对所述靶标的稳健免疫应答。本发明还提供了一种将生物聚合物(诸如蛋白质和核酸)递送至胞质溶胶而不降解的方式。
因此，在第一方面，本发明提供了一种将生物聚合物递送至细胞的胞质溶胶的方法，其中所述细胞表达包含细胞内结构域的跨膜蛋白，所述细胞内结构域包含来源于DNGR-1的胞质尾区的信号传导结构域的Syk结合序列，其中所述生物聚合物包含可以特异性地结合所述跨膜蛋白的细胞外部分的结合结构域，并且其中所述方法包括使所述细胞与所述生物聚合物接触以允许所述结合结构域与所述跨膜蛋白的细胞外部分结合，使得所述生物聚合物被内化并易位到所述胞质溶胶中而不在吞噬体中降解。优选地，所述生物聚合物进一步包含编码基因产物的核酸。
因此，本发明提供了一种将有效载荷递送至胞质溶胶的方式。所述有效载荷可以是生物聚合物本身和/或与所述生物聚合物共价或非共价缔合的另外部分，诸如编码基因产物的核酸。此类有效载荷的递送可用于研究、诊断和医疗应用。因此，本发明提供了本文定义的生物聚合物，其用于医学方法，诸如疫苗接种。体外方法特别适合于研究和/或诊断应用。
在一些实施方案中，所述生物聚合物包含第二结构域，所述第二结构域不直接结合所述跨膜蛋白的细胞外部分。此第二结构域与结合结构域共价接合，例如经由接头接合。在一些实施方案中，所述接头可以被细胞的胞质溶胶中存在的蛋白酶切割，允许所述第二结构域与胞质溶胶中的所述结合结构域解离。(所述第二生物聚合物结构域可以称为有效载荷。)所述第二结构域可以是编码基因产物的核酸。所述基因产物可以是如本文所述的促凋亡蛋白、酶或细胞毒性肽。
在一些实施方案中，(第一)生物聚合物(其包含结合结构域)与第二生物聚合物非共价缔合。此第二生物聚合物在本文中可以称为“第二结构域”，并且还可以称为有效载荷。非共价缔合可以例如通过将所述第一生物聚合物融合至抗生物素蛋白或链霉亲和素部分，并且将生物素“标签”共价连接至所述第二生物聚合物来实现，或反之亦然。所述抗生物素蛋白/链霉亲和素非共价结合所述生物素标签，从而使所述第一生物聚合物与所述第二生物聚合物彼此缔合。
因此，所述生物聚合物的结合结构域可以与第二生物聚合物结构域偶联，其中所述偶联可以是共价的或非共价的。
多肽生物聚合物、多核苷酸生物聚合物和多糖生物聚合物都在设想范围内。
所述生物聚合物可以是多肽，并且所述结合结构域可以包含抗体的抗原结合片段的至少一部分。例如，所述多肽可以包含抗体VH结构域；并且这可以与作为单独多肽提供的抗体VL结构域配对。可替代地，所述多肽可以包含抗体VL结构域；并且这可以与作为单独多肽提供的抗体VH结构域配对。在一些实施方案中，所述多肽可以包含抗体VH结构域和VL结构域两者，所述结构域存在于单条多肽链上，例如呈scFV形式。
所述生物聚合物可以是核酸，并且所述结合结构域可以是适配体。
对于所述结合结构域以及对于所述第二结构域，还设想了不同类别的生物聚合物的组合。例如，作为抗体(多肽)的结合结构域可以与非肽第二结构域偶联。不同类别的生物聚合物结构域的这种偶联可以共价或非共价地实现，例如如本文所述。本文所用的术语“生物聚合物”和递送所述生物聚合物的方法旨在涵盖生物聚合物配对，其中所述结合结构域是一种类型的生物聚合物并且所述有效载荷是另一种类型的生物聚合物。
所述生物聚合物可以包含多个结合结构域。例如，两种或更多种抗体和一种或多种第二生物聚合物结构域可以全部固定在单个载体颗粒(例如乳胶珠)上。
在所述生物聚合物包含多核苷酸的实施方案中，所述多核苷酸可以编码基因产物，并且能够在宿主细胞中表达所述基因产物。编码的基因产物可以是本文公开的多肽。在其他实施方案中，所述多核苷酸是或编码干扰所述细胞中另一种蛋白质的表达的RNA序列(例如RNAi分子，诸如siRNA)。所述多核苷酸可以是DNA。所述DNA可能能够激活STING途径。所述多核苷酸可以是RNA。所述RNA可能能够激活RIG-I和/或MDA5途径。
在一些实施方案中，所述第二结构域包含细胞毒素或促凋亡蛋白。例如，所述第二结构域可以包含细胞色素C、半胱天冬酶、类美登素、尾海兔素、澳瑞他汀药物类似物、念珠藻素、倍癌霉素衍生物、烯二炔抗生素或吡咯并苯二氮
。在一些实施方案中，所述第二结构域包含与细胞内靶标结合的抗体。在一些实施方案中，所述生物聚合物包含与抗DNGR-1抗体(第一结构域)缀合的肿瘤抗原(第二结构域)。在一些实施方案中，所述第二结构域不是肽抗原。换句话说，在一些实施方案中，所述第二结构域是完整蛋白质或蛋白质的完整结构域。在一些实施方案中，所述第二结构域包含多于50个氨基酸残基。
在一些实施方案中，所述第二结构域是编码细胞毒素或促凋亡蛋白的核酸。例如，所述第二结构域可以编码细胞色素C、半胱天冬酶、类美登素、尾海兔素、澳瑞他汀药物类似物、念珠藻素、倍癌霉素衍生物、烯二炔抗生素或吡咯并苯二氮
。在一些实施方案中，所述第二结构域编码与细胞内靶标结合的抗体。在一些实施方案中，所述生物聚合物编码与抗DNGR-1抗体(第一结构域)缀合的肿瘤抗原(第二结构域)。在一些实施方案中，所述第二结构域不是肽抗原。换句话说，在一些实施方案中，所述第二结构域是完整蛋白质或蛋白质的完整结构域。在一些实施方案中，所述第二结构域编码多于50个氨基酸残基。
所述生物聚合物的一个或多个结合结构域和/或第二结构域可以经由接头(例如肽接头)彼此连接。肽接头在本领域中是熟知的。技术人员可以从选择的接头序列中选择，如：GGGSGGG(SEQ ID NO:92)、GGGGSGGGGS(SEQ ID NO:93)、PGPG(SEQ ID NO:94)和GSAGSAAGSGEF(SEQ ID NO:95)。也可以使用这些接头序列的组合和重复。
所述细胞可以是肿瘤细胞。所述细胞可以是免疫细胞。在一些实施方案中，所述细胞表达包含NOX2的NADPH氧化酶。
由第一方面的细胞表达的跨膜蛋白可以是本发明的嵌合受体，如本文别处更详细描述的。第一方面的方法可以包括在细胞与生物聚合物接触之前在所述细胞中表达所述跨膜蛋白的步骤。所述表达方法可以包括将包含核酸的载体递送至所述细胞。所述载体可以是非病毒基因治疗载体，例如质粒(任选地与脂质体或递送剂复合)，或者所述载体可以是病毒基因治疗载体，例如病毒载体。慢病毒载体、腺病毒载体和基于腺病毒相关病毒(AAV)的载体都在设想范围内。
在其他实施方案中，由所述细胞表达的跨膜蛋白是DNGR-1。在这些实施方案中，所述生物聚合物的结合结构域将结合DNGR-1的细胞外结构域。例如，所述生物聚合物的结合结构域可以是包含F-肌动蛋白/肌球蛋白复合物的DNGR-1结合片段的多肽。可替代地，所述生物聚合物的结合结构域可以是特异性结合DNGR-1的抗体或适配体。
在第二方面，本发明提供了一种治疗有需要的患者的疾病的方法，所述方法通过使用第一方面的方法将生物聚合物递送至所述患者的细胞的胞质溶胶来进行。在相关的第三方面，本发明提供了一种用于治疗有需要的患者的疾病的方法的生物聚合物，所述方法包括使用第一方面的方法将生物聚合物递送至所述患者的细胞的胞质溶胶。在相关的第四方面，本发明提供了一种生物聚合物在制造用于治疗有需要的患者的疾病的药物中的用途，所述治疗包括使用第一方面的方法将生物聚合物递送至所述患者的细胞的胞质溶胶。
通过向所述患者施用所述生物聚合物，可以在所述患者的细胞仍在所述患者体内时将所述生物聚合物递送至所述细胞。所述细胞可以是癌细胞。所述生物聚合物可以通过注射施用于癌症或周围组织。可替代地，可以在收获所述细胞的步骤后，将所述生物聚合物离体递送至所述患者的细胞。所述细胞可以是免疫细胞。在这些实施方案中，可以将所述细胞作为细胞治疗再引入所述患者。
在一些实施方案中，将所述生物聚合物施用于癌症患者以引发抗癌Th1反应。在一些实施方案中，所述生物聚合物包含自身抗原，并且将所述生物聚合物施用于患有自身免疫性疾病的患者，以引发对所述自身抗原的耐受原反应。
在第五方面，本发明提供了一种如本文所述的生物聚合物。还提供了编码本发明的多肽生物聚合物的核酸分子。在一些实施方案中，这些核酸形成载体的一部分。
在第六方面，本发明提供了一种嵌合受体，其包含细胞外靶结合结构域、跨膜结构域和细胞内结构域，所述细胞内结构域包含来源于DNGR-1的胞质尾区的信号传导结构域的Syk结合序列，其中所述Syk结合序列含有酪氨酸残基。所述Syk结合序列可以包含hemITAM。在一些实施方案中，所述hemITAM包含SEQ ID NO:14(EXXYXXL；其中X表示任何氨基酸残基)中所示的氨基酸序列。
在一些实施方案中，所述Syk结合序列包含如SEQ ID NO:15(MHAEXXYXXLQWD)中所示的氨基酸序列，其中“X”表示任何氨基酸残基。在一些实施方案中，所述Syk结合序列包含如SEQ ID NO:15(MHAEXXYXXLQWD)中所示的氨基酸序列，其中“X”表示任何氨基酸残基，并且其中N末端的一个、两个或所有三个残基可以被去除或被另一个氨基酸残基取代，和/或其中C末端的一个、两个或所有三个残基可以被去除或被另一个氨基酸残基取代。在一些实施方案中，所述Syk结合序列包含如SEQ ID NO:90(MHEEXXYXXLQWD)中所示的氨基酸序列。在一些实施方案中，所述Syk结合序列包含如SEQ ID NO:90(MHEEXXYXXLQWD)中所示的氨基酸序列，其中“X”表示任何氨基酸残基，并且其中N末端的一个、两个或所有三个残基可以被去除或被另一个氨基酸残基取代，和/或其中C末端的一个、两个或所有三个残基可以被去除或被另一个氨基酸残基取代。在一些实施方案中，所述Syk结合序列包含如SEQ ID NO:11(MHAEEIYTSLQWD)中所示的氨基酸序列，任选地其中一个、两个或三个氨基酸残基被另一个氨基酸残基取代。优选地，所述细胞内信号传导结构域的N末端氨基酸残基位于本发明的嵌合受体的N末端。在一些实施方案中，所述Syk结合序列包含如SEQ ID NO:89(MHEEEIYTSLQWD)中所示的氨基酸序列。
SEQ ID NO:11来源于小鼠DNGR-1，而SEQ ID NO:69来源于人DNGR-1。这些序列的彼此不同之处在于第三氨基酸残基，所述第三氨基酸残基在小鼠中是丙氨酸，并且在人中是谷氨酸。因此，所述Syk结合序列可以包含如SEQ ID NO:91(其为MHXEEIYTSLQWD)中所示的氨基酸序列，其中X表示任何氨基酸。优选地，SEQ ID NO:91中的X是丙氨酸(A)或谷氨酸(E)。
在一些实施方案中，所述靶结合结构域结合存在于病原体、致病细胞、死细胞或患病细胞上的靶标。靶抗原可以存在于癌细胞上。所述靶抗原可以是肿瘤抗原，例如新抗原。例如，所述肿瘤抗原可以是CEA、ERBB2、EGFR、GD2、间皮素、MUC1、PSMA、CAIX、CD133、c-Met、EGFR、EGFRvIII、Epcam、EphA2、FRα、CD19、CD20、GPC3、GUCY2C、HER1、HER2、ICAM-1、MAGE或MET。
所述靶抗原可以存在于病毒感染的细胞上，例如在细胞表面处。所述靶抗原可以是病毒抗原，例如HCMV gB、流感病毒A血凝素、流感病毒基质2蛋白M2e、RSV糖蛋白F、SARS-Cov-2刺突蛋白、HIV gp120或HIV Env。
在一些实施方案中，所述嵌合受体的靶结合结构域来源于非DNGR-1凝集素，或者来源于转铁蛋白受体。非DNGR-1凝集素的一个例子是Dectin-1。例如，所述嵌合受体的靶结合结构域可以来源于小鼠Dectin-1。
在其他实施方案中，所述靶结合结构域包含抗体可变区重链(VH)和/或可变区轻链(VL)。例如，所述嵌合受体可以包含抗体VH结构域；并且这可以与单独表达的抗体VL结构域配对。可替代地，所述嵌合受体可以包含抗体VL结构域；并且这可以与单独表达的抗体VH结构域配对。在一些实施方案中，所述嵌合受体可以包含抗体VH结构域和VL结构域两者，所述结构域存在于单条多肽链上，例如呈单链可变片段(scFv)形式。
所述嵌合受体的靶结合结构域可以包含核酸受体(例如模式识别受体)的配体结合结构域，使得所述嵌合受体能够与核酸靶标结合并促进将所述核酸运输到胞质溶胶。
所述嵌合受体的靶结合结构域、跨膜结构域和/或胞质溶胶结构域可以经由接头(例如肽接头)彼此连接。肽接头在本领域中是熟知的。技术人员可以从选择的接头序列中选择，如：GGGSGGG(SEQ ID NO:92)、GGGGSGGGGS(SEQ ID NO:93)、PGPG(SEQ ID NO:94)和GSAGSAAGSGEF(SEQ ID NO:95)。也可以使用这些接头序列的组合和重复。
在一些实施方案中，所述嵌合受体的跨膜结构域来自I型跨膜蛋白。在一些实施方案中，所述嵌合受体的细胞外结构域来自I型跨膜蛋白。在一些实施方案中，所述嵌合受体的跨膜结构域来自II型跨膜蛋白。在一些实施方案中，所述嵌合受体的细胞外结构域来自II型跨膜蛋白。
相关方面提供了一种包含本发明的嵌合受体的细胞。所述嵌合受体可以在细胞的表面处，即在细胞膜处表达。所述细胞可以是非专职APC，例如肿瘤细胞。所述细胞可以是免疫细胞。所述细胞可以是专职APC，诸如巨噬细胞或DC。在一些实施方案中，所述细胞表达包含NOX2的NADPH氧化酶。
本文公开了跨膜受体的氨基酸序列及其组成结构域。根据本发明，清楚的是包含Syk结合序列(来源于DNGR-1的胞质尾区的信号传导结构域)的细胞内结构域可以与来自这些示例性序列中的任一个的跨膜结构域和细胞外/配体结合结构域组合，以提供一大系列的嵌合受体。这个原理可以用于提供具有所需靶特异性的嵌合受体，以促进目的靶标易位至细胞的胞质溶胶。
本发明的嵌合受体的非限制性示例性氨基酸序列在此提供：
MHAEEIYTSLQWDIPTSEASQKCQSPSKCSGAVGLGILCFVVVVVAAVLGALAFWRHNSGRNPEEKDSFLSRNKENHKPTESSLDEKVAPSKASQTTGGFSQSCLPNWIMHGKSCYLFSFSGNSWYGSKRHCSQLGAHLLKIDNSKEFEFIESQTSSHRINAFWIGLSRNQSEGPWFWEDGSAFFPNSFQVRNAVPQESLLHNCVWIHGSEVYNQICNTSSYSICE(SEQ ID NO:96)
SEQ ID NO:96的示例性嵌合受体由与小鼠Dectin-1的跨膜和细胞外结构域融合的小鼠DNGR-1的胞质结构域(如SEQ ID NO:7中所示)组成。SEQ ID NO:18是所述跨膜结构域。所述细胞外结构域包含颈部区域(SEQ ID NO:19)和C型凝集素结构域CTLD(SEQ ID NO:20)。
MHEEEIYTSLQWDSPAPDTYQKCLSSNKCSGACCLVMVISCVFCMGLLTASIFLGVCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF(SEQ ID NO:97)
SEQ ID NO:97的示例性嵌合受体由与人转铁蛋白受体的细胞外结构域(如SEQ IDNO:67中所示)融合的人DNGR-1的胞质结构域和跨膜结构域(分别如SEQ ID NO:2和3中所示)组成。
MILTSFGDDMWLLTTLLLWVPVGGEVVNATKAVITLQPPWVSIFQKENVTLWCEGPHLPGDSSTQWFINGTAVQISTPSYSIPEASFQDSGEYRCQIGSSMPSDPVQLQIHNDWLLLQASRRVLTEGEPLALRCHGWKNKLVYNVVFYRNGKSFQFSSDSEVAILKTNLSHSGIYHCSGTGRHRYTSAGVSITVKELFTTPVLRASVSSPFPEGSLVTLNCETNLLLQRPGLQLHFSFYVGSKILEYRNTSSEYHIARAEREDAGFYWCEVATEDSSVLKRSPELELQVLGPQSSAPVWFHILFYLSVGIMFSLNTVLYVGGGSGGGMHEEEIYTSLQWD(SEQ ID NO:98)
SEQ ID NO:98的示例性嵌合受体由与人DNGR-1的Syk结合序列(如SEQ ID NO:69中所示)融合的小鼠FcγRI的细胞外结构域和跨膜结构域(分别如SEQ ID NO:36和35中所示)组成，它们由短接头序列GGGSGGG(SEQ ID NO:92)隔开。
肽接头在本领域中是熟知的。技术人员可以从选择的接头序列中选择，如：GGGSGGG(SEQ ID NO:92)、GGGGSGGGGS(SEQ ID NO:93)、PGPG(SEQ ID NO:94)和GSAGSAAGSGEF(SEQ ID NO:95)。也可以使用这些接头序列的组合和重复。
在另一方面，本发明提供了编码本发明的嵌合受体的核酸分子。在一些实施方案中，该核酸形成载体的一部分。
本发明进一步提供了包含编码所述嵌合受体的核酸或载体的宿主细胞。在优选的实施方案中，所述宿主细胞表达所述嵌合受体，并且因此能够交叉呈递与所述嵌合受体结合的外源抗原。
在一些实施方案中，表达所述嵌合受体的细胞是髓样细胞，例如巨噬细胞、单核细胞或树突细胞。在一些实施方案中，表达所述嵌合受体的细胞是淋巴细胞。在一些实施方案中，表达所述嵌合受体的细胞不是专职抗原呈递细胞。在一些实施方案中，表达所述嵌合受体的细胞不是树突细胞。
本发明进一步提供了一种产生表达本发明的嵌合受体的细胞的方法，所述方法包括：
a.提供前体细胞；
b.将编码所述嵌合受体的核酸或载体引入所述前体细胞中以产生宿主细胞；以及
c.在促进由所述核酸编码的嵌合受体的表达的条件下繁殖步骤b.的宿主细胞，使得所述宿主细胞表达所述嵌合受体，并且因此变得能够交叉呈递外源抗原。
在本发明的一些实施方案中，由表达所述嵌合受体的细胞呈递的外源抗原是由所述嵌合受体的靶结合结构域结合的靶标。在其他实施方案中，由表达所述嵌合受体的细胞呈递的外源抗原与由所述嵌合受体的靶结合结构域结合的靶标缔合。
本发明进一步提供了包含本发明的载体的药物组合物。本发明还提供了包含表达本发明的嵌合受体的细胞的药物组合物。
本发明的药物组合物适用于治疗癌症的方法，其中所述方法包括向癌症患者施用所述药物组合物。所述癌症可以是实体瘤，并且所述方法可以任选地包括将所述药物组合物注射到所述实体瘤中，或注射到紧邻所述实体瘤周围的组织中。本发明的药物组合物可以用于药物中。
本发明的药物组合物适用于治疗感染性疾病的方法，其中所述方法包括向受感染的患者施用所述药物组合物。
本发明的药物组合物还适合用作疫苗或用作疫苗接种程序的一部分。
DNGR-1结合剂；DNGR-1结合剂复合物和缀合物
诸位发明人发现具有包含DNGR-1的Syk结合序列的细胞内结构域的跨膜受体可以促进大分子从细胞外间隙易位至胞质溶胶，而不会持续实质性降解。这提供了经由靶向所述跨膜受体的细胞外部分将功能性大分子(诸如蛋白质和核酸)运输到胞质溶胶的方式。所述跨膜受体可以是野生型DNGR-1，或者其可以是本发明的嵌合受体。因此，本发明提供了与有效载荷缔合的结合剂，其中所述结合剂能够结合所述跨膜受体的细胞外结构域。这种结合剂-有效载荷复合物/缀合物可以进入表达所述跨膜受体的细胞的胞质溶胶。
所述有效载荷可以是生物大分子，例如蛋白质或核酸。所述有效载荷可以经由化学缀合(例如经由表达为融合蛋白)或通过高亲和力非共价相互作用(诸如在生物素与链霉亲和素或抗生物素蛋白之间)与所述结合剂缔合。所述结合剂可以是抗DNGR-1抗体、DNGR-1结合适配体或作为DNGR-1的天然配体的F-肌动蛋白/肌球蛋白复合物的DNGR-1结合片段。在一些实施方案中，所述结合剂充当DNGR-1激动剂，强烈刺激本文所述的XP途径。在一些实施方案中，所述有效载荷与多种结合剂(例如两种或更多种抗体)缔合。此类“多价”缀合物可以在刺激本文所述的XP途径方面特别有效。
本发明的这个方面可用于将生物活性有效载荷递送至细胞中，例如在未复合的有效载荷不易穿透细胞膜的情况下。因此，在一些实施方案中，所述有效载荷是蛋白质或细胞毒素，例如类美登素、尾海兔素、澳瑞他汀药物类似物、念珠藻素、倍癌霉素、倍癌霉素衍生物、烯二炔抗生素或吡咯并苯二氮
。在一些实施方案中，所述有效载荷是遗传活性核酸(即，与翻译和/或转录机构相互作用的核酸)。因此，在一些实施方案中，所述核酸不充当辅助剂。在这些实施方案中，所述核酸不是TLR激动剂。更特别地，在这些实施方案中，所述核酸不是结合TLR3的多聚I:C(聚肌苷-聚胞苷酸)；不是结合TLR7的多聚U RNA(1-(2-甲基丙基)-1H-咪唑并(4,5-c)喹啉-4-胺)；并且不是结合TLR9的CpG(DNA CpG基序)。相反，所述核酸可以编码基因产物，或者其可以编码(或者是)干扰RNA。
在所述核酸编码基因产物的实施方案中，所述基因产物可以是抗原，例如肿瘤抗原或病毒抗原，并且所述结合剂/有效载荷复合物可以用作疫苗。
序列
鉴于本公开文本，以下受体的结构域的氨基酸序列可以被剪接在一起以形成本发明的嵌合受体：
人DNGR-1(CLEC9A)
人DNGR-1是II型跨膜蛋白。人DNGR-1(CLEC9A)的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_997228.1获得，并且在此由以下SEQ IDNO:1提供：
MHEEEIYTSLQWDSPAPDTYQKCLSSNKCSGACCLVMVISCVFCMGLLTASIFLGVKLLQVSTIAMQQQEKLIQQERALLNFTEWKRSCALQMKYCQAFMQNSLSSAHNSSPCPNNWIQNRESCYYVSEIWSIWHTSQENCLKEGSTLLQIESKEEMDFITGSLRKIKGSYDYWVGLSQDGHSGRWLWQDGSSPSPGLLPAERSQSANQVCGYVKSNSLLSSNCSTWKYFICEKYALRSSV(SEQ ID NO:1)
(存在人DNGR-1的一些剪接变体，然而它们在胞质结构域方面没有变化。)
人DNGR-1的胞质结构域
人DNGR-1的氨基酸序列中作为N末端胞质结构域的部分示于以下SEQ ID NO:2中：
MHEEEIYTSLQWDSPAPDTYQKCLSSNKCSGA(SEQ ID NO:2)
人DNGR-1的跨膜结构域
人DNGR-1的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:3中：
CCLVMVISCVFCMGLLTASIFLGV(SEQ ID NO:3)
人DNGR-1的颈部结构域
人DNGR-1的氨基酸序列中作为颈部结构域的部分示于以下SEQ ID NO:4中：
KLLQVSTIAMQQQEKLIQQERALLNFTEWKRSCALQMKYCQAFMQNSLSSAHNSS(SEQ ID NO:4)
人DNGR-1的C型凝集素结构域(CTLD)
人DNGR-1的氨基酸序列中作为CTLD的部分示于以下SEQ ID NO:5中：
PCPNNWIQNRESCYYVSEIWSIWHTSQENCLKEGSTLLQIESKEEMDFITGSLRKIKGSYDYWVGLSQDGHSGRWLWQDGSSPSPGLLPAERSQSANQVCGYVKSNSLLSSNCSTWKYFICEKYA(SEQ ID NO:5)
小鼠DNGR-1(CLEC9A)
小鼠DNGR-1是II型跨膜蛋白。小鼠DNGR-1(CLEC9A)的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_001192292.1获得，并且在此由以下SEQ ID NO:6提供：
MHAEEIYTSLQWDIPTSEASQKCQSPSKCSGAWCVVTMISCVVCMGLLATSIFLGIKFFQVSSLVLEQQERLIQQDTALVNLTQWQRKYTLEYCQALLQRSLHSGTDASTGPVLLTSPQMVPQTLDSKETGSDCSPCPHNWIQNGKSCYYVFERWEMWNISKKSCLKEGASLFQIDSKEEMEFISSIGKLKGGNKYWVGVFQDGISGSWFWEDGSSPLSDLLPAERQRSAGQICGYLKDSTLISDKCDSWKYFICEKKAFGSCI(SEQ ID NO:6)
小鼠DNGR-1的胞质结构域
小鼠DNGR-1的氨基酸序列中作为N末端胞质结构域的部分示于以下SEQ ID NO:7中：
MHAEEIYTSLQWDIPTSEASQKCQSPSKCSGA(SEQ ID NO:7)
小鼠DNGR-1的跨膜结构域
小鼠DNGR-1的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:8中：
WCVVTMISCVVCMGLLATSIFLGI(SEQ ID NO:8)
小鼠DNGR-1的颈部结构域
小鼠DNGR-1的氨基酸序列中作为颈部结构域的部分示于以下SEQ ID NO:9中：
KFFQVSSLVLEQQERLIQQDTALVNLTQWQRKYTLEYCQALLQRSLHSGTDASTGPVLLTSPQMVPQTLDSKETGSDCS(SEQ ID NO:9)
小鼠DNGR-1的C型凝集素结构域(CTLD)
小鼠DNGR-1的氨基酸序列中作为CTLD的部分示于以下SEQ ID NO:10中：PCPHNWIQNGKSCYYVFERWEMWNISKKSCLKEGASLFQIDSKEEMEFISSIGKLKGGNKYWVGVFQDGISGSWFWEDGSSPLSDLLPAERQRSAGQICGYLKDSTLISDKCDSWKYFICEKKA(SEQ ID NO:10)
DNGR-1的胞质尾区的信号传导结构域
本公开文本显示DNGR-1的胞质尾区是XP的关键介体，并且其介导XP的能力仅需要DNGR-1信号传导结构域。这个序列包含“HemITAM”54序列，其在与小鼠Dectin-1融合时允许/介导Syk结合。
DNGR-1胞质结构域的信号传导部分位于所述胞质结构域的前十三个残基中，如SEQ ID NO:11中所示：
MHAEEIYTSLQWD(SEQ ID NO:11)。这个序列是小鼠DNGR-1的前十三个残基。
人DNGR-1胞质结构域的信号传导部分位于所述胞质结构域的前十三个残基中，如SEQ ID NO:89中所示：
MHEEEIYTSLQWD(SEQ ID NO:89)。
hemITAM结构域
DNGR-1胞质结构域的上述13个氨基酸部分包含hemITAM结构域，如SEQ ID NO:12中所示：
EEIYTSL(SEQ ID NO:12)
其他hemITAM序列已有报道(Baur和Steinle,2017，将其通过引用以其整体特此并入)，诸如以下SEQ ID NO:13中所示的hemITAM序列
DEDGYXXL(SEQ ID NO:13)
hemITAM序列的必需氨基酸残基是首个谷氨酸(E)、中心酪氨酸(Y)和亮氨酸(L)，因为这些残基在不同蛋白质和不同物种的hemITAM序列中是保守的。因此，本发明的嵌合受体的信号传导结构域可以包含如以下SEQ ID NO:14中所示的hemITAM：EXXYXXL(SEQ IDNO:14)，其中“X”表示任何氨基酸残基。
细胞内信号传导结构域
诸位发明人认为，在DNGR-1的hemITAM的每一侧的上的1-3个氨基酸对于促进XP可能是重要的。因此，本发明的嵌合受体可以包含细胞内信号传导结构域(其可以表示为“来源于DNGR-1的胞质尾区的信号传导结构域的Syk结合序列”)，所述细胞内信号传导结构域包含如以下SEQ ID NO:15中所示的序列：
MHAEXXYXXLQWD(SEQ ID NO:15)，其中“X”表示任何氨基酸残基。
小鼠Dectin-1(CLEC7A)
小鼠Dectin-1是II型跨膜蛋白。小鼠Dectin-1(CLEC7A)的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符AAS37670.1获得，并且在此由以下SEQ ID NO:16提供：
MKYHSHIENLDEDGYTQLDFSTQDIHKRPRGSEKGSRAPSSPWRPIAVGLGILCFVVVVVAAVLGALAFWRHNSGRNPEEKDSFLSRNKENHKPTESSLDEKVAPSKASQTTGGFSQSCLPNWIMHGKSCYLFSFSGNSWYGSKRHCSQLGAHLLKIDNSKEFEFIESQTSSHRINAFWIGLSRNQSEGPWFWEDGSAFFPNSFQVRNAVPQESLLHNCVWIHGSEVYNQICNTSSYSICEKEL(SEQ ID NO:16)
小鼠Dectin-1的胞质结构域
小鼠Dectin-1的氨基酸序列中作为N末端胞质结构域的部分示于以下SEQ ID NO:17中：
MKYHSHIENLDEDGYTQLDFSTQDIHKRPRGSEKGSRAPSSPWR(SEQ ID NO:17)
小鼠Dectin-1的跨膜结构域
小鼠Dectin-1的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:18中：
VGLGILCFVVVVVAAVLGALAFW(SEQ ID NO:18)
小鼠Dectin-1的颈部结构域
小鼠Dectin-1的氨基酸序列中作为颈部结构域的部分示于以下SEQ ID NO:19中：
RHNSGRNPEEKDSFLSRNKENHKPTESSLDEKVAPSKASQTTGGFSQSCLPNWIM(SEQ ID NO:19)
小鼠Dectin-1的C型凝集素结构域(CTLD)
小鼠Dectin-1的氨基酸序列中作为CTLD的部分示于以下SEQ ID NO:20中：
HGKSCYLFSFSGNSWYGSKRHCSQLGAHLLKIDNSKEFEFIESQTSSHRINAFWIGLSR NQSEGPWFWEDGSAFFPNSFQVRNAVPQESLLHNCVWIHGSEVYNQICNTSSYSICE(SEQ ID NO:20)
人FcRγ链
人FcRγ链是I型跨膜蛋白。人FcRγ链的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_004097获得，并且在此由以下SEQ ID NO:21提供：MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ(SEQ ID NO:21)
人FcRγ链的胞质结构域
人FcRγ链的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ ID NO:22中：RLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ(SEQ ID NO:22)
人FcRγ链的跨膜结构域
人FcRγ链的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:23中：LCYILDAILFLYGIVLTLLYC(SEQ ID NO:23)
人FcRγ链的细胞外结构域
人FcRγ链的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:24中：LGEPQ(SEQ ID NO:24)
小鼠FcRγ链
小鼠FcRγ链是I型跨膜蛋白。小鼠FcRγ链的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_034315.1获得，并且在此由以下SEQ ID NO:25提供：
MISAVILFLLLLVEQAAALGEPQLCYILDAVLFLYGIVLTLLYCRLKIQVRKAAIASREKADAVYTGLNTRSQETYETLKHEKPPQ(SEQ ID NO:25)
小鼠FcRγ链的胞质结构域
小鼠FcRγ链的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ ID NO:26中：RLKIQVRKAAIASREKADAVYTGLNTRSQETYETLKHEKPPQ(SEQ ID NO:26)
小鼠FcRγ链的跨膜结构域
小鼠FcRγ链的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:27中：LCYILDAVLFLYGIVLTLLYC(SEQ ID NO:27)
小鼠FcRγ链的细胞外结构域
小鼠FcRγ链的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:28中：LGEPQ(SEQ ID NO:28)
人FcγRI
人FcγRI是I型跨膜蛋白。人FcγRI的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_001365733获得，并且在此由以下SEQ ID NO:29提供：MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFHVLFYLAVGIMFLVNTVLWVTIRKELKRKKKWDLEISLDSGHEKKVISSLQEDRHLEEELKCQEQKEEQLQEGVHRKEPQGAT(SEQID NO:29)
人FcγRI的胞质结构域
人FcγRI的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ ID NO:30中：RKELKRKKKWDLEISLDSGHEKKVISSLQEDRHLEEELKCQEQKEEQLQEGVHRKEPQGAT(SEQ ID NO:30)
人FcγRI的跨膜结构域
人FcγRI的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:31中：VLFYLAVGIMFLVNTVLWVTI(SEQ ID NO:31)
人FcγRI的细胞外结构域
人FcγRI的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:32中：QVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFH(SEQ ID NO:32)
小鼠FcγRI
小鼠FcγRI是I型跨膜蛋白。小鼠FcγRI的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_034316获得，并且在此由以下SEQ ID NO:33提供：MILTSFGDDMWLLTTLLLWVPVGGEVVNATKAVITLQPPWVSIFQKENVTLWCEGPHLPGDSSTQWFINGTAVQISTPSYSIPEASFQDSGEYRCQIGSSMPSDPVQLQIHNDWLLLQASRRVLTEGEPLALRCHGWKNKLVYNVVFYRNGKSFQFSSDSEVAILKTNLSHSGIYHCSGTGRHRYTSAGVSITVKELFTTPVLRASVSSPFPEGSLVTLNCETNLLLQRPGLQLHFSFYVGSKILEYRNTSSEYHIARAEREDAGFYWCEVATEDSSVLKRSPELELQVLGPQSSAPVWFHILFYLSVGIMFSLNTVLYVKIHRLQREKKYNLEVPLVSEQGKKANSFQQVRSDGVYEEVTATASQTTPKEAPDGPRSSVGDCGPEQPEPLPPSDSTGAQTSQS(SEQ ID NO:33)
小鼠FcγRI的胞质结构域
小鼠FcγRI的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ ID NO:34中：KIHRLQREKKYNLEVPLVSEQGKKANSFQQVRSDGVYEEVTATASQTTPKEAPDGPRSSVGDCGPEQPEPLPPSDSTGAQTSQS(SEQ ID NO:34)
小鼠FcγRI的跨膜结构域
小鼠FcγRI的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:35中：VWFHILFYLSVGIMFSLNTVLYV(SEQ ID NO:35)
小鼠FcγRI的细胞外结构域
小鼠FcγRI的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:36中：EVVNATKAVITLQPPWVSIFQKENVTLWCEGPHLPGDSSTQWFINGTAVQISTPSYSIPEASFQDSGEYRCQIGSSMPSDPVQLQIHNDWLLLQASRRVLTEGEPLALRCHGWKNKLVYNVVFYRNGKSFQFSSDSEVAILKTNLSHSGIYHCSGTGRHRYTSAGVSITVKELFTTPVLRASVSSPFPEGSLVTLNCETNLLLQRPGLQLHFSFYVGSKILEYRNTSSEYHIARAEREDAGFYWCEVATEDSSVLKRSPELELQVLGPQSSAP(SEQ ID NO:36)
人FcγRIIA
人FcγRIIA是I型跨膜蛋白。人FcγRIIA的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_001129691获得，并且在此由以下SEQ ID NO:37提供：MTMETQMSQNVCPRNLWLLQPLTVLLLLASADSQAAAPPKAVLKLEPPWINVLQEDSVTLTCQGARSPESDSIQWFHNGNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWLVLQTPHLEFQEGETIMLRCHSWKDKPLVKVTFFQNGKSQKFSHLDPTFSIPQANHSHSGDYHCTGNIGYTLFSSKPVTITVQVPSMGSSSPMGIIVAVVIATAVAAIVAAVVALIYCRKKRISANSTDPVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHVNSNN(SEQ ID NO:37)
人FcγRIIA的胞质结构域
人FcγRIIA的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ ID NO:38中：CRKKRISANSTDPVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHVNSNN(SEQ ID NO:38)
人FcγRIIA的跨膜结构域
人FcγRIIA的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:39中：IIVAVVIATAVAAIVAAVVALIY(SEQ ID NO:39)
人FcγRIIA的细胞外结构域
人FcγRIIA的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:40中：QAAAPPKAVLKLEPPWINVLQEDSVTLTCQGARSPESDSIQWFHNGNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWLVLQTPHLEFQEGETIMLRCHSWKDKPLVKVTFFQNGKSQKFSHLDPTFSIPQANHSHSGDYHCTGNIGYTLFSSKPVTITVQVPSMGSSSPMG(SEQ ID NO:40)
人TIMD4
人TIMD4是I型跨膜蛋白。人TIMD4的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_612388获得，并且在此由以下SEQ ID NO:41提供：MSKEPLILWLMIEFWWLYLTPVTSETVVTEVLGHRVTLPCLYSSWSHNSNSMCWGKDQCPYSGCKEALIRTDGMRVTSRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCRIEVPGWFNDVKINVRLNLQRASTTTHRTATTTTRRTTTTSPTTTRQMTTTPAALPTTVVTTPDLTTGTPLQMTTIAVFTTANTCLSLTPSTLPEEATGLLTPEPSKEGPILTAESETVLPSDSWSSVESTSADTVLLTSKESKVWDLPSTSHVSMWKTSDSVSSPQPGASDTAVPEQNKTTKTGQMDGIPMSMKNEMPISQLLMIIAPSLGFVLFALFVAFLLRGKLMETYCSQKHTRLDYIGDSKNVLNDVQHGREDEDGLFTL(SEQID NO:41)
人TIMD4的胞质结构域
人TIMD4的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ ID NO:42中：LRGKLMETYCSQKHTRLDYIGDSKNVLNDVQHGREDEDGLFTL(SEQ ID NO:42)
人TIMD4的跨膜结构域
人TIMD4的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:43中：LLMIIAPSLGFVLFALFVAFL(SEQ ID NO:43)
人TIMD4的细胞外结构域
人TIMD4的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:44中：ETVVTEVLGHRVTLPCLYSSWSHNSNSMCWGKDQCPYSGCKEALIRTDGMRVTSRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCRIEVPGWFNDVKINVRLNLQRASTTTHRTATTTTRRTTTTSPTTTRQMTTTPAALPTTVVTTPDLTTGTPLQMTTIAVFTTANTCLSLTPSTLPEEATGLLTPEPSKEGPILTAESETVLPSDSWSSVESTSADTVLLTSKESKVWDLPSTSHVSMWKTSDSVSSPQPGASDTAVPEQNKTTKTGQMDGIPMSMKNEMPISQ(SEQ ID NO:44)
小鼠TIMD4
小鼠TIMD4是I型跨膜蛋白。小鼠TIMD4的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_848874获得，并且在此由以下SEQ ID NO:45提供：MSKGLLLLWLVTELWWLYLTPAASEDTIIGFLGQPVTLPCHYLSWSQSRNSMCWGKGSCPNSKCNAELLRTDGTRIISRKSTKYTLLGKVQFGEVSLTISNTNRGDSGVYCCRIEVPGWFNDVKKNVRLELRRATTTKKPTTTTRPTTTPYVTTTTPELLPTTVMTTSVLPTTTPPQTLATTAFSTAVTTCPSTTPGSFSQETTKGSAFTTESETLPASNHSQRSMMTISTDIAVLRPTGSNPGILPSTSQLTTQKTTLTTSESLQKTTKSHQINSRQTILIIACCVGFVLMVLLFLAFLLRGKVTGANCLQRHKRPDNTEDSDSVLNDMSHGRDDEDGIFTL(SEQ ID NO:45)
小鼠TIMD4的胞质结构域
小鼠TIMD4的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ ID NO:46中：LRGKVTGANCLQRHKRPDNTEDSDSVLNDMSHGRDDEDGIFTL(SEQ ID NO:46)
小鼠TIMD4的跨膜结构域
小鼠TIMD4的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:88中：ILIIACCVGFVLMVLLFLAFL(SEQ ID NO:88)
小鼠TIMD4的细胞外结构域
小鼠TIMD4的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:47中：ASEDTIIGFLGQPVTLPCHYLSWSQSRNSMCWGKGSCPNSKCNAELLRTDGTRIISRKSTKYTLLGKVQFGEVSLTISNTNRGDSGVYCCRIEVPGWFNDVKKNVRLELRRATTTKKPTTTTRPTTTPYVTTTTPELLPTTVMTTSVLPTTTPPQTLATTAFSTAVTTCPSTTPGSFSQETTKGSAFTTESETLPASNHSQRSMMTISTDIAVLRPTGSNPGILPSTSQLTTQKTTLTTSESLQKTTKSHQINSRQT(SEQ ID NO:47)
人Megf10
人Megf10是I型跨膜蛋白。人Megf10的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_001243474获得，并且在此由以下SEQ ID NO:48提供：MVISLNSCLSFICLLLCHWIGTASPLNLEDPNVCSHWESYSVTVQESYPHPFDQIYYTSCTDILNWFKCTRHRVSYRTAYRHGEKTMYRRKSQCCPGFYESGEMCVPHCADKCVHGRCIAPNTCQCEPGWGGTNCSSACDGDHWGPHCTSRCQCKNGALCNPITGACHCAAGFRGWRCEDRCEQGTYGNDCHQRCQCQNGATCDHVTGECRCPPGYTGAFCEDLCPPGKHGPQCEQRCPCQNGGVCHHVTGECSCPSGWMGTVCGQPCPEGRFGKNCSQECQCHNGGTCDAATGQCHCSPGYTGERCQDECPVGTYGVLCAETCQCVNGGKCYHVSGACLCEAGFAGERCEARLCPEGLYGIKCDKRCPCHLENTHSCHPMSGECACKPGWSGLYCNETCSPGFYGEACQQICSCQNGADCDSVTGKCTCAPGFKGIDCSTPCPLGTYGINCSSRCGCKNDAVCSPVDGSCTCKAGWHGVDCSIRCPSGTWGFGCNLTCQCLNGGACNTLDGTCTCAPGWRGEKCELPCQDGTYGLNCAERCDCSHADGCHPTTGHCRCLPGWSGVHCDSVCAEGRWGPNCSLPCYCKNGASCSPDDGICECAPGFRGTTCQRICSPGFYGHRCSQTCPQCVHSSGPCHHITGLCDCLPGFTGALCNEVCPSGRFGKNCAGICTCTNNGTCNPIDRSCQCYPGWIGSDCSQPCPPAHWGPNCIHTCNCHNGAFCSAYDGECKCTPGWTGLYCTQRCPLGFYGKDCALICQCQNGADCDHISGQCTCRTGFMGRHCEQKCPSGTYGYGCRQICDCLNNSTCDHITGTCYCSPGWKGARCDQAGVIIVGNLNSLSRTSTALPADSYQIGAIAGIIILVLVVLFLLALFIIYRHKQKGKESSMPAVTYTPAMRVVNADYTISGTLPHSNGGNANSHYFTNPSYHTLTQCATSPHVNNRDRMTVTKSKNNQLFVNLKNVNPGKRGPVGDCTGTLPADWKHGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSNCSLSSSENPYATIKDPPVLIPKSSECGYVEMKSPARRDSPYAEINNSTSANRNVYEVEPTVSVVQGVFSNNGRLSQDPYDLPKNSHIPCHYDLLPVRDSSSSPKQEDSGGSSSNSSSSSE(SEQ ID NO:48)
人Megf10的胞质结构域
人Megf10的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ ID NO:49中：YRHKQKGKESSMPAVTYTPAMRVVNADYTISGTLPHSNGGNANSHYFTNPSYHTLTQCATSPHVNNRDRMTVTKSKNNQLFVNLKNVNPGKRGPVGDCTGTLPADWKHGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSNCSLSSSENPYATIKDPPVLIPKSSECGYVEMKSPARRDSPYAEINNSTSANRNVYEVEPTVSVVQGVFSNNGRLSQDPYDLPKNSHIPCHYDLLPVRDSSSSPKQEDSGGSSSNSSSSSE(SEQ ID NO:49)
人Megf10的跨膜结构域
人Megf10的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:50中：AIAGIIILVLVVLFLLALFII(SEQ ID NO:50)
人Megf10的细胞外结构域
人Megf10的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:51中：LNLEDPNVCSHWESYSVTVQESYPHPFDQIYYTSCTDILNWFKCTRHRVSYRTAYRHGEKTMYRRKSQCCPGFYESGEMCVPHCADKCVHGRCIAPNTCQCEPGWGGTNCSSACDGDHWGPHCTSRCQCKNGALCNPITGACHCAAGFRGWRCEDRCEQGTYGNDCHQRCQCQNGATCDHVTGECRCPPGYTGAFCEDLCPPGKHGPQCEQRCPCQNGGVCHHVTGECSCPSGWMGTVCGQPCPEGRFGKNCSQECQCHNGGTCDAATGQCHCSPGYTGERCQDECPVGTYGVLCAETCQCVNGGKCYHVSGACLCEAGFAGERCEARLCPEGLYGIKCDKRCPCHLENTHSCHPMSGECACKPGWSGLYCNETCSPGFYGEACQQICSCQNGADCDSVTGKCTCAPGFKGIDCSTPCPLGTYGINCSSRCGCKNDAVCSPVDGSCTCKAGWHGVDCSIRCPSGTWGFGCNLTCQCLNGGACNTLDGTCTCAPGWRGEKCELPCQDGTYGLNCAERCDCSHADGCHPTTGHCRCLPGWSGVHCDSVCAEGRWGPNCSLPCYCKNGASCSPDDGICECAPGFRGTTCQRICSPGFYGHRCSQTCPQCVHSSGPCHHITGLCDCLPGFTGALCNEVCPSGRFGKNCAGICTCTNNGTCNPIDRSCQCYPGWIGSDCSQPCPPAHWGPNCIHTCNCHNGAFCSAYDGECKCTPGWTGLYCTQRCPLGFYGKDCALICQCQNGADCDHISGQCTCRTGFMGRHCEQKCPSGTYGYGCRQICDCLNNSTCDHITGTCYCSPGWKGARCDQAGVIIVGNLNSLSRTSTALPADSYQIG(SEQID NO:51)
小鼠Megf10
小鼠Megf10是I型跨膜蛋白。小鼠Megf10的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_001001979获得，并且在此由以下SEQ ID NO:52提供：
MAISSSSCLGLICSLLCHWVGTASSLNLEDPNVCSHWESYSVTVQESYPHPFDQIYYTSCTDILNWFKCTRHRISYRTAYRHGEKTMYRRKSQCCPGFYESRDMCVPHCADKCVHGRCIAPNTCQCEPGWGGTNCSSACDGDHWGPHCSSRCQCKNRALCNPITGACHCAAGYRGWRCEDRCEQGTYGNDCHQRCQCQNGATCDHITGECRCSPGYTGAFCEDLCPPGKHGPHCEQRCPCQNGGVCHHVTGECSCPSGWMGTVCGQPCPEGRFGKNCSQECQCHNGGTCDAATGQCHCSPGYTGERCQDECPVGSYGVRCAEACRCVNGGKCYHVSGTCLCEAGFSGELCEARLCPEGLYGIKCDKRCPCHLDNTHSCHPMSGECGCKPGWSGLYCNETCSPGFYGEACQQICSCQNGADCDSVTGRCACAPGFKGTDCSTPCPLGRYGINCSSRCGCKNDAVCSPVDGSCICKAGWHGVDCSIRCPSGTWGFGCNLTCQCLNGGACNTLDGTCTCAPGWRGAKCEFPCQDGTYGLNCAERCDCSHADGCHPTTGHCRCLPGWSGVHCDSVCAEGRWGPNCSLPCYCKNGASCSPDDGICECAPGFRGTTCQRICSPGFYGHRCSQTCPQCVHSSGPCHHITGLCDCLPGFTGALCNEVCPSGRFGKNCAGVCTCTNNGTCNPIDRSCQCYPGWIGSDCSQPCPPAHWGPNCIHTCNCHNGAFCSAYDGECKCTPGWTGLYCTQRCPLGFYGKDCALICQCQNGADCDHISGQCTCRTGFMGRHCEQKCPAGTYGYGCRQICDCLNNSTCDHITGTCYCSPGWKGARCDQAGVIIVGNLNSLSRTSTALPADSYQIGAIAGIVVLVLVVLFLLALFIIYRHKQKRKESSMPAVTYTPAMRVINADYTIAETLPHSNGGNANSHYFTNPSYHTLSQCATSPHVNNRDRMTIAKSKNNQLFVNLKNVNPGKRGTLVDCTGTLPADWKQGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSTCSLSSSENPYATIKDPPALLPKSSECGYVEMKSPARRDSPYAEINNSTPANRNVYEVEPTVSVVQGVFSNSGHVTQDPYDLPKNSHIPCHYDLLPVRDSSSSPKREDGGGSNSTSSNSTSSSSSSSE(SEQ ID NO:52)
小鼠Megf10的胞质结构域
小鼠Megf10的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ ID NO:53中：YRHKQKRKESSMPAVTYTPAMRVINADYTIAETLPHSNGGNANSHYFTNPSYHTLSQCATSPHVNNRDRMTIAKSKNNQLFVNLKNVNPGKRGTLVDCTGTLPADWKQGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSTCSLSSSENPYATIKDPPALLPKSSECGYVEMKSPARRDSPYAEINNSTPANRNVYEVEPTVSVVQGVFSNSGHVTQDPYDLPKNSHIPCHYDLLPVRDSSSSPKREDGGGSNSTSSNSTSSSSSSSE(SEQ ID NO:53)
小鼠Megf10的跨膜结构域
小鼠Megf10的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:54中：AIAGIVVLVLVVLFLLALFII(SEQ ID NO:54)
小鼠Megf10的细胞外结构域
小鼠Megf10的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:55中：LNLEDPNVCSHWESYSVTVQESYPHPFDQIYYTSCTDILNWFKCTRHRISYRTAYRHGEKTMYRRKSQCCPGFYESRDMCVPHCADKCVHGRCIAPNTCQCEPGWGGTNCSSACDGDHWGPHCSSRCQCKNRALCNPITGACHCAAGYRGWRCEDRCEQGTYGNDCHQRCQCQNGATCDHITGECRCSPGYTGAFCEDLCPPGKHGPHCEQRCPCQNGGVCHHVTGECSCPSGWMGTVCGQPCPEGRFGKNCSQECQCHNGGTCDAATGQCHCSPGYTGERCQDECPVGSYGVRCAEACRCVNGGKCYHVSGTCLCEAGFSGELCEARLCPEGLYGIKCDKRCPCHLDNTHSCHPMSGECGCKPGWSGLYCNETCSPGFYGEACQQICSCQNGADCDSVTGRCACAPGFKGTDCSTPCPLGRYGINCSSRCGCKNDAVCSPVDGSCICKAGWHGVDCSIRCPSGTWGFGCNLTCQCLNGGACNTLDGTCTCAPGWRGAKCEFPCQDGTYGLNCAERCDCSHADGCHPTTGHCRCLPGWSGVHCDSVCAEGRWGPNCSLPCYCKNGASCSPDDGICECAPGFRGTTCQRICSPGFYGHRCSQTCPQCVHSSGPCHHITGLCDCLPGFTGALCNEVCPSGRFGKNCAGVCTCTNNGTCNPIDRSCQCYPGWIGSDCSQPCPPAHWGPNCIHTCNCHNGAFCSAYDGECKCTPGWTGLYCTQRCPLGFYGKDCALICQCQNGADCDHISGQCTCRTGFMGRHCEQKCPAGTYGYGCRQICDCLNNSTCDHITGTCYCSPGWKGARCDQAGVIIVGNLNSLSRTSTALPADSYQIG(SEQID NO:55)
人CD3ζ链
人CD3ζ链是I型跨膜蛋白。人CD3ζ链的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_932170获得，并且在此由以下SEQ ID NO:56提供：MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:56)
人CD3ζ链的胞质结构域
人CD3ζ链的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ ID NO:57中：RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:57)
人CD3ζ链的跨膜结构域
人CD3ζ链的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:58中：LCYLLDGILFIYGVILTALFL(SEQ ID NO:58)
人CD3ζ链的细胞外结构域
人CD3ζ链的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:59中：QSFGLLDPK(SEQ ID NO:59)
小鼠CD3ζ链
小鼠CD3ζ链是I型跨膜蛋白。小鼠CD3ζ链的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_001106862获得，并且在此由以下SEQ ID NO:60提供：
MKWKVSVLACILHVRFPGAEAQSFGLLDPKLCYLLDGILFIYGVIITALYLRAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR(SEQ ID NO:60)
小鼠CD3ζ链的胞质结构域
小鼠CD3ζ链的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ ID NO:61中：RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR(SEQ ID NO:61)
小鼠CD3ζ链的跨膜结构域
小鼠CD3ζ链的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:62中：LCYLLDGILFIYGVIITALYL(SEQ ID NO:62)
小鼠CD3ζ链的细胞外结构域
小鼠CD3ζ链的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:63中：QSFGLLDPK(SEQ ID NO:63)
人转铁蛋白受体
人转铁蛋白受体是II型跨膜蛋白。人转铁蛋白受体的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_001121620获得，并且在此由以下SEQID NO:64提供：
MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF(SEQ ID NO:64)
人转铁蛋白受体的胞质结构域
人转铁蛋白受体的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ IDNO:65中：
MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSIC(SEQ ID NO:65)
人转铁蛋白受体的跨膜结构域
人转铁蛋白受体的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:66中：YGTIAVIVFFLIGFMIGYLGY(SEQ ID NO:66)
人转铁蛋白受体的细胞外结构域
人转铁蛋白受体的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:67中：CKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF(SEQ IDNO:67)
人转铁蛋白受体的细胞外结构域
人转铁蛋白受体的氨基酸序列中作为配体结合结构域的部分示于以下SEQ IDNO:68中：EDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF(SEQ ID NO:68)
人Clec-2受体
人Clec-2受体是II型跨膜蛋白。人Clec-2受体的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_057593获得，并且在此由以下SEQ ID NO:69提供：
MQDEDGYITLNIKTRKPALISVGSASSSWWRVMALILLILCVGMVVGLVALGIWSVMQRNYLQGENENRTGTLQQLAKRFCQYVVKQSELKGTFKGHKCSPCDTNWRYYGDSCYGFFRHNLTWEESKQYCTDMNATLLKIDNRNIVEYIKARTHLIRWVGLSRQKSNEVWKWEDGSVISENMFEFLEDGKGNMNCAYFHNGKMHPTFCENKHYLMCERKAGMTKVDQLP(SEQ ID NO:69)
人Clec-2受体的胞质结构域
人Clec-2受体的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ ID NO:70中：MQDEDGYITLNIKTRKPALISVGSASSSWWRVM(SEQ ID NO:70)
人Clec-2受体的跨膜结构域
人Clec-2受体的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:71中：ALILLILCVGMVVGLVALGIW(SEQ ID NO:71)
人Clec-2受体的细胞外结构域
人Clec-2受体的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:72中：SVMQRNYLQGENENRTGTLQQLAKRFCQYVVKQSELKGTFKGHKCSPCDTNWRYYGDSCYGFFRHNLTWEESKQYCTDMNATLLKIDNRNIVEYIKARTHLIRWVGLSRQKSNEVWKWEDGSVISENMFEFLEDGKGNMNCAYFHNGKMHPTFCENKHYLMCERKAGMTKVDQLP(SEQ ID NO:72)
人KLRF1受体
人KLRF1受体是II型跨膜蛋白。人KLRF1受体的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符Q9NZS2获得，并且在此由以下SEQ ID NO:73提供：
MQDEERYMTLNVQSKKRSSAQTSQLTFKDYSVTLHWYKILLGISGTVNGILTLTLISLILLVSQGVLLKCQKGSCSNATQYEDTGDLKVNNGTRRNISNKDLCASRSADQTVLCQSEWLKYQGKCYWFSNEMKSWSDSYVYCLERKSHLLIIHDQLEMAFIQKNLRQLNYVWIGLNFTSLKMTWTWVDGSPIDSKIFFIKGPAKENSCAAIKESKIFSETCSSVFKWICQY(SEQ ID NO:73)
人KLRF1受体的胞质结构域
人KLRF1受体的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ ID NO:74中：MQDEERYMTLNVQSKKRSSAQTSQLTFKDYSVTLHWYK(SEQ ID NO:74)
人KLRF1受体的跨膜结构域
人KLRF1受体的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:75中：ILLGISGTVNGILTLTLISLI(SEQ ID NO:75)
人KLRF1受体的细胞外结构域
人KLRF1受体的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:76中：LLVSQGVLLKCQKGSCSNATQYEDTGDLKVNNGTRRNISNKDLCASRSADQTVLCQSEWLKYQGKCYWFSNEMKSWSDSYVYCLERKSHLLIIHDQLEMAFIQKNLRQLNYVWIGLNFTSLKMTWTWVDGSPIDSKIFFIKGPAKENSCAAIKESKIFSETCSSVFKWICQY(SEQ ID NO:76)
人Dectin-1受体
人Dectin-1受体是II型跨膜蛋白。人Dectin-1受体的全长氨基酸序列可在公共蛋白质数据库中获得，例如在NCBI数据库中以标识符NP_922938获得，并且在此由以下SEQ IDNO:77提供：
MEYHPDLENLDEDGYTQLHFDSQSNTRIAVVSEKGSCAASPPWRLIAVILGILCLVILVIAVVLGTMAIWRSNSGSNTLENGYFLSRNKENHSQPTQSSLEDSVTPTKAVKTTGVLSSPCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFIVKQVSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLCSVPSYSICEKKFSM(SEQ ID NO:77)
人Dectin-1受体的胞质结构域
人Dectin-1受体的氨基酸序列中作为C末端胞质结构域的部分示于以下SEQ IDNO:78中：
MEYHPDLENLDEDGYTQLHFDSQSNTRIAVVSEKGSCAASPPWR(SEQ ID NO:78)
人Dectin-1受体的跨膜结构域
人Dectin-1受体的氨基酸序列中作为跨膜结构域的部分示于以下SEQ ID NO:79中：LIAVILGILCLVILVIAVVLG(SEQ ID NO:79)
人Dectin-1受体的细胞外结构域
人Dectin-1受体的氨基酸序列中作为细胞外结构域的部分示于以下SEQ ID NO:80中：TMAIWRSNSGSNTLENGYFLSRNKENHSQPTQSSLEDSVTPTKAVKTTGVLSSPCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFIVKQVSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLCSVPSYSICEKKFSM(SEQ ID NO:80)
本文公开的序列信息提供了示例性胞质溶胶结构域、跨膜结构域和细胞外结构域，这些结构域可以组合以制备本发明的另外示例性嵌合受体。然而，本发明的嵌合受体由权利要求定义，并且不限于上文公开的示例性序列，除非在权利要求中如此指定。
嵌合蛋白
嵌合蛋白(诸如本发明提供的那些)包含来源于多于一种野生型蛋白的序列。例如，本发明的嵌合蛋白具有细胞内序列，所述细胞内序列包含与来源于其他(非DNGR-1)蛋白的序列融合的来源于DNGR-1蛋白的氨基酸序列。因此，本发明的嵌合蛋白的细胞外结构域在一些方面与野生型DNGR-1的细胞外结构域在功能上不同。例如，本发明的嵌合蛋白可能不与F-肌动蛋白/肌球蛋白复合物结合，所述F-肌动蛋白/肌球蛋白复合物是野生型人DNGR-1的天然配体(并且结合在DNGR-1的细胞外结构域处的CTLD)。例如，特异性结合野生型人DNGR-1的细胞外结构域的抗DNGR-1抗体可能不特异性结合本发明的嵌合蛋白。除了这些功能差异之外，因为本发明的嵌合蛋白的细胞外结构域并非来源于DNGR-1，所以其具有与DNGR-1的细胞外结构域的低序列同一性。例如，本发明的嵌合蛋白的细胞外结构域可以具有与野生型人DNGR-1的细胞外结构域小于50％的序列同一性，如在DNGR-1细胞外结构域的长度上所测量的。在一些实施方案中，本发明的嵌合蛋白的细胞外结构域具有与野生型人DNGR-1的细胞外结构域小于40％的序列同一性、小于30％的序列同一性、小于25％的序列同一性或小于20％的序列同一性，如在DNGR-1细胞外结构域的长度上所测量的。
序列关系
鉴定相关序列是生物化学和分子生物学领域内的常规操作。技术人员可以容易地确定受试序列是否与参考序列相关。如果所述序列彼此相关，则可以称所述序列中的一个“来源于”另一个。
一旦技术人员理解哪些氨基酸残基对于第一蛋白质的功能是重要的，她/他就可以确定第二序列是否具有适当定位的这些氨基酸残基，并且因此将可能共享第一蛋白质的功能；如果是这样，则所述序列在结构上和功能上是相关的。在本发明的氨基酸序列的情况下，“来源于DNGR-1的胞质尾区的信号传导结构域的Syk结合序列”在结构上和功能上与DNGR-1的胞质尾区的信号传导结构域相关，因为它共享DNGR-1的胞质尾区的关键氨基酸序列基序，并且因此它共享结合Syk并促进经由胞质溶胶途径进行XP的能力。在由SEQ ID NO:15(MHAEXXYXXLQWD)或SEQ ID NO:90(MHEEXXYXXLQWD)表示的Syk结合序列的中间的酪氨酸残基对于Syk结合功能是重要的。相比之下，位置1处的甲硫氨酸残基对于功能而言不太可能是重要的，尽管在Syk结合序列位于嵌合蛋白的N末端处的情况下所述甲硫氨酸残基是构建体的翻译所需要的。在N末端存在另外的前导序列或接头的嵌合构建体中，可以省略所述甲硫氨酸残基。这些构建体因此可以包含SEQ ID NO:81(HAEXXYXXLQWD)或SEQ ID NO:82(HEEXXYXXLQWD)。另外，认为在Syk结合序列的C末端的三个氨基酸残基是不重要的。因此，本发明的嵌合蛋白可以包含SEQ ID NO:83(MHAEXXYXXL)、SEQ ID NO:84(MHEEXXYXXL)、SEQID NO:85(HAEXXYXXL)或SEQ ID NO:86(HEEXXYXXL)。本发明的嵌合蛋白可以包含SEQ IDNO:87(HXEXXYXXL)。
另外地或可替代地，可以指定序列同一性程度以定义在两个序列之间的结构关系。例如，可以指定序列同一性程度为从至少60％至100％序列同一性。更优选地，指定的序列同一性程度可以是至少65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性中的一种。
本发明包括所描述的方面和优选特征的组合，除非明显不允许或明确避免这样的组合。
附图说明
现在将参考附图讨论说明本发明原理的实施方案和实验，在附图中：
图1.吞噬体损伤。在用WT或突变体(Y7F)DNGR-1重建的DNGR-1缺陷型MuTuDC中表达mCherry-半乳凝素-3，并且将细胞与FM-OVA珠一起孵育。计数半乳凝素-3吞噬体+细胞并将其绘制为与珠+细胞的比率(指数)。半乳凝素-3结合附接至受损内体和吞噬体的腔侧上的膜蛋白的糖部分。在表达野生型(WT)而非酪氨酸-苯丙氨酸(Y7F)突变体的细胞中，mCherry-半乳凝素-3被募集至吞噬体。
图2.在表达本发明的嵌合受体“C9/C7”(示为“C9::C7”)的非专职APC中的有效XP。来自B3Z杂交瘤和用酵母聚糖-OVA刺激的HEK293T C7、C9/C7或C9(Y7F)/C7(示为“C9(Y7F)::C7”)细胞共培养上清液的IL-2ELISA。数据绘制为一式三份实验的平均值±标准差。
图3.Syk磷酸化。在HEK293T C7、C7(Y15F)、C9/C7(示为“C9::C7”)和C9(Y7F)/C7(示为“C9(Y7F)::C7”)细胞中对吞噬体(n＞50个吞噬体)的磷酸-SYK染色的富集倍数的定量。p值通过双尾Mann-Whitney检验计算。代表性图(n＝2)。
具体实施方式
本公开文本表明cDC1在XP中的优势不完全依赖于独特的细胞生物学，而且还依赖于受体(诸如DNGR-1)的表达，所述受体检测相关XP底物并起始细胞内信号传导，从而允许吞噬体破坏并使得能够实现有效XP。诸位发明人表明在吞噬体水平上的配体依赖型DNGR-1信号传导诱导局部NADPH依赖型氧化爆发，使吞噬体膜去稳定，从而导致破裂和腔内容物大量进入胞质区室，在那里它们可以进入内源MHC I类加工途径。值得注意的是，DNGR-1传导信号以致吞噬体破裂的能力是其胞质信号传导结构域固有的，并且可以移植到其他受体和其他细胞类型上。因此，XP依赖于内体中用于活性氧类产生的普遍存在的机构，这种机构可以被特化受体破坏，以故意激起液泡膜损伤和P2C。
这些结果不排除cDC1具有有利于XP的细胞生物特化的事实7、30、36、40-42。实际上，我们在这些细胞中鉴定了可以长期保留未降解的货物(已知这有利于XP)的缓慢成熟的吞噬体区室22、23。DNGR-1优先定位于这些早期吞噬体但不影响它们成熟的事实与这样的观点一致，即所述受体的主要功能是调查液泡区室中暴露的F-肌动蛋白/肌球蛋白复合物的存在，表明推定的抗原货物相对完整。然后受体接合导致ROS的Syk依赖型局部产生和膜损伤。任何给定吞噬体的破裂可能是随机事件，部分取决于损伤程度，可能通过膜修复来补偿。未破裂的吞噬体可以继续成熟，产生LAMP+DNGR-1-降解性晚期吞噬体库，我们也在我们的测定中检测到所述库。破裂事件的有限性质和它被局限于早期非降解内体的事实可以有助于防止预期通过将蛋白水解酶引入胞质溶胶中而诱导的细胞毒性49。然而，破裂的概率足够高，以致所有表达C9/C7的HEK293T细胞在与细胞色素c浸泡的酵母聚糖一起孵育过夜后都死亡，这表明在24h时段内，在每个细胞中发生至少一次吞噬体破裂事件。可以经由Syk传导信号的其他受体(例如，整联蛋白)也可以以不同效率插入吞噬体损伤途径中，这可以解释用配体(诸如乳胶珠)进行XP的情况，已经显示所述配体参与Vav-Rac-NADPH氧化酶依赖型XP途径35。然而，可以靶向用于cDC1的XP的抗原的一些受体(诸如甘露糖受体)可能采用不同的P2C机制44、50。此外，清楚的是，不是所有的Syk激活性受体都导致吞噬体损伤和P2C，这支持在Syk激活水平下的信号发散。最值得注意的是，Dectin-1(典型的Syk偶联的带有hemITAM的受体51)不诱导XP，而是促进DC激活和炎性基因表达52。相反，DNGR-1信号传导触发XP，但不诱导DC激活4，这意味着DNGR-1仅充当受体对内化货物的抗原性进行解码。因此，需要从死细胞发出的另外信号来激活交叉呈递cDC1，并使它们有能力使CD8+T细胞致敏(例如，用于抗肿瘤免疫)，如先前在抗体介导的抗原靶向DNGR-1的情况下所述，其中辅助剂是诱导生产性CTL反应所必需的11。因为激活信号也可以影响XP53，所以对它们如何与由专门的XP促进性受体(诸如DNGR-1)发出的信号协同作用的进一步理解，为设计利用CD8+T细胞的能力的免疫疗法和疫苗提供了很大的希望。
专职/非专职抗原呈递细胞
一些免疫细胞(诸如树突细胞和巨噬细胞)被认为是“专职”抗原呈递细胞(专职APC)，因为它们被特化以执行这种功能。虽然许多细胞类型可以在MHC I类分子上执行一定程度的抗原呈递(在某些条件下，特别是当抗原已经在细胞内合成时)，但是除了在MHC I类上呈递抗原之外，专职APC还可以在MHC II类分子上呈递抗原。通过表达DNGR-1或本发明的嵌合受体，可以使并非专职APC的细胞能够在MHC I上交叉呈递抗原。例如，通过在成纤维细胞或肌肉细胞(其是非专职APC)中表达DNGR-1或本发明的嵌合受体，可以使这些细胞能够在MHC I上交叉呈递抗原。
生物聚合物
生物聚合物是聚合生物分子，其在自然界中在生物系统(诸如细胞)中产生，并且其也可以通过生物技术系统(诸如无细胞表达系统)产生。生物聚合物包括多肽、多核苷酸和多糖。包含多肽、多核苷酸和/或多糖结构域的分子被认为是生物聚合物，即使在自然界中没有发现所述肽、核苷酸或糖序列和/或即使所述分子包含另外的非生物分子和/或非聚合物结构域。
药物组合物
药物组合物可以使用由被认为安全且有效的材料构成的药学上可接受的“载体”制备。“药学上可接受的”是指分子实体和组合物在被施用于人时“大体上被视为安全的”，例如，在生理上可耐受并且典型地不产生过敏反应或类似的不良反应，诸如胃部不适等。在一些实施方案中，这个术语是指由美国联邦或州政府的管理机构批准的分子实体和组合物，如联邦食品、药品和化妆品法案第204(s)和409节的GRAS列表(其经历FDA的上市前审查和批准)，或者类似列表、美国药典或用于动物且更特别地用于人的另一公认药典。
术语“载体”是指稀释剂、粘合剂、润滑剂和崩解剂。本领域技术人员熟悉此类药物载体和使用此类载体配混药物组合物的方法。
本文提供的药物组合物可以包含一种或多种赋形剂，例如溶剂、溶解度增强剂、悬浮剂、缓冲剂、等渗剂、抗氧化剂或抗微生物防腐剂。当使用时，组合物的赋形剂不会对组合物中使用的活性成分(即载体、细胞和或嵌合受体)的稳定性、生物利用度、安全性和/或功效造成不良影响。因此，技术人员将理解，提供了其中在剂型的任何组分之间没有不相容性的组合物。赋形剂可以选自缓冲剂、增溶剂、张度剂、螯合剂、抗氧化剂、抗微生物剂和防腐剂。
受试者
待治疗的受试者可以是任何动物或人。所述受试者优选是哺乳动物，更优选是人。所述受试者可以是非人哺乳动物，但更优选是人。所述受试者可以是雄性或雌性。所述受试者可以是患者。治疗用途可以是在人或动物(兽医用途)中。
癌症
“癌症”可以包括以下中的任何一种或多种：急性淋巴细胞白血病(ALL)、急性髓系白血病(AML)、肾上腺皮质癌、肛门癌、膀胱癌、血癌、骨癌、脑瘤、乳腺癌、女性生殖系统癌、男性生殖系统癌、中枢神经系统淋巴瘤、宫颈癌、儿童横纹肌肉瘤、儿童肉瘤、慢性淋巴细胞白血病(CLL)、慢性髓系白血病(CML)、结直肠癌、结肠癌、子宫内膜癌、子宫内膜肉瘤、食管癌、眼癌、胆囊癌、胃癌、胃肠道癌、毛细胞白血病、头颈癌、肝细胞癌、霍奇金病、下咽肿瘤、卡波西肉瘤、肾癌、喉癌、白血病、白血病、肝癌、肺癌、恶性纤维组织细胞瘤、恶性胸腺瘤、黑色素瘤、间皮瘤、多发性骨髓瘤、骨髓瘤、鼻腔和鼻旁窦癌、鼻咽癌、神经系统癌、神经母细胞瘤、非霍奇金淋巴瘤、口腔癌、口咽癌、骨肉瘤、卵巢癌、胰腺癌、甲状旁腺癌、阴茎癌、咽喉癌、垂体瘤、浆细胞瘤、原发性CNS淋巴瘤、前列腺癌、直肠癌、呼吸系统、视网膜母细胞瘤、唾液腺癌、皮肤癌、小肠癌、软组织肉瘤、胃癌、胃癌、睾丸癌、甲状腺癌、泌尿系统癌、子宫肉瘤、阴道癌、血管系统、瓦尔登斯特伦巨球蛋白血症和肾母细胞瘤。
癌症可以是特定类型的。癌症类型的例子包括星形细胞瘤、癌(例如腺癌、肝细胞癌、髓样癌、乳头状癌、鳞状细胞癌)、神经胶质瘤、淋巴瘤、髓母细胞瘤、黑色素瘤、骨髓瘤、脑膜瘤、神经母细胞瘤、肉瘤(例如血管肉瘤、软骨肉瘤、骨肉瘤)。
一些癌症引起实体瘤。此类实体瘤可以位于任何组织中，例如胰腺、肺、乳腺、子宫、胃、肾或睾丸中。相比之下，血液癌症(诸如白血病)可能不会引起实体瘤，并且可以被称为液体瘤。
载体
如本文所用的“载体”是用作将外来遗传物质转移到细胞中的媒介物的寡核苷酸分子(DNA或RNA)。载体可以是用于在细胞中表达外来遗传物质的表达载体。此类载体可以包括可操作地连接到编码待表达的基因序列的核苷酸序列的启动子序列。载体还可以包含终止密码子和表达增强子。本领域已知的任何合适的载体、启动子、增强子和终止密码子可以用于在细胞或组织中表达本发明的嵌合受体。
技术人员将理解，基因治疗载体可以用于将本发明的核酸引入接受者细胞或组织中。在一些实施方案中，所述基因治疗载体是病毒载体。所述病毒载体可以是腺病毒载体、AAV或慢病毒载体。对于一些应用，使用用促进造血干细胞和/或祖细胞转导的包膜蛋白假型化的病毒载体是有利的。在一些实施方案中，使用CRISPR-CAS9系统将核酸引入哺乳动物细胞中。
基于抗体的靶结合结构域
将与本文讨论的靶标结合的抗体是已知的。考虑到目前与单克隆抗体技术相关的技术，可以制备针对大多数抗原的抗体。
靶结合结构域可以是抗体的一部分(例如Fab片段)或合成的抗体片段(例如单链Fv片段[ScFv])。针对所选择的抗原的合适单克隆抗体可以通过已知技术制备，例如以下文献中披露的那些技术：“Monoclonal Antibodies:A manual of techniques”,H Zola(CRCPress,1988)和“Monoclonal Hybridoma Antibodies:Techniques and Applications”,JG R Hurrell(CRC Press,1982)。Neuberger等人(1988,8th InternationalBiotechnology Symposium Part 2,792-799)讨论了嵌合抗体。
单克隆抗体(mAb)可用于本发明的方法中，并且是特异性靶向在抗原上的单个表位的抗体的同质群体。合适的单克隆抗体可以使用本领域熟知的方法制备(例如，参见
G.；Milstein,C.(1975)."Continuous cultures of fused cells secretingantibody of predefined specificity".Nature 256(5517):495；Siegel DL(2002)."Recombinant monoclonal antibody technology"；Schmitz U,Versmold A,Kaufmann P,Frank HG(2000)；"Phage display:a molecular tool for the generation ofantibodies--a review".Placenta.21增刊A:S106-12；Helen E.Chadd和StevenM.Chamow；“Therapeutic antibody expression technology,”Current Opinion inBiotechnology 12,第2期(2001年4月1日):188-194；McCafferty,J.；Griffiths,A.；Winter,G.；Chiswell,D.(1990)."Phage antibodies:filamentous phage displayingantibody variable domains".Nature 348(6301):552-554；"Monoclonal Antibodies:Amanual of techniques",H Zola(CRC Press,1988)；以及"Monoclonal HybridomaAntibodies:Techniques and Applications",J G R Hurrell(CRC Press,1982)。Neuberger等人(1988,8th International Biotechnology Symposium Part 2,792-799))讨论了嵌合抗体。
多克隆抗体可用于本发明的方法中。单特异性多克隆抗体是优选的。合适的多克隆抗体可以使用本领域熟知的方法制备。也可以使用抗体片段(诸如Fab和Fab2片段)作为基因工程化抗体和抗体片段。抗体的可变重链(VH)和可变轻链(VL)结构域参与抗原识别，这个事实是通过早期蛋白酶消化实验首次认识到的。通过啮齿类抗体的“人源化”得到了进一步证实。啮齿类来源的可变结构域可以与人来源的恒定结构域融合，使得所得抗体保留啮齿类亲本抗体的抗原特异性(Morrison等人(1984)Proc.Natl.Acad.Sd.USA 81,6851-6855)。
从涉及抗体片段(都含有一个或多个可变结构域)的细菌表达的实验得知，抗原特异性由可变结构域赋予并且与恒定结构域无关。这些分子包括Fab样分子(Better等人(1988)Science 240,1041)；Fv分子(Skerra等人(1988)Science 240,1038)；单链Fv(ScFv)分子，其中VH和VL配偶体结构域经由柔性寡肽连接(Bird等人(1988)Science 242,423；Huston等人(1988)Proc.Natl.Acad.Sd.USA 85,5879)以及包含分离的V结构域的单结构域抗体(dAb)(Ward等人(1989)Nature 341,544)。保留其特异性结合位点的抗体片段的合成中所涉及技术的一般综述可参见Winter&Milstein(1991)Nature 349,293-299。
“ScFv分子”意指这样的分子，其中VH和VL配偶体结构域共价连接，例如直接连接，通过肽连接或通过柔性寡肽连接。Fab、Fv、ScFv和dAb抗体片段都可以在大肠杆菌中表达并且从大肠杆菌中分泌，从而允许容易地产生大量的所述片段。
完整抗体和F(ab')2片段是“二价的”。“二价的”意指所述抗体和F(ab')2片段具有两个抗原结合位点。相比之下，Fab、Fv、ScFv和dAb片段是单价的，它们仅具有一个抗原结合位点。与本文讨论的靶标结合的合成抗体也可以使用本身为本领域所熟知的噬菌体展示技术来制备(例如参见"Phage display:a molecular tool for the generation ofantibodies--a review".Placenta.21增刊A:S106-12.Helen E.Chadd和StevenM.Chamow；"Phage antibodies:filamentous phage displaying antibody variabledomains".Nature 348(6301):552-554)。
通过在编码抗体链之一(例如重链)的核苷酸序列的3'端可操作地连接编码所需肽或蛋白质的核苷酸序列，可以容易地将抗体与肽或蛋白质部分缀合。因此，可以表达融合抗体，其中抗体多肽的C末端与所需肽或蛋白质融合，通常经由接头序列融合。抗体融合物是本领域熟知的。
基于适配体的靶结合结构域
适配体是短DNA/RNA/肽分子，其可以与靶分子特异性地结合(Pan&Clawson,2009)。对特定靶标具有特异性的适配体通常选自随机产生的分子文库的大库，例如通过使用指数富集的配体系统进化(SELEX)方法来选择。SELEX方法包括将随机序列文库暴露于特定靶标并扩增结合的分子，随后对其进行另外轮次的选择。在多轮选择之后，可以对经鉴定与靶分子结合的特定适配体进行另外轮次的修饰以改进其结合亲和力和稳定性。适配体可以容易地与另外的核酸部分缀合，从而促进所述另外的核酸部分与所述适配体的特异性结合靶标的靶向结合。
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现将参考附图讨论本发明的各方面和实施方案。其他方面和实施方案对于本领域技术人员而言将是清楚的。将本文本中提及的所有文献都通过引用并入本文。
在前述说明书、或所附权利要求书、或附图中公开的特征以其特定形式或在执行所公开的功能的手段、或用于获得所公开的结果的方法或工序方面进行表达，可以酌情单独地或以这样的特征的任何组合加以利用，以其多样的形式来实现本发明。
虽然已经结合上述示例性实施方案描述了本发明，但是当给出本公开文本时，许多等同的修改和变化对于本领域技术人员来说是清楚的。因此，上述本发明的示例性实施方案被认为是说明性的而非限制性的。在不脱离本发明的精神和范围的情况下，可以对所描述的实施方案进行各种改变。
为了避免任何疑问，本文提供的任何理论解释都是为了改善读者的理解。诸位发明人不希望受任何这些理论解释的束缚。
本文使用的任何章节标题仅用于组织目的，不应被解释为限制所描述的主题。
在整个说明书中，包括随后的权利要求，除非上下文另有要求，否则词语“包括”和“包含”，以及诸如“其包括”、“包含了”和“包括了”的变化将被理解为暗示包括所述的整体或步骤或一组整体或步骤，但不排除任何其他整体或步骤或其他组整体或步骤。
必须指出，如在说明书和所附权利要求书中所使用，单数形式“一个/一种(a/an)”和“所述(the)”包括复数指示物，除非上下文另外清楚地规定。范围可以在本文中被表述为从“约”一个特定值和/或至“约”另一个特定值。在表示这样的范围时，另一个实施方案包括从一个特定值和/或至另一个特定值。类似地，当通过使用先行词“约”将值表示为近似值时，将理解特定值形成另一个实施方案。与数值相关的术语“约”是任选的，并且例如意指+/-10％。
实施例
实施例1-DNGR-1敲除的cDC1细胞
为了研究DNGR-1对cDC1的死细胞相关抗原的XP12,15的贡献，诸位发明人使用了cDC1细胞系MuTuDC1940(此后称为MuTuDC)17。将MuTuDC用UV照射的表达卵清蛋白(OVA)的H-2Kbm1小鼠胚胎成纤维细胞(OVA死细胞)进行脉冲处理，然后用预激活的OVA特异性(OT-I)CD8+T细胞培养。培养物中的干扰素-γ(IFN-γ)积累表明H2Kb/OVA复合物的OT-I受体触发，因此是MuTuDC的OVA XP的间接量度。如所报道的18，与使用野生型MuTuDC(WT)的培养物相比，使用DNGR-1缺陷型MuTuDC(KO)的OT-I T培养物积累更低水平的IFN-γ。这种缺陷通过所述受体在DNGR-1缺陷型MuTuDC(KO-WT)中的异位再表达来校正，并且不归因于对抗原摄取的影响，因为KO、WT和KO-WT MuTuDC都展示出类似的内化染料标记的死细胞物质的能力。相比之下，当研究OVA包被的乳胶珠(OVA珠)的摄取时注意到，另外用DNGR-1的生理配体F-肌动蛋白/肌球蛋白II复合物包被16(FM-OVA珠)确实导致更大程度的珠内化(p＜0.0001)，这增强了OVA XP(通过在与预激活的OT-I CD8+T细胞共培养(这增强了OVA XP)后的IFN-γ产生来测量)。因此，DNGR-1可以充当带有配体的颗粒的吞噬受体，但它对于cDC1的死细胞碎片摄取是冗余的，可能是因为所述死细胞碎片含有可以接合另外的cDC1吞噬受体的配体。
为了将DNGR-1对XP的作用与其对配体摄取的贡献分开，诸位发明人用OVA珠或FM-OVA珠饲喂MuTuDC，并且分选已经吞噬单个珠的细胞，之后将其在XP测定中进行测试19。发现含有单个FM-OVA珠的MuTuDC比具有单个OVA珠的细胞更有效地刺激CD8+OT-I T细胞。表明这种作用对XP是特异性的，因为两组分选的MuTuDC(FM-OVA珠摄取；和OVA珠摄取)以相同程度刺激CD4+OT-II T细胞。
对再表达野生型受体(KO-WT)或不能结合F-肌动蛋白的突变受体(W155A/W250A；称为KO-2WA)的DNGR-1缺陷型MuTuDC进行比较，表明了KO-2WA细胞中死细胞相关抗原的XP中的缺陷，但是交叉呈递OVA珠或蛋清(无内毒素的可溶性OVA抗原的来源)的能力未改变。KO-WA MuTuDC内化的FM-OVA珠比KO-WT细胞少，并且展示出刺激OT-I细胞的能力显著降低。因此，如上所述，采用单个FM-OVA珠+MuTuDC，诸位发明人探究DNGR-1的F-肌动蛋白结合区中的突变是否与颗粒摄取无关地影响XP。他们再次观察到在带有不能接合其配体的突变体DNGR-1的细胞中XP的降低。总之，这些数据表明DNGR-1接合对配体相关抗原的XP的特异性作用，这可以在形式上与受体对配体摄取的贡献向区分。
实施例2-交叉呈递的胞质溶胶途径
已经在多种细胞类型的研究中形成XP的两种基本模型，在一种模型中，抗原加工和MHC I类分子加载完全发生在APC的吞噬体/内体区室内(“液泡”途径)，并且在另一种模型中，外源抗原以某种方式得以进入APC胞质溶胶(“胞质溶胶”或“吞噬体至胞质溶胶”(P2C)途径)并如同内源抗原21一样由蛋白酶体进行加工。阻断液泡途径的亮抑蛋白酶肽或胃蛋白酶抑制剂对溶酶体蛋白酶的抑制没有减少可溶性抗原、珠结合的OVA或死细胞相关OVA的XP。相比之下，蛋白酶体抑制剂乳胞素抑制珠结合抗原和死细胞相关抗原两者的XP，但是对可溶性抗原的XP没有影响(除了在高浓度下)或者对用预加工的OVA肽SIINFEKL刺激的T细胞没有不利影响。根据先前的报道22-24，通过氯喹阻断溶酶体酸化或通过E64阻断半胱氨酸蛋白酶增强了FM-OVA珠和死细胞相关抗原的XP。
总之，这些结果表明，通过溶酶体蛋白酶进行的抗原降解对DNGR-1依赖型XP是有害的，并且在cDC1中DNGR-1参与胞质溶胶XP途径而不是液泡XP途径。
实施例3-DNGR-1+吞噬体区室
为了研究DNGR-1如何影响含抗原的吞噬体的特性，我们从MuTuDC中分离了FM-OVA珠吞噬体，并且通过流式细胞术(PhagoFACS)对其进行表征。有趣的是，DNGR-1和溶酶体标记物LAMP-2标记了两个相互排斥的吞噬体群体，通过显微术发现它们在单独细胞中共存。进一步的分析揭示，DNGR-1+吞噬体针对MHC I和II共染色，而LAMP-2+吞噬体是MHC II+，但含有较低水平的MHC I。
重要的是，两个吞噬体群体显示出不同的降解抗原的能力，因为DNGR-1+吞噬体在流式细胞术和显微术测定两者中都展示出高抗OVA染色，而LAMP-2+吞噬体仅弱染色。在长追踪期之后，一些DNGR-1+MHC I+OVA高吞噬体最终丧失DNGR-1染色，获得LAMP并降解OVA，表明它们没有完全停止成熟。DNGR-1+MHC I+OVA高吞噬体不是MuTu DC的异常区室，因为在来源于原代cDC1的吞噬体制剂中也发现了它们，所述原代cDC1从在Flt3L中和在KID细胞(第二cDC1细胞系)中培养的骨髓获得26。此外，DNGR-1在巨噬细胞系RAW264.7中的异位表达还允许鉴定不同于LAMP-2+OVA低吞噬体的DNGR-1+OVA高吞噬体。为了探究形成MHC I+OVA高吞噬体区室是否需要DNGR-1，诸位发明人比较了来自DNGR-1缺陷型(KO)和野生型(WT)MuTuDC的FM-OVA珠吞噬体。有趣的是，在两种细胞中以类似频率鉴定出MHC I+LAMP-2-和MHC I+OVA高吞噬体。我们进一步比较了来自已经用OVA珠与FM-OVA珠饲喂的野生型MuTuDC的DNGR-1+吞噬体，并且发现在OVA降解或MHC I募集方面没有差异。
总之，这些数据表明，DNGR-1标记在cDC1中(以及当异位表达时，在RAW264.7巨噬细胞中)的MHC I+吞噬体区室，所述区室具有低降解潜力和保存未降解抗原的能力，至少是暂时地。然而，DNGR-1或其配体的存在不影响吞噬货物进入这种低降解区室的能力。
实施例4-吞噬体至胞质溶胶的转移(P2C)
我们搜索XP中可能被DNGR-1配体调节的后续步骤。由于DNGR-1依赖型XP是通过胞质溶胶途径介导的(见上文)，我们集中于抗原的吞噬体至胞质溶胶的转移。通过测量胞质溶胶半乳凝素-3或8的局部募集，我们研究了DNGR-1+吞噬体是否显示与破坏一致的膜损伤迹象，所述半乳凝素与附接至在受损内体和吞噬体的腔侧上的膜蛋白的糖部分结合27。引人注目的是，我们通过PhagoFACS发现，4小时之后，对于DNGR-1+吞噬体，与LAMP-2+吞噬体相比，半乳凝素-8+吞噬体在MuTuDC中的频率更高。类似地，通过共聚焦显微术可以发现，当用FM-OVA珠饲喂MuTuDC时，mCherry-半乳凝素-3修饰吞噬体，但在用不触发DNGR-1的OVA珠的情况下频率低得多。
DNGR-1是II型跨膜蛋白，其具有带有单个hemITAM信号传导基序的短N末端细胞内尾28。为了测试DNGR-1hemITAM的作用，我们在用野生型(KO-WT)或没有hemITAM信号传导能力的DNGR-1(在位置7处的酪氨酸至苯丙氨酸突变，即KO-Y7F)重建的DNGR-1缺陷型MuTuDC中表达mCherry-半乳凝素-3。当用FM-OVA珠饲喂细胞时，mCherry-半乳凝素-3被募集到表达WT但不表达Y7F受体的细胞中的吞噬体(图1)。
为了验证这些观察结果，我们使用了内体膜损伤的不同胞质溶胶传感器。鞘磷脂在细胞膜中不对称地分布，并且仅在吞噬体损伤时暴露于胞质溶胶。因此，含有与mCherry融合的鞘磷脂结合蛋白胞溶素(lysenin)形式的探针的胞质溶胶表达允许对受损吞噬体膜进行特异性标记(Ellison等人,已提交)。与使用半乳凝素的结果一致，当表达野生型DNGR-1而不是Y7F DNGR-1时，mCherry-胞溶素探针在FM-OVA珠吞噬体上特异性地积累。诸位发明人推断，配体依赖型DNGR-1经由其hemITAM的信号传导在MutuDC中诱导吞噬体膜损伤。
实施例5-嵌合受体促进P2C
诸位发明人研究了DNGR-1hemITAM信号传导可以在异源系统中介导吞噬体损伤的可能性。用Dectin-1(亦称CLEC7A；一种在结构上与DNGR-1同源的受体)或包含与DNGR-1的胞质尾区的变体融合的Dectin-1的细胞外结构域和跨膜区的嵌合体转染HEK293T细胞。Dectin-1与酵母β-葡聚糖结合并用作酵母吞噬受体，允许我们分析酵母聚糖颗粒(即，酵母细胞壁)而不是乳胶珠的摄取。实际上，Dectin-1(C7)、与野生型(C9/C7)或DNGR-1的hemITAM酪氨酸突变的胞质尾区(C9(Y7F)/C7)融合的Dectin-1都赋予HEK293T摄取酵母聚糖的能力，而Dectin-1的无尾突变体则不能。
值得注意的是，当在HEK293T中共表达mCherry-胞溶素探针和嵌合受体时，在表达C9/C7嵌合体的细胞中观察到胞溶素+吞噬体，但在表达野生型C7(p＜0.0001)或无信号传导能力的嵌合体C9(Y7F)/C7(p＝0.03)的细胞中未观察到胞溶素+吞噬体。与乳胶珠相比，酵母聚糖颗粒是多孔的并且不具有固体核，因此充当进入吞噬体腔的任何探针的海绵。引人注目的是，我们注意到表达C9/C7嵌合体的HEK293T细胞中的吞噬体变为对胞质溶胶中表达的GFP呈阳性。相比之下，在表达C7或C9(Y7F)/C7的HEK293T中，含酵母聚糖的吞噬体中基本上不存在吞噬体内GFP，表明了对DNGR-1hemITAM信号传导的特定需要。
与胞溶素-吞噬体相比，胞溶素+吞噬体显示对GFP的更高平均荧光强度(MFI)，表明胞质溶胶GFP进入吞噬体与膜损伤偶联。这也根据活细胞成像而变得清楚，其表明胞溶素募集预示着GFP流入，但先于GFP流入。这些结果表明，DNGR-1hemITAM信号传导对吞噬体进行渗透化处理，使得胞质溶胶蛋白可进入它们的腔。
为了评估渗透性是否是双向的并且允许吞噬货物释放到胞质溶胶中，将表达C9/C7或C9(Y7F)/C7的HEK293T用浸泡在磺酰罗丹明B(SRB)中的酵母聚糖进行脉冲处理。在表达C9/C7的HEK293T的胞质溶胶中检测到SRB荧光的显著增加，但在表达C9(Y7F)/C7的HEK293T的胞质溶胶中未检测到。
先前报道的P2C测定29也用于证实，与在表达C9(Y7F)/C7的HEK293T细胞中相比，在表达C9/C7的HEK293T细胞中酵母聚糖吸附的β-内酰胺酶可以从吞噬体释放到胞质溶胶中的功效更大。
将酵母聚糖浸泡的细胞色素c颗粒(酵母聚糖-cyt.c颗粒)添加到表达C7、C9/C7和C9(Y7F)/C7的HEK293T细胞中(图3g)。当与酵母聚糖-cyt.c颗粒一起孵育时，表达C7或C9(Y7F)/C7的HEK293T细胞内化所述颗粒，但存活期不受影响。完全相反，所有表达C9/C7的细胞都在24h时段内死亡，证明DNGR-1hemITAM依赖型P2C基本上发生在受体信号传导被触发的所有细胞中。总之，这些结果表明，hemITAM信号传导对吞噬体进行渗透化处理以便允许腔内容物流出到胞质溶胶中，并且当在除DNGR-1外的其他跨膜蛋白的胞质尾区中存在hemITAM基序时，这种作用得以保留。
实施例6-表征hemITAM诱导的吞噬体渗透性
为了检查DNGR-1hemITAM诱导的吞噬体渗透性的性质和持久性，诸位发明人对用胞溶素探针染色的GFP+吞噬体进行了FRAP实验。在对溶胞素+吞噬体腔内的GFP进行光漂白之后，在2分钟内重新观察到信号，表明持续的GFP流入并且表明不可逆的吞噬体膜损伤。作为对照，对胞溶素信号的漂白没有导致荧光恢复。
通过光电联用显微术对表达C9/C7的细胞中的GFP+胞溶素+酵母聚糖吞噬体的超微结构的分析揭示，吞噬体膜含有直径为大约1-1.5μm的大孔。因此，DNGR-1信号传导可以引起吞噬体的大规模破裂，这将允许甚至相当大的腔内容物被释放到胞质溶胶中。
实施例7-HEK293T细胞中的XP
进一步转染稳定表达鼠H-2Kb和β-2微球蛋白的HEK293T细胞系以表达C7、C9/C7或C9(Y7F)/C7嵌合体，克隆并选择单细胞用于相等的H-2Kb和Dectin-1细胞外结构域表达水平。当用外源SIINFEKL肽进行脉冲处理时，所有三种细胞系都显示相当的刺激B3Z(OVA/H-2Kb特异性T细胞杂交瘤)的能力，并且当被转染以表达Venus-SIINFEKL(内源OVA的融合蛋白模拟物)时具有相同的呈递内源抗原的能力。将这些细胞暴露于浸泡在蛋清中的酵母聚糖(酵母聚糖-OVA)。C7、C9/C7和C9(Y7F)/C7 HEK293T细胞系均以类似功效内化酵母聚糖-OVA。然而，如在HEK293T细胞固定之后通过B3Z激活所测量的，仅在使用表达C9/C7嵌合体的细胞时观察到有效的XP(图2)。
然后，用在蛋清和细胞色素c中双重浸泡的酵母聚糖饲喂表达C9/C7的HEK293T细胞。优化暴露时间以仅杀死一部分细胞。在与用仅用OVA浸泡的酵母聚糖颗粒饲喂细胞相比时，这导致交叉呈递活性完全丧失。培养物中细胞色素c的总体细胞毒性被如下事实排除：在将酵母聚糖-cyt.c颗粒与表达C9/C7嵌合体和Venus-SIINFEKL的HEK293T细胞一起孵育时，没有观察到B3Z激活的降低。这些数据在形式上表明，DNGR-1hemITAM信号传导诱导的吞噬体破裂负责XP。
实施例8-hemITAM基序与酪氨酸激酶Syk的相互作用
DNGR-1的hemITAM基序可以响应于DNGR-1配体接合而募集和激活Syk或SHP-112、33。因此，在用抗DNGR-1交联抗体或F-肌动蛋白/肌球蛋白II复合物(DNGR-1配体；DNGR-1L)处理的野生型MuTuDC中，在两个不同位点处观察到Syk磷酸化。诸位发明人现在证实，C9/C7嵌合体也响应于酵母聚糖处理诱导HEK293T中的Syk磷酸化。这是在吞噬体水平上观察到的，并且是hemITAM依赖型的，因为在表达C9(Y7F)/C7的细胞中未观察到(图3)。
为了确定Syk的磷酸化是否在吞噬体破裂的上游，使用CRISPR/Cas9产生表达C9/C7的Syk缺陷型HEK293T细胞(SykCRISPR)。在SykCRISPR细胞中完全没有胞质GFP流入酵母聚糖吞噬体中。然而，通过用(CRISPR抗性)野生型小鼠Syk(Syk WT)而不是催化缺陷型突变体(K396R-激酶死亡；Syk KD)重建表达C9/C7的Syk缺陷型HEK293T细胞恢复了该流入。类似地，当用Syk抑制剂R406处理表达C9/C7的HEK293T细胞时，即使酵母聚糖摄取不受影响，也未观察到吞噬体GFP流入。在MuTu DC中，R406和另一种Syk抑制剂(抑制剂IV)两者都消除了胞溶素探针对吞噬体的染色，并阻断了FM-OVA珠的XP。这表明，诱导吞噬体破裂和XP需要Syk激活和DNGR-1下游的激酶活性。
实施例9-Syk经由活性氧类介导的膜去稳定和破裂
诸位发明人检查了Syk下游可能导致膜去稳定和破裂的可能机制。由NADPH氧化酶产生的活性氧类(ROS)引起脂质过氧化，从而导致膜去稳定和内体内容物泄漏34-37。此外，Syk经由Vav和Rac激活NADPH氧化酶的激活，所有这些都涉及髓样细胞对微粒抗原的XP34-37。诸位发明人注意到，暴露于酵母聚糖导致在异位表达C9/C7而不是C9(Y7F)/C7的RAW264.7细胞中的非常有效的氧化爆发。使用荧光探针，诸位发明人证实这种爆发在单独吞噬体水平上发生，并且在表达C9(Y7F)/C7的RAW264.7细胞中或在用NADPH抑制剂DPI处理的细胞中减少。类似地，在表达DNGR-1(C9)并用固定的和渗透化处理的绵羊红细胞(带有可以被DNGR-1识别的暴露的皮质F-肌动蛋白的吞噬靶标14)饲喂的RAW264.7细胞中，诸位发明人观察到吞噬体周围的氧化爆发，其因SYK抑制或DPI处理而减少。在表达C9/C7的HEK293T细胞中获得了类似的结果。由NADPH产生的活性氧类引起脂质过氧化并导致膜去稳定和内体内容物泄漏37。与这种观点一致，DPI对NADPH氧化酶的抑制在RAW264.7细胞中防止胞溶素在吞噬体上积累。值得注意的是，其还降低了HEK293T细胞对酵母聚糖-OVA的XP，而不影响Venus-SIINFEKL的呈递。最后，在MuTu DC中，DPI以及活性氧类(ROS)清除剂α-生育酚(维生素E)消除了FM-OVA珠或带有OVA的死细胞的XP，但不影响SIINFEKL肽的呈递。这些结果表明NADPH的DNGR-1/Syk依赖型激活引起脂质过氧化，从而允许吞噬体破裂和P2C。这使得内化的配体相关抗原非选择性地释放到cDC1的胞质溶胶中，并允许进入内源MHC I类加工和呈递途径。
由NADPH氧化酶产生的活性氧类(ROS)可以引起脂质过氧化并导致膜去稳定和内体内容物泄漏37。与ROS在膜损伤中的作用一致，DPI对NADPH氧化酶的抑制在表达C9/C7的RAW264.7细胞中以与Y7F突变相同的水平阻断胞溶素在吞噬体上的积累。此外，DPI以及ROS清除剂α-生育酚(维生素E)极大地降低MutuDC中FM-OVA珠和带有OVA的死细胞两者的XP，而不影响SIINFEKL肽的呈递。类似地，DPI降低了表达C9/C7的HEK293T细胞对酵母聚糖-OVA的XP，但没有减少内源Venus-SIINFEKL抗原的呈递。最后，siRNA介导的NOX2(CYBB)(髓样细胞中NADPH氧化酶的主要催化亚基)的沉默降低表达C9/C7和H-2Kb的RAW264.7对酵母聚糖-OVA的XP，但不影响SIINFEKL肽的呈递。
通过用来自野生型、DNGR-1KO和NOX2 KO小鼠的骨髓和通过磁富集纯化的cDC1建立Flt3L培养物，将这些数据扩展至原代cDC1。NOX2 KO cDC1在响应于DNGR-1刺激产生吞噬体ROS方面是有缺陷的，并且DNGR-1KO和NOX2 KO cDC1两者都相对于WT cDC1展示出带有OVA的死细胞的XP的降低，尽管它们在呈递SIINFEKL肽和内化死细胞物质方面同样有效。为了评估NOX2在体内对于DNGR-1依赖型XP的重要性，我们首先用FM-OVA珠+多聚I:C对野生型、DNGR-1KO、BATF3 KO或NOX2 KO小鼠进行，并通过H-2Kb/OVA五聚体染色测量OVA特异性CD8+T细胞应答。如所报道的16，WT小鼠对FM-OVA珠+多聚I:C产生稳健反应，所述反应在DNGR-1KO小鼠和BATF3 KO小鼠两者中显著降低。重要的是，NOX2 KO小鼠还展示出OVA特异性CD8+T细胞交叉致敏的减少。为了证实这反映了cDC1中的NOX2功能并且为了将数据扩展至针对死细胞相关抗原的交叉致敏，我们使用与来自BATF3 KO、野生型、DNGR-1KO或NOX2KO CD45.2小鼠的骨髓以80:20的比率混合的来自BATF3 KO CD45.1小鼠的骨髓产生了辐射嵌合体。诸位发明人使用BATF3 KO CD45.1小鼠作为接受者，进一步确保从CD45.2供体骨髓仅产生在重建之后发育的cDC1，并且我们仅分析CD45.1 T细胞的应答，以排除淋巴细胞中NOX2缺失的任何细胞固有效应。在用OVA+多聚I:C-脉冲处理的死细胞进行免疫后，BATF3KO:BATF3 KO嵌合体未能产生OVA特异性CD8+T细胞，如所预期的18。相比之下，如通过H-2Kb/OVA-四聚体染色或通过响应用SIINFEKL肽的离体脾细胞再刺激针对IFNγ的细胞内染色所测量的，在BATF3 KO:WT嵌合体中观察到对OVA的稳健交联致敏。与先前在DNGR-1KO小鼠中的观察结果3、11、12、44一致，OVA特异性CD8+T细胞应答在BATF3KO:DNGR-1KO嵌合体中显著减少。值得注意的是，其在BATF3 KO:NOX2 KO嵌合体中也显著减少。
巨噬细胞、单核细胞来源的树突细胞和其他髓样细胞类型以及非免疫细胞已广泛用于剖析XP中涉及的一些机制7、23、45。相对少的论文集中在XP机制上，特别是在cDC1中。在此，诸位发明人集中于如下可能性：cDC1在XP中的优势不仅取决于独特的细胞生物学，而且取决于检测相关XP底物的受体(诸如DNGR-1)的表达。诸位发明人表明在吞噬体水平上的配体依赖型DNGR-1信号传导诱导局部NADPH依赖型氧化爆发，所述局部NADPH依赖型氧化爆发使吞噬体膜去稳定，导致破裂和腔内容物大量进入胞质区室，在那里它们可以进入内源MHCI加工途径。值得注意的是，DNGR-1传导信号以致吞噬体破裂的能力是其胞质信号传导结构域固有的，并且可以移植到其他受体上并且可以在其他细胞类型(包括非免疫细胞)中起作用。因此，XP依赖于内体中活性氧类产生的机构。这种机构可以被特化受体破坏，以故意激起液泡膜损伤和P2C。
编号的段落
1.一种嵌合受体，所述嵌合受体包含细胞外靶结合结构域、跨膜结构域和细胞内结构域，所述细胞内结构域包含来源于DNGR-1的胞质尾区的信号传导结构域的Syk结合序列，其中所述Syk结合序列含有酪氨酸残基。
2.根据段落1所述的嵌合受体，其中所述Syk结合序列包含hemITAM。
3.根据段落2所述的嵌合受体，其中所述hemITAM包含SEQ ID NO:14(EXXYXXL；其中X表示任何氨基酸残基)中所示的氨基酸序列。
4.根据段落3所述的嵌合受体，其中所述Syk结合序列包含如SEQ ID NO:15(MHAEXXYXXLQWD)中所示或如SEQ ID NO:90(MHEEXXYXXLQWD)中所示的氨基酸序列；任选地，其中在所述MHAEXXYXXLQWD(SEQ ID NO:15)或MHEEXXYXXLQWD(SEQ ID NO:90)序列的N末端的一个、两个或所有三个氨基酸残基被去除或被另一氨基酸残基取代，或者其中在所述MHAEXXYXXLQWD(SEQ ID NO:15)序列的C末端的一个、两个或所有三个氨基酸残基被去除或被另一氨基酸残基取代。
5.根据段落1-4中任一项所述的嵌合受体，其中所述Syk结合序列包含如SEQ IDNO:11(MHAEEIYTSLQWD)中所示的氨基酸序列或如SEQ ID NO:89(MHEEEIYTSLQWD)中所示的氨基酸序列，任选地其中一个、两个或三个氨基酸残基被另一氨基酸残基取代。
6.根据前述段落中任一项所述的嵌合受体，其中所述靶结合结构域结合存在于病原体、致病细胞、死细胞或患病细胞上的靶标。
7.根据段落6所述的嵌合受体，其中所述靶标是存在于肿瘤细胞上的抗原，任选地其中所述靶标是肿瘤新抗原。
8.根据段落7所述的嵌合受体，其中所述存在于肿瘤细胞上的抗原是CEA、ERBB2、EGFR、GD2、间皮素、MUC1、PSMA、CAIX、CD133、c-Met、EGFR、EGFRvIII、Epcam、EphA2、FRα、CD19、CD20、GPC3、GUCY2C、HER1、HER2、ICAM-1、MAGE或MET。
9.根据段落6所述的嵌合受体，其中所述靶标是存在于病毒颗粒表面上或存在于病毒感染的细胞上的病毒抗原。
10.根据段落9所述的嵌合受体，其中所述病毒抗原是HCMV gB、流感病毒A血凝素、流感病毒基质2蛋白M2e、RSV糖蛋白F、SARS-Cov-2刺突蛋白、HIV gp120或HIV Env。
11.根据前述段落中任一项所述的嵌合受体，其中所述靶结合结构域来源于非DNGR-1凝集素、转铁蛋白受体、FcR、FcγRI、FcγRIIA、TIMD4、Megf10或CD3ζ链的配体结合结构域。
12.根据段落11所述的嵌合受体，其中所述靶结合结构域来源于小鼠Dectin-1。
13.根据前述段落中任一项所述的嵌合受体，其中所述靶结合结构域包含抗体可变区重链(VH)和/或轻链(VL)。
14.根据段落13所述的嵌合受体，其中所述靶结合结构域包含单链可变片段(scFv)。
15.根据前述段落中任一项所述的嵌合受体，其中所述靶结合结构域包含核酸受体的配体结合结构域，并且其中所述靶标是核酸。
16.一种核酸，所述核酸编码根据前述段落中任一项所述的嵌合受体。
17.一种载体，所述载体包含根据段落16所述的核酸。
18.一种宿主细胞，所述宿主细胞包含根据段落16所述的核酸或根据段落17所述的载体。
19.一种能够交叉呈递外源抗原的细胞，所述细胞包含根据段落18所述的宿主细胞和在细胞表面上表达的根据段落1-15中任一项所述的嵌合受体。
20.根据段落18或段落19所述的细胞，其中所述细胞是髓样细胞。
21.根据段落20所述的细胞，其中所述细胞是巨噬细胞、单核细胞或树突细胞。
22.根据段落18或段落19所述的细胞，其中所述细胞是淋巴细胞。
23.根据段落18或段落19所述的细胞，其中所述细胞不是树突细胞。
24.根据段落19所述的细胞，其中所述细胞不是专职抗原呈递细胞。
25.根据段落19至24中任一项所述的细胞，其中所述细胞是成纤维细胞或肌肉细胞。
26.一种产生根据段落19-25中任一项所述的细胞的方法，所述方法包括：
a.提供前体细胞；
b.将根据段落16所述的核酸或根据段落17所述的载体引入所述前体细胞以产生根据段落18所述的宿主细胞
c.在促进由所述核酸编码的嵌合受体的表达的条件下繁殖步骤b.的宿主细胞，使得所述宿主细胞表达所述嵌合受体，并且因此变得能够交叉呈递外源抗原。
27.根据段落19-26中任一项所述的方法，其中所述外源抗原是被所述嵌合受体的靶结合结构域结合的靶标。
28.根据段落19-26中任一项所述的方法，其中所述外源抗原与由所述嵌合受体的靶结合结构域结合的靶标相关。
29.一种药物组合物，所述药物组合物包含根据段落17所述的载体或根据段落19-28中任一项所述的细胞。
30.根据段落29所述的药物组合物，其用于治疗癌症的方法中，所述方法包括向患者施用所述药物组合物。
31.根据段落29所述的药物组合物，其用于治疗患者的感染性疾病的方法中，所述方法包括向所述患者施用所述药物组合物。
32.一种治疗有需要的患者的癌症的方法，所述方法包括向所述患者施用根据段落29所述的药物组合物。
33.一种治疗有需要的患者的感染性疾病的方法，所述方法包括向所述患者施用根据段落29所述的药物组合物。
34.根据段落30所述的用于所述用途的药物组合物或根据段落32所述的治疗癌症的方法，其中所述癌症是实体瘤。
35.根据段落34所述的用于所述用途的药物组合物或治疗癌症的方法，其中所述方法包括将所述药物组合物注射到所述实体瘤中或注射到紧邻所述实体瘤周围的组织中。
36.根据段落29所述的药物组合物，其用作疫苗。
37.一种对受试者进行疫苗接种的方法，所述方法包括向需要疫苗接种的受试者施用根据段落28所述的药物组合物。
38.一种将生物聚合物递送至细胞的胞质溶胶的方法，其中所述细胞表达包含细胞内结构域的跨膜蛋白，所述细胞内结构域包含来源于DNGR-1的胞质尾区的信号传导结构域的Syk结合序列，其中所述生物聚合物包含可以特异性地结合所述跨膜蛋白的细胞外部分的结合结构域，并且其中所述方法包括使所述细胞与所述生物聚合物接触以允许所述结合结构域与所述跨膜蛋白的细胞外部分结合，使得所述生物聚合物被内化并易位到所述胞质溶胶中而不在吞噬体中降解。
39.根据段落38所述的方法，其中所述生物聚合物包含与所述结合结构域共价接合的第二结构域。
40.根据段落39所述的方法，其中所述结合结构域通过接头与所述第二结构域共价接合，所述接头可以被所述细胞的胞质溶胶中存在的蛋白酶切割。
41.根据段落38所述的方法，其中所述生物聚合物与第二生物聚合物非共价缔合，所述第二生物聚合物构成第二结构域。
42.根据段落38-41中任一项所述的方法，其中所述生物聚合物是蛋白质。
43.根据段落38-42中任一项所述的方法，其中所述结合结构域是抗体。
44.根据段落38-43中任一项所述的方法，其中所述第二结构域是任选地编码抗原的核酸。
45.根据段落44所述的方法，其中所述核酸包含能够经由STING途径激活所述细胞的DNA。
46.根据段落44所述的方法，其中所述核酸包含能够经由RIG-I和/或MDA5途径激活所述细胞的RNA。
47.根据段落38-42中任一项所述的方法，其中所述第二结构域是促凋亡蛋白或细胞毒素。
48.根据段落47所述的方法，其中所述促凋亡蛋白或细胞毒素选自细胞色素C、半胱天冬酶、类美登素、尾海兔素、澳瑞他汀药物类似物、念珠藻素、倍癌霉素衍生物、烯二炔抗生素和吡咯并苯二氮
。
49.根据段落38-48中任一项所述的方法，其中所述跨膜蛋白是根据段落1-15中任一项所述的嵌合受体。
50.根据段落38-49中任一项所述的方法，其中所述细胞是肿瘤细胞。
51.根据段落38-49中任一项所述的方法，其中所述细胞是免疫细胞。
52.根据段落38-51中任一项所述的方法，其中所述方法包括在所述细胞与所述生物聚合物接触之前在所述细胞中表达所述跨膜蛋白的步骤。
53.一种治疗方法，所述方法包括根据段落38-52中任一项所述的方法。
54.根据段落53所述的治疗方法，其中所述生物聚合物包含与抗DNGR-1抗体缀合的肿瘤抗原。
55.根据段落54所述的治疗方法，其中将所述生物聚合物施用于癌症患者以引发抗癌Th1反应。
56.根据段落55所述的治疗方法，其中所述生物聚合物包含自身抗原，并且其中将所述生物聚合物施用于患有自身免疫性疾病的患者以引发对所述自身抗原的耐受原反应。
参考文献
上面引用了许多出版物，以便更全面地描述和公开本发明以及本发明所属领域的现状。下面提供了这些参考文献中的一些的完整引用。在本文件中任何地方提到的每个参考文献的整体都通过引用明确地并入本文，如同每个参考文献都以其整体复制一样。
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SEQUENCE LISTING
<110> 弗朗西斯克里克研究所有限公司
<120> 胞质溶胶递送
<130> 007931314
<140>
<141> 2021-07-30
<150> GB 2011859.2
<151> 2020-07-30
<160> 98
<170> PatentIn version 3.5
<210> 1
<211> 241
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Human DNGR-1 (CLEC9A)
<400> 1
Met His Glu Glu Glu Ile Tyr Thr Ser Leu Gln Trp Asp Ser Pro Ala
1 5 10 15
Pro Asp Thr Tyr Gln Lys Cys Leu Ser Ser Asn Lys Cys Ser Gly Ala
20 25 30
Cys Cys Leu Val Met Val Ile Ser Cys Val Phe Cys Met Gly Leu Leu
35 40 45
Thr Ala Ser Ile Phe Leu Gly Val Lys Leu Leu Gln Val Ser Thr Ile
50 55 60
Ala Met Gln Gln Gln Glu Lys Leu Ile Gln Gln Glu Arg Ala Leu Leu
65 70 75 80
Asn Phe Thr Glu Trp Lys Arg Ser Cys Ala Leu Gln Met Lys Tyr Cys
85 90 95
Gln Ala Phe Met Gln Asn Ser Leu Ser Ser Ala His Asn Ser Ser Pro
100 105 110
Cys Pro Asn Asn Trp Ile Gln Asn Arg Glu Ser Cys Tyr Tyr Val Ser
115 120 125
Glu Ile Trp Ser Ile Trp His Thr Ser Gln Glu Asn Cys Leu Lys Glu
130 135 140
Gly Ser Thr Leu Leu Gln Ile Glu Ser Lys Glu Glu Met Asp Phe Ile
145 150 155 160
Thr Gly Ser Leu Arg Lys Ile Lys Gly Ser Tyr Asp Tyr Trp Val Gly
165 170 175
Leu Ser Gln Asp Gly His Ser Gly Arg Trp Leu Trp Gln Asp Gly Ser
180 185 190
Ser Pro Ser Pro Gly Leu Leu Pro Ala Glu Arg Ser Gln Ser Ala Asn
195 200 205
Gln Val Cys Gly Tyr Val Lys Ser Asn Ser Leu Leu Ser Ser Asn Cys
210 215 220
Ser Thr Trp Lys Tyr Phe Ile Cys Glu Lys Tyr Ala Leu Arg Ser Ser
225 230 235 240
Val
<210> 2
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of human DNGR-1
<400> 2
Met His Glu Glu Glu Ile Tyr Thr Ser Leu Gln Trp Asp Ser Pro Ala
1 5 10 15
Pro Asp Thr Tyr Gln Lys Cys Leu Ser Ser Asn Lys Cys Ser Gly Ala
20 25 30
<210> 3
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of human DNGR-1
<400> 3
Cys Cys Leu Val Met Val Ile Ser Cys Val Phe Cys Met Gly Leu Leu
1 5 10 15
Thr Ala Ser Ile Phe Leu Gly Val
20
<210> 4
<211> 55
<212> PRT
<213> Artificial Sequence
<220>
<223> The neck domain of human DNGR-1
<400> 4
Lys Leu Leu Gln Val Ser Thr Ile Ala Met Gln Gln Gln Glu Lys Leu
1 5 10 15
Ile Gln Gln Glu Arg Ala Leu Leu Asn Phe Thr Glu Trp Lys Arg Ser
20 25 30
Cys Ala Leu Gln Met Lys Tyr Cys Gln Ala Phe Met Gln Asn Ser Leu
35 40 45
Ser Ser Ala His Asn Ser Ser
50 55
<210> 5
<211> 125
<212> PRT
<213> Artificial Sequence
<220>
<223> The C-type lectin domain (CTLD) of human DNGR-1
<400> 5
Pro Cys Pro Asn Asn Trp Ile Gln Asn Arg Glu Ser Cys Tyr Tyr Val
1 5 10 15
Ser Glu Ile Trp Ser Ile Trp His Thr Ser Gln Glu Asn Cys Leu Lys
20 25 30
Glu Gly Ser Thr Leu Leu Gln Ile Glu Ser Lys Glu Glu Met Asp Phe
35 40 45
Ile Thr Gly Ser Leu Arg Lys Ile Lys Gly Ser Tyr Asp Tyr Trp Val
50 55 60
Gly Leu Ser Gln Asp Gly His Ser Gly Arg Trp Leu Trp Gln Asp Gly
65 70 75 80
Ser Ser Pro Ser Pro Gly Leu Leu Pro Ala Glu Arg Ser Gln Ser Ala
85 90 95
Asn Gln Val Cys Gly Tyr Val Lys Ser Asn Ser Leu Leu Ser Ser Asn
100 105 110
Cys Ser Thr Trp Lys Tyr Phe Ile Cys Glu Lys Tyr Ala
115 120 125
<210> 6
<211> 264
<212> PRT
<213> Mus musculus
<220>
<221> misc_feature
<223> Mouse DNGR-1 (CLEC9A)
<400> 6
Met His Ala Glu Glu Ile Tyr Thr Ser Leu Gln Trp Asp Ile Pro Thr
1 5 10 15
Ser Glu Ala Ser Gln Lys Cys Gln Ser Pro Ser Lys Cys Ser Gly Ala
20 25 30
Trp Cys Val Val Thr Met Ile Ser Cys Val Val Cys Met Gly Leu Leu
35 40 45
Ala Thr Ser Ile Phe Leu Gly Ile Lys Phe Phe Gln Val Ser Ser Leu
50 55 60
Val Leu Glu Gln Gln Glu Arg Leu Ile Gln Gln Asp Thr Ala Leu Val
65 70 75 80
Asn Leu Thr Gln Trp Gln Arg Lys Tyr Thr Leu Glu Tyr Cys Gln Ala
85 90 95
Leu Leu Gln Arg Ser Leu His Ser Gly Thr Asp Ala Ser Thr Gly Pro
100 105 110
Val Leu Leu Thr Ser Pro Gln Met Val Pro Gln Thr Leu Asp Ser Lys
115 120 125
Glu Thr Gly Ser Asp Cys Ser Pro Cys Pro His Asn Trp Ile Gln Asn
130 135 140
Gly Lys Ser Cys Tyr Tyr Val Phe Glu Arg Trp Glu Met Trp Asn Ile
145 150 155 160
Ser Lys Lys Ser Cys Leu Lys Glu Gly Ala Ser Leu Phe Gln Ile Asp
165 170 175
Ser Lys Glu Glu Met Glu Phe Ile Ser Ser Ile Gly Lys Leu Lys Gly
180 185 190
Gly Asn Lys Tyr Trp Val Gly Val Phe Gln Asp Gly Ile Ser Gly Ser
195 200 205
Trp Phe Trp Glu Asp Gly Ser Ser Pro Leu Ser Asp Leu Leu Pro Ala
210 215 220
Glu Arg Gln Arg Ser Ala Gly Gln Ile Cys Gly Tyr Leu Lys Asp Ser
225 230 235 240
Thr Leu Ile Ser Asp Lys Cys Asp Ser Trp Lys Tyr Phe Ile Cys Glu
245 250 255
Lys Lys Ala Phe Gly Ser Cys Ile
260
<210> 7
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of mouse DNGR-1
<400> 7
Met His Ala Glu Glu Ile Tyr Thr Ser Leu Gln Trp Asp Ile Pro Thr
1 5 10 15
Ser Glu Ala Ser Gln Lys Cys Gln Ser Pro Ser Lys Cys Ser Gly Ala
20 25 30
<210> 8
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of mouse DNGR-1
<400> 8
Trp Cys Val Val Thr Met Ile Ser Cys Val Val Cys Met Gly Leu Leu
1 5 10 15
Ala Thr Ser Ile Phe Leu Gly Ile
20
<210> 9
<211> 79
<212> PRT
<213> Artificial Sequence
<220>
<223> The neck domain of mouse DNGR-1
<400> 9
Lys Phe Phe Gln Val Ser Ser Leu Val Leu Glu Gln Gln Glu Arg Leu
1 5 10 15
Ile Gln Gln Asp Thr Ala Leu Val Asn Leu Thr Gln Trp Gln Arg Lys
20 25 30
Tyr Thr Leu Glu Tyr Cys Gln Ala Leu Leu Gln Arg Ser Leu His Ser
35 40 45
Gly Thr Asp Ala Ser Thr Gly Pro Val Leu Leu Thr Ser Pro Gln Met
50 55 60
Val Pro Gln Thr Leu Asp Ser Lys Glu Thr Gly Ser Asp Cys Ser
65 70 75
<210> 10
<211> 124
<212> PRT
<213> Artificial Sequence
<220>
<223> The C-type lectin domain (CTLD) of mouse DNGR-1
<400> 10
Pro Cys Pro His Asn Trp Ile Gln Asn Gly Lys Ser Cys Tyr Tyr Val
1 5 10 15
Phe Glu Arg Trp Glu Met Trp Asn Ile Ser Lys Lys Ser Cys Leu Lys
20 25 30
Glu Gly Ala Ser Leu Phe Gln Ile Asp Ser Lys Glu Glu Met Glu Phe
35 40 45
Ile Ser Ser Ile Gly Lys Leu Lys Gly Gly Asn Lys Tyr Trp Val Gly
50 55 60
Val Phe Gln Asp Gly Ile Ser Gly Ser Trp Phe Trp Glu Asp Gly Ser
65 70 75 80
Ser Pro Leu Ser Asp Leu Leu Pro Ala Glu Arg Gln Arg Ser Ala Gly
85 90 95
Gln Ile Cys Gly Tyr Leu Lys Asp Ser Thr Leu Ile Ser Asp Lys Cys
100 105 110
Asp Ser Trp Lys Tyr Phe Ile Cys Glu Lys Lys Ala
115 120
<210> 11
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> The signalling domain of the cytoplasmic tail of DNGR-1
<400> 11
Met His Ala Glu Glu Ile Tyr Thr Ser Leu Gln Trp Asp
1 5 10
<210> 12
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> hemITAM domain
<400> 12
Glu Glu Ile Tyr Thr Ser Leu
1 5
<210> 13
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> hemITAM domain
<220>
<221> misc_feature
<222> (6)..(7)
<223> Xaa can be any naturally occurring amino acid
<400> 13
Asp Glu Asp Gly Tyr Xaa Xaa Leu
1 5
<210> 14
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> hemITAM domain
<220>
<221> misc_feature
<222> (2)..(3)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (5)..(6)
<223> Xaa can be any naturally occurring amino acid
<400> 14
Glu Xaa Xaa Tyr Xaa Xaa Leu
1 5
<210> 15
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> intracellular signalling domain
<220>
<221> misc_feature
<222> (5)..(6)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (8)..(9)
<223> Xaa can be any naturally occurring amino acid
<400> 15
Met His Ala Glu Xaa Xaa Tyr Xaa Xaa Leu Gln Trp Asp
1 5 10
<210> 16
<211> 244
<212> PRT
<213> Mus musculus
<220>
<221> misc_feature
<223> Mouse Dectin-1 (CLEC7A)
<400> 16
Met Lys Tyr His Ser His Ile Glu Asn Leu Asp Glu Asp Gly Tyr Thr
1 5 10 15
Gln Leu Asp Phe Ser Thr Gln Asp Ile His Lys Arg Pro Arg Gly Ser
20 25 30
Glu Lys Gly Ser Arg Ala Pro Ser Ser Pro Trp Arg Pro Ile Ala Val
35 40 45
Gly Leu Gly Ile Leu Cys Phe Val Val Val Val Val Ala Ala Val Leu
50 55 60
Gly Ala Leu Ala Phe Trp Arg His Asn Ser Gly Arg Asn Pro Glu Glu
65 70 75 80
Lys Asp Ser Phe Leu Ser Arg Asn Lys Glu Asn His Lys Pro Thr Glu
85 90 95
Ser Ser Leu Asp Glu Lys Val Ala Pro Ser Lys Ala Ser Gln Thr Thr
100 105 110
Gly Gly Phe Ser Gln Ser Cys Leu Pro Asn Trp Ile Met His Gly Lys
115 120 125
Ser Cys Tyr Leu Phe Ser Phe Ser Gly Asn Ser Trp Tyr Gly Ser Lys
130 135 140
Arg His Cys Ser Gln Leu Gly Ala His Leu Leu Lys Ile Asp Asn Ser
145 150 155 160
Lys Glu Phe Glu Phe Ile Glu Ser Gln Thr Ser Ser His Arg Ile Asn
165 170 175
Ala Phe Trp Ile Gly Leu Ser Arg Asn Gln Ser Glu Gly Pro Trp Phe
180 185 190
Trp Glu Asp Gly Ser Ala Phe Phe Pro Asn Ser Phe Gln Val Arg Asn
195 200 205
Ala Val Pro Gln Glu Ser Leu Leu His Asn Cys Val Trp Ile His Gly
210 215 220
Ser Glu Val Tyr Asn Gln Ile Cys Asn Thr Ser Ser Tyr Ser Ile Cys
225 230 235 240
Glu Lys Glu Leu
<210> 17
<211> 44
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of mouse Dectin-1
<400> 17
Met Lys Tyr His Ser His Ile Glu Asn Leu Asp Glu Asp Gly Tyr Thr
1 5 10 15
Gln Leu Asp Phe Ser Thr Gln Asp Ile His Lys Arg Pro Arg Gly Ser
20 25 30
Glu Lys Gly Ser Arg Ala Pro Ser Ser Pro Trp Arg
35 40
<210> 18
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of mouse Dectin-1
<400> 18
Val Gly Leu Gly Ile Leu Cys Phe Val Val Val Val Val Ala Ala Val
1 5 10 15
Leu Gly Ala Leu Ala Phe Trp
20
<210> 19
<211> 55
<212> PRT
<213> Artificial Sequence
<220>
<223> The neck domain of mouse Dectin-1
<400> 19
Arg His Asn Ser Gly Arg Asn Pro Glu Glu Lys Asp Ser Phe Leu Ser
1 5 10 15
Arg Asn Lys Glu Asn His Lys Pro Thr Glu Ser Ser Leu Asp Glu Lys
20 25 30
Val Ala Pro Ser Lys Ala Ser Gln Thr Thr Gly Gly Phe Ser Gln Ser
35 40 45
Cys Leu Pro Asn Trp Ile Met
50 55
<210> 20
<211> 116
<212> PRT
<213> Artificial Sequence
<220>
<223> The C-type lectin domain (CTLD) of mouse Dectin-1
<400> 20
His Gly Lys Ser Cys Tyr Leu Phe Ser Phe Ser Gly Asn Ser Trp Tyr
1 5 10 15
Gly Ser Lys Arg His Cys Ser Gln Leu Gly Ala His Leu Leu Lys Ile
20 25 30
Asp Asn Ser Lys Glu Phe Glu Phe Ile Glu Ser Gln Thr Ser Ser His
35 40 45
Arg Ile Asn Ala Phe Trp Ile Gly Leu Ser Arg Asn Gln Ser Glu Gly
50 55 60
Pro Trp Phe Trp Glu Asp Gly Ser Ala Phe Phe Pro Asn Ser Phe Gln
65 70 75 80
Val Arg Asn Ala Val Pro Gln Glu Ser Leu Leu His Asn Cys Val Trp
85 90 95
Ile His Gly Ser Glu Val Tyr Asn Gln Ile Cys Asn Thr Ser Ser Tyr
100 105 110
Ser Ile Cys Glu
115
<210> 21
<211> 86
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Human FcR gamma chain
<400> 21
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> 22
<211> 42
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of human FcR gamma chain
<400> 22
Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys
1 5 10 15
Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr
20 25 30
Glu Thr Leu Lys His Glu Lys Pro Pro Gln
35 40
<210> 23
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of human FcR gamma chain
<400> 23
Leu Cys Tyr Ile Leu Asp Ala Ile Leu Phe Leu Tyr Gly Ile Val Leu
1 5 10 15
Thr Leu Leu Tyr Cys
20
<210> 24
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of human FcR gamma chain
<400> 24
Leu Gly Glu Pro Gln
1 5
<210> 25
<211> 86
<212> PRT
<213> Mus musculus
<220>
<221> misc_feature
<223> Mouse FcR gamma chain
<400> 25
Met Ile Ser Ala Val Ile Leu Phe 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 Val 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 Ala Ser Arg Glu Lys Ala Asp Ala Val
50 55 60
Tyr Thr Gly Leu Asn Thr Arg Ser Gln Glu Thr Tyr Glu Thr Leu Lys
65 70 75 80
His Glu Lys Pro Pro Gln
85
<210> 26
<211> 42
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of mouse FcR gamma chain
<400> 26
Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile Ala Ser Arg Glu Lys
1 5 10 15
Ala Asp Ala Val Tyr Thr Gly Leu Asn Thr Arg Ser Gln Glu Thr Tyr
20 25 30
Glu Thr Leu Lys His Glu Lys Pro Pro Gln
35 40
<210> 27
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of mouse FcR gamma chain
<400> 27
Leu Cys Tyr Ile Leu Asp Ala Val Leu Phe Leu Tyr Gly Ile Val Leu
1 5 10 15
Thr Leu Leu Tyr Cys
20
<210> 28
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of mouse FcR gamma chain
<400> 28
Leu Gly Glu Pro Gln
1 5
<210> 29
<211> 374
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Human Fc gamma RI
<400> 29
Met Trp Phe Leu Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln
1 5 10 15
Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser
20 25 30
Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu
35 40 45
Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln
50 55 60
Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser
65 70 75 80
Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile
85 90 95
Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg
100 105 110
Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys
115 120 125
Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe
130 135 140
Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile
145 150 155 160
Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr
165 170 175
Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro
180 185 190
Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val
195 200 205
Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln
210 215 220
Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn
225 230 235 240
Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly
245 250 255
Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg
260 265 270
Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro
275 280 285
Val Trp Phe His Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu
290 295 300
Val Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys
305 310 315 320
Lys Lys Trp Asp Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys
325 330 335
Val Ile Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys
340 345 350
Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys
355 360 365
Glu Pro Gln Gly Ala Thr
370
<210> 30
<211> 61
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of human Fc gamma RI
<400> 30
Arg Lys Glu Leu Lys Arg Lys Lys Lys Trp Asp Leu Glu Ile Ser Leu
1 5 10 15
Asp Ser Gly His Glu Lys Lys Val Ile Ser Ser Leu Gln Glu Asp Arg
20 25 30
His Leu Glu Glu Glu Leu Lys Cys Gln Glu Gln Lys Glu Glu Gln Leu
35 40 45
Gln Glu Gly Val His Arg Lys Glu Pro Gln Gly Ala Thr
50 55 60
<210> 31
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of human Fc gamma RI
<400> 31
Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu Val Asn Thr Val
1 5 10 15
Leu Trp Val Thr Ile
20
<210> 32
<211> 277
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of human Fc gamma RI
<400> 32
Gln Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val
1 5 10 15
Ser Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His
20 25 30
Leu Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr
35 40 45
Gln Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp
50 55 60
Ser Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro
65 70 75 80
Ile Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser
85 90 95
Arg Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp
100 105 110
Lys Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala
115 120 125
Phe Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn
130 135 140
Ile Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg
145 150 155 160
Tyr Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala
165 170 175
Pro Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu
180 185 190
Val Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu
195 200 205
Gln Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg
210 215 220
Asn Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser
225 230 235 240
Gly Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys
245 250 255
Arg Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr
260 265 270
Pro Val Trp Phe His
275
<210> 33
<211> 404
<212> PRT
<213> Mus musculus
<220>
<221> misc_feature
<223> Mouse Fc gamma RI
<400> 33
Met Ile Leu Thr Ser Phe Gly Asp Asp Met Trp Leu Leu Thr Thr Leu
1 5 10 15
Leu Leu Trp Val Pro Val Gly Gly Glu Val Val Asn Ala Thr Lys Ala
20 25 30
Val Ile Thr Leu Gln Pro Pro Trp Val Ser Ile Phe Gln Lys Glu Asn
35 40 45
Val Thr Leu Trp Cys Glu Gly Pro His Leu Pro Gly Asp Ser Ser Thr
50 55 60
Gln Trp Phe Ile Asn Gly Thr Ala Val Gln Ile Ser Thr Pro Ser Tyr
65 70 75 80
Ser Ile Pro Glu Ala Ser Phe Gln Asp Ser Gly Glu Tyr Arg Cys Gln
85 90 95
Ile Gly Ser Ser Met Pro Ser Asp Pro Val Gln Leu Gln Ile His Asn
100 105 110
Asp Trp Leu Leu Leu Gln Ala Ser Arg Arg Val Leu Thr Glu Gly Glu
115 120 125
Pro Leu Ala Leu Arg Cys His Gly Trp Lys Asn Lys Leu Val Tyr Asn
130 135 140
Val Val Phe Tyr Arg Asn Gly Lys Ser Phe Gln Phe Ser Ser Asp Ser
145 150 155 160
Glu Val Ala Ile Leu Lys Thr Asn Leu Ser His Ser Gly Ile Tyr His
165 170 175
Cys Ser Gly Thr Gly Arg His Arg Tyr Thr Ser Ala Gly Val Ser Ile
180 185 190
Thr Val Lys Glu Leu Phe Thr Thr Pro Val Leu Arg Ala Ser Val Ser
195 200 205
Ser Pro Phe Pro Glu Gly Ser Leu Val Thr Leu Asn Cys Glu Thr Asn
210 215 220
Leu Leu Leu Gln Arg Pro Gly Leu Gln Leu His Phe Ser Phe Tyr Val
225 230 235 240
Gly Ser Lys Ile Leu Glu Tyr Arg Asn Thr Ser Ser Glu Tyr His Ile
245 250 255
Ala Arg Ala Glu Arg Glu Asp Ala Gly Phe Tyr Trp Cys Glu Val Ala
260 265 270
Thr Glu Asp Ser Ser Val Leu Lys Arg Ser Pro Glu Leu Glu Leu Gln
275 280 285
Val Leu Gly Pro Gln Ser Ser Ala Pro Val Trp Phe His Ile Leu Phe
290 295 300
Tyr Leu Ser Val Gly Ile Met Phe Ser Leu Asn Thr Val Leu Tyr Val
305 310 315 320
Lys Ile His Arg Leu Gln Arg Glu Lys Lys Tyr Asn Leu Glu Val Pro
325 330 335
Leu Val Ser Glu Gln Gly Lys Lys Ala Asn Ser Phe Gln Gln Val Arg
340 345 350
Ser Asp Gly Val Tyr Glu Glu Val Thr Ala Thr Ala Ser Gln Thr Thr
355 360 365
Pro Lys Glu Ala Pro Asp Gly Pro Arg Ser Ser Val Gly Asp Cys Gly
370 375 380
Pro Glu Gln Pro Glu Pro Leu Pro Pro Ser Asp Ser Thr Gly Ala Gln
385 390 395 400
Thr Ser Gln Ser
<210> 34
<211> 84
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of mouse Fc gamma RI
<400> 34
Lys Ile His Arg Leu Gln Arg Glu Lys Lys Tyr Asn Leu Glu Val Pro
1 5 10 15
Leu Val Ser Glu Gln Gly Lys Lys Ala Asn Ser Phe Gln Gln Val Arg
20 25 30
Ser Asp Gly Val Tyr Glu Glu Val Thr Ala Thr Ala Ser Gln Thr Thr
35 40 45
Pro Lys Glu Ala Pro Asp Gly Pro Arg Ser Ser Val Gly Asp Cys Gly
50 55 60
Pro Glu Gln Pro Glu Pro Leu Pro Pro Ser Asp Ser Thr Gly Ala Gln
65 70 75 80
Thr Ser Gln Ser
<210> 35
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of mouse Fc gamma RI
<400> 35
Val Trp Phe His Ile Leu Phe Tyr Leu Ser Val Gly Ile Met Phe Ser
1 5 10 15
Leu Asn Thr Val Leu Tyr Val
20
<210> 36
<211> 273
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of mouse Fc gamma RI
<400> 36
Glu Val Val Asn Ala Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp
1 5 10 15
Val Ser Ile Phe Gln Lys Glu Asn Val Thr Leu Trp Cys Glu Gly Pro
20 25 30
His Leu Pro Gly Asp Ser Ser Thr Gln Trp Phe Ile Asn Gly Thr Ala
35 40 45
Val Gln Ile Ser Thr Pro Ser Tyr Ser Ile Pro Glu Ala Ser Phe Gln
50 55 60
Asp Ser Gly Glu Tyr Arg Cys Gln Ile Gly Ser Ser Met Pro Ser Asp
65 70 75 80
Pro Val Gln Leu Gln Ile His Asn Asp Trp Leu Leu Leu Gln Ala Ser
85 90 95
Arg Arg Val Leu Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Gly
100 105 110
Trp Lys Asn Lys Leu Val Tyr Asn Val Val Phe Tyr Arg Asn Gly Lys
115 120 125
Ser Phe Gln Phe Ser Ser Asp Ser Glu Val Ala Ile Leu Lys Thr Asn
130 135 140
Leu Ser His Ser Gly Ile Tyr His Cys Ser Gly Thr Gly Arg His Arg
145 150 155 160
Tyr Thr Ser Ala Gly Val Ser Ile Thr Val Lys Glu Leu Phe Thr Thr
165 170 175
Pro Val Leu Arg Ala Ser Val Ser Ser Pro Phe Pro Glu Gly Ser Leu
180 185 190
Val Thr Leu Asn Cys Glu Thr Asn Leu Leu Leu Gln Arg Pro Gly Leu
195 200 205
Gln Leu His Phe Ser Phe Tyr Val Gly Ser Lys Ile Leu Glu Tyr Arg
210 215 220
Asn Thr Ser Ser Glu Tyr His Ile Ala Arg Ala Glu Arg Glu Asp Ala
225 230 235 240
Gly Phe Tyr Trp Cys Glu Val Ala Thr Glu Asp Ser Ser Val Leu Lys
245 250 255
Arg Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Pro Gln Ser Ser Ala
260 265 270
Pro
<210> 37
<211> 317
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Human Fc gamma RIIA
<400> 37
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> 38
<211> 77
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of human Fc gamma RIIA
<400> 38
Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala
1 5 10 15
Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg
20 25 30
Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr
35 40 45
Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr
50 55 60
Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn
65 70 75
<210> 39
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of human Fc gamma RIIA
<400> 39
Ile Ile Val Ala Val Val Ile Ala Thr Ala Val Ala Ala Ile Val Ala
1 5 10 15
Ala Val Val Ala Leu Ile Tyr
20
<210> 40
<211> 184
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of human Fc gamma RIIA
<400> 40
Gln Ala Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp
1 5 10 15
Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly Ala
20 25 30
Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn Leu
35 40 45
Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn
50 55 60
Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp
65 70 75 80
Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro
85 90 95
His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser
100 105 110
Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys
115 120 125
Ser Gln Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile Pro Gln Ala
130 135 140
Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr
145 150 155 160
Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro Ser
165 170 175
Met Gly Ser Ser Ser Pro Met Gly
180
<210> 41
<211> 378
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Human TIMD4
<400> 41
Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp
1 5 10 15
Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu
20 25 30
Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn
35 40 45
Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys
50 55 60
Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys
65 70 75 80
Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser
85 90 95
Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys
100 105 110
Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg
115 120 125
Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr
130 135 140
Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met
145 150 155 160
Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp
165 170 175
Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr
180 185 190
Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu
195 200 205
Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu
210 215 220
Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val
225 230 235 240
Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys
245 250 255
Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser
260 265 270
Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro
275 280 285
Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met
290 295 300
Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala
305 310 315 320
Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu
325 330 335
Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg Leu
340 345 350
Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His Gly
355 360 365
Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu
370 375
<210> 42
<211> 43
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of human TIMD4
<400> 42
Leu Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg
1 5 10 15
Leu Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His
20 25 30
Gly Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu
35 40
<210> 43
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of human TIMD4
<400> 43
Leu Leu Met Ile Ile Ala Pro Ser Leu Gly Phe Val Leu Phe Ala Leu
1 5 10 15
Phe Val Ala Phe Leu
20
<210> 44
<211> 290
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of human TIMD4
<400> 44
Glu Thr Val Val Thr Glu Val Leu Gly His Arg Val Thr Leu Pro Cys
1 5 10 15
Leu Tyr Ser Ser Trp Ser His Asn Ser Asn Ser Met Cys Trp Gly Lys
20 25 30
Asp Gln Cys Pro Tyr Ser Gly Cys Lys Glu Ala Leu Ile Arg Thr Asp
35 40 45
Gly Met Arg Val Thr Ser Arg Lys Ser Ala Lys Tyr Arg Leu Gln Gly
50 55 60
Thr Ile Pro Arg Gly Asp Val Ser Leu Thr Ile Leu Asn Pro Ser Glu
65 70 75 80
Ser Asp Ser Gly Val Tyr Cys Cys Arg Ile Glu Val Pro Gly Trp Phe
85 90 95
Asn Asp Val Lys Ile Asn Val Arg Leu Asn Leu Gln Arg Ala Ser Thr
100 105 110
Thr Thr His Arg Thr Ala Thr Thr Thr Thr Arg Arg Thr Thr Thr Thr
115 120 125
Ser Pro Thr Thr Thr Arg Gln Met Thr Thr Thr Pro Ala Ala Leu Pro
130 135 140
Thr Thr Val Val Thr Thr Pro Asp Leu Thr Thr Gly Thr Pro Leu Gln
145 150 155 160
Met Thr Thr Ile Ala Val Phe Thr Thr Ala Asn Thr Cys Leu Ser Leu
165 170 175
Thr Pro Ser Thr Leu Pro Glu Glu Ala Thr Gly Leu Leu Thr Pro Glu
180 185 190
Pro Ser Lys Glu Gly Pro Ile Leu Thr Ala Glu Ser Glu Thr Val Leu
195 200 205
Pro Ser Asp Ser Trp Ser Ser Val Glu Ser Thr Ser Ala Asp Thr Val
210 215 220
Leu Leu Thr Ser Lys Glu Ser Lys Val Trp Asp Leu Pro Ser Thr Ser
225 230 235 240
His Val Ser Met Trp Lys Thr Ser Asp Ser Val Ser Ser Pro Gln Pro
245 250 255
Gly Ala Ser Asp Thr Ala Val Pro Glu Gln Asn Lys Thr Thr Lys Thr
260 265 270
Gly Gln Met Asp Gly Ile Pro Met Ser Met Lys Asn Glu Met Pro Ile
275 280 285
Ser Gln
290
<210> 45
<211> 343
<212> PRT
<213> Mus musculus
<220>
<221> misc_feature
<223> Mouse TIMD4
<400> 45
Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp
1 5 10 15
Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu
20 25 30
Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser
35 40 45
Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys
50 55 60
Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys
65 70 75 80
Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser
85 90 95
Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys
100 105 110
Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg
115 120 125
Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr
130 135 140
Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu
145 150 155 160
Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro
165 170 175
Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro
180 185 190
Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala
195 200 205
Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg
210 215 220
Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly
225 230 235 240
Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys
245 250 255
Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His
260 265 270
Gln Ile Asn Ser Arg Gln Thr Ile Leu Ile Ile Ala Cys Cys Val Gly
275 280 285
Phe Val Leu Met Val Leu Leu Phe Leu Ala Phe Leu Leu Arg Gly Lys
290 295 300
Val Thr Gly Ala Asn Cys Leu Gln Arg His Lys Arg Pro Asp Asn Thr
305 310 315 320
Glu Asp Ser Asp Ser Val Leu Asn Asp Met Ser His Gly Arg Asp Asp
325 330 335
Glu Asp Gly Ile Phe Thr Leu
340
<210> 46
<211> 43
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of mouse TIMD4
<400> 46
Leu Arg Gly Lys Val Thr Gly Ala Asn Cys Leu Gln Arg His Lys Arg
1 5 10 15
Pro Asp Asn Thr Glu Asp Ser Asp Ser Val Leu Asn Asp Met Ser His
20 25 30
Gly Arg Asp Asp Glu Asp Gly Ile Phe Thr Leu
35 40
<210> 47
<211> 257
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of mouse TIMD4
<400> 47
Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu Gly Gln Pro Val Thr Leu
1 5 10 15
Pro Cys His Tyr Leu Ser Trp Ser Gln Ser Arg Asn Ser Met Cys Trp
20 25 30
Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys Asn Ala Glu Leu Leu Arg
35 40 45
Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys Ser Thr Lys Tyr Thr Leu
50 55 60
Leu Gly Lys Val Gln Phe Gly Glu Val Ser Leu Thr Ile Ser Asn Thr
65 70 75 80
Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys Arg Ile Glu Val Pro Gly
85 90 95
Trp Phe Asn Asp Val Lys Lys Asn Val Arg Leu Glu Leu Arg Arg Ala
100 105 110
Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr Arg Pro Thr Thr Thr Pro
115 120 125
Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu Pro Thr Thr Val Met Thr
130 135 140
Thr Ser Val Leu Pro Thr Thr Thr Pro Pro Gln Thr Leu Ala Thr Thr
145 150 155 160
Ala Phe Ser Thr Ala Val Thr Thr Cys Pro Ser Thr Thr Pro Gly Ser
165 170 175
Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala Phe Thr Thr Glu Ser Glu
180 185 190
Thr Leu Pro Ala Ser Asn His Ser Gln Arg Ser Met Met Thr Ile Ser
195 200 205
Thr Asp Ile Ala Val Leu Arg Pro Thr Gly Ser Asn Pro Gly Ile Leu
210 215 220
Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys Thr Thr Leu Thr Thr Ser
225 230 235 240
Glu Ser Leu Gln Lys Thr Thr Lys Ser His Gln Ile Asn Ser Arg Gln
245 250 255
Thr
<210> 48
<211> 1140
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Human Megf10
<400> 48
Met Val Ile Ser Leu Asn Ser Cys Leu Ser Phe Ile Cys Leu Leu Leu
1 5 10 15
Cys His Trp Ile Gly Thr Ala Ser Pro Leu Asn Leu Glu Asp Pro Asn
20 25 30
Val Cys Ser His Trp Glu Ser Tyr Ser Val Thr Val Gln Glu Ser Tyr
35 40 45
Pro His Pro Phe Asp Gln Ile Tyr Tyr Thr Ser Cys Thr Asp Ile Leu
50 55 60
Asn Trp Phe Lys Cys Thr Arg His Arg Val Ser Tyr Arg Thr Ala Tyr
65 70 75 80
Arg His Gly Glu Lys Thr Met Tyr Arg Arg Lys Ser Gln Cys Cys Pro
85 90 95
Gly Phe Tyr Glu Ser Gly Glu Met Cys Val Pro His Cys Ala Asp Lys
100 105 110
Cys Val His Gly Arg Cys Ile Ala Pro Asn Thr Cys Gln Cys Glu Pro
115 120 125
Gly Trp Gly Gly Thr Asn Cys Ser Ser Ala Cys Asp Gly Asp His Trp
130 135 140
Gly Pro His Cys Thr Ser Arg Cys Gln Cys Lys Asn Gly Ala Leu Cys
145 150 155 160
Asn Pro Ile Thr Gly Ala Cys His Cys Ala Ala Gly Phe Arg Gly Trp
165 170 175
Arg Cys Glu Asp Arg Cys Glu Gln Gly Thr Tyr Gly Asn Asp Cys His
180 185 190
Gln Arg Cys Gln Cys Gln Asn Gly Ala Thr Cys Asp His Val Thr Gly
195 200 205
Glu Cys Arg Cys Pro Pro Gly Tyr Thr Gly Ala Phe Cys Glu Asp Leu
210 215 220
Cys Pro Pro Gly Lys His Gly Pro Gln Cys Glu Gln Arg Cys Pro Cys
225 230 235 240
Gln Asn Gly Gly Val Cys His His Val Thr Gly Glu Cys Ser Cys Pro
245 250 255
Ser Gly Trp Met Gly Thr Val Cys Gly Gln Pro Cys Pro Glu Gly Arg
260 265 270
Phe Gly Lys Asn Cys Ser Gln Glu Cys Gln Cys His Asn Gly Gly Thr
275 280 285
Cys Asp Ala Ala Thr Gly Gln Cys His Cys Ser Pro Gly Tyr Thr Gly
290 295 300
Glu Arg Cys Gln Asp Glu Cys Pro Val Gly Thr Tyr Gly Val Leu Cys
305 310 315 320
Ala Glu Thr Cys Gln Cys Val Asn Gly Gly Lys Cys Tyr His Val Ser
325 330 335
Gly Ala Cys Leu Cys Glu Ala Gly Phe Ala Gly Glu Arg Cys Glu Ala
340 345 350
Arg Leu Cys Pro Glu Gly Leu Tyr Gly Ile Lys Cys Asp Lys Arg Cys
355 360 365
Pro Cys His Leu Glu Asn Thr His Ser Cys His Pro Met Ser Gly Glu
370 375 380
Cys Ala Cys Lys Pro Gly Trp Ser Gly Leu Tyr Cys Asn Glu Thr Cys
385 390 395 400
Ser Pro Gly Phe Tyr Gly Glu Ala Cys Gln Gln Ile Cys Ser Cys Gln
405 410 415
Asn Gly Ala Asp Cys Asp Ser Val Thr Gly Lys Cys Thr Cys Ala Pro
420 425 430
Gly Phe Lys Gly Ile Asp Cys Ser Thr Pro Cys Pro Leu Gly Thr Tyr
435 440 445
Gly Ile Asn Cys Ser Ser Arg Cys Gly Cys Lys Asn Asp Ala Val Cys
450 455 460
Ser Pro Val Asp Gly Ser Cys Thr Cys Lys Ala Gly Trp His Gly Val
465 470 475 480
Asp Cys Ser Ile Arg Cys Pro Ser Gly Thr Trp Gly Phe Gly Cys Asn
485 490 495
Leu Thr Cys Gln Cys Leu Asn Gly Gly Ala Cys Asn Thr Leu Asp Gly
500 505 510
Thr Cys Thr Cys Ala Pro Gly Trp Arg Gly Glu Lys Cys Glu Leu Pro
515 520 525
Cys Gln Asp Gly Thr Tyr Gly Leu Asn Cys Ala Glu Arg Cys Asp Cys
530 535 540
Ser His Ala Asp Gly Cys His Pro Thr Thr Gly His Cys Arg Cys Leu
545 550 555 560
Pro Gly Trp Ser Gly Val His Cys Asp Ser Val Cys Ala Glu Gly Arg
565 570 575
Trp Gly Pro Asn Cys Ser Leu Pro Cys Tyr Cys Lys Asn Gly Ala Ser
580 585 590
Cys Ser Pro Asp Asp Gly Ile Cys Glu Cys Ala Pro Gly Phe Arg Gly
595 600 605
Thr Thr Cys Gln Arg Ile Cys Ser Pro Gly Phe Tyr Gly His Arg Cys
610 615 620
Ser Gln Thr Cys Pro Gln Cys Val His Ser Ser Gly Pro Cys His His
625 630 635 640
Ile Thr Gly Leu Cys Asp Cys Leu Pro Gly Phe Thr Gly Ala Leu Cys
645 650 655
Asn Glu Val Cys Pro Ser Gly Arg Phe Gly Lys Asn Cys Ala Gly Ile
660 665 670
Cys Thr Cys Thr Asn Asn Gly Thr Cys Asn Pro Ile Asp Arg Ser Cys
675 680 685
Gln Cys Tyr Pro Gly Trp Ile Gly Ser Asp Cys Ser Gln Pro Cys Pro
690 695 700
Pro Ala His Trp Gly Pro Asn Cys Ile His Thr Cys Asn Cys His Asn
705 710 715 720
Gly Ala Phe Cys Ser Ala Tyr Asp Gly Glu Cys Lys Cys Thr Pro Gly
725 730 735
Trp Thr Gly Leu Tyr Cys Thr Gln Arg Cys Pro Leu Gly Phe Tyr Gly
740 745 750
Lys Asp Cys Ala Leu Ile Cys Gln Cys Gln Asn Gly Ala Asp Cys Asp
755 760 765
His Ile Ser Gly Gln Cys Thr Cys Arg Thr Gly Phe Met Gly Arg His
770 775 780
Cys Glu Gln Lys Cys Pro Ser Gly Thr Tyr Gly Tyr Gly Cys Arg Gln
785 790 795 800
Ile Cys Asp Cys Leu Asn Asn Ser Thr Cys Asp His Ile Thr Gly Thr
805 810 815
Cys Tyr Cys Ser Pro Gly Trp Lys Gly Ala Arg Cys Asp Gln Ala Gly
820 825 830
Val Ile Ile Val Gly Asn Leu Asn Ser Leu Ser Arg Thr Ser Thr Ala
835 840 845
Leu Pro Ala Asp Ser Tyr Gln Ile Gly Ala Ile Ala Gly Ile Ile Ile
850 855 860
Leu Val Leu Val Val Leu Phe Leu Leu Ala Leu Phe Ile Ile Tyr Arg
865 870 875 880
His Lys Gln Lys Gly Lys Glu Ser Ser Met Pro Ala Val Thr Tyr Thr
885 890 895
Pro Ala Met Arg Val Val Asn Ala Asp Tyr Thr Ile Ser Gly Thr Leu
900 905 910
Pro His Ser Asn Gly Gly Asn Ala Asn Ser His Tyr Phe Thr Asn Pro
915 920 925
Ser Tyr His Thr Leu Thr Gln Cys Ala Thr Ser Pro His Val Asn Asn
930 935 940
Arg Asp Arg Met Thr Val Thr Lys Ser Lys Asn Asn Gln Leu Phe Val
945 950 955 960
Asn Leu Lys Asn Val Asn Pro Gly Lys Arg Gly Pro Val Gly Asp Cys
965 970 975
Thr Gly Thr Leu Pro Ala Asp Trp Lys His Gly Gly Tyr Leu Asn Glu
980 985 990
Leu Gly Ala Phe Gly Leu Asp Arg Ser Tyr Met Gly Lys Ser Leu Lys
995 1000 1005
Asp Leu Gly Lys Asn Ser Glu Tyr Asn Ser Ser Asn Cys Ser Leu
1010 1015 1020
Ser Ser Ser Glu Asn Pro Tyr Ala Thr Ile Lys Asp Pro Pro Val
1025 1030 1035
Leu Ile Pro Lys Ser Ser Glu Cys Gly Tyr Val Glu Met Lys Ser
1040 1045 1050
Pro Ala Arg Arg Asp Ser Pro Tyr Ala Glu Ile Asn Asn Ser Thr
1055 1060 1065
Ser Ala Asn Arg Asn Val Tyr Glu Val Glu Pro Thr Val Ser Val
1070 1075 1080
Val Gln Gly Val Phe Ser Asn Asn Gly Arg Leu Ser Gln Asp Pro
1085 1090 1095
Tyr Asp Leu Pro Lys Asn Ser His Ile Pro Cys His Tyr Asp Leu
1100 1105 1110
Leu Pro Val Arg Asp Ser Ser Ser Ser Pro Lys Gln Glu Asp Ser
1115 1120 1125
Gly Gly Ser Ser Ser Asn Ser Ser Ser Ser Ser Glu
1130 1135 1140
<210> 49
<211> 262
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of human Megf10
<400> 49
Tyr Arg His Lys Gln Lys Gly Lys Glu Ser Ser Met Pro Ala Val Thr
1 5 10 15
Tyr Thr Pro Ala Met Arg Val Val Asn Ala Asp Tyr Thr Ile Ser Gly
20 25 30
Thr Leu Pro His Ser Asn Gly Gly Asn Ala Asn Ser His Tyr Phe Thr
35 40 45
Asn Pro Ser Tyr His Thr Leu Thr Gln Cys Ala Thr Ser Pro His Val
50 55 60
Asn Asn Arg Asp Arg Met Thr Val Thr Lys Ser Lys Asn Asn Gln Leu
65 70 75 80
Phe Val Asn Leu Lys Asn Val Asn Pro Gly Lys Arg Gly Pro Val Gly
85 90 95
Asp Cys Thr Gly Thr Leu Pro Ala Asp Trp Lys His Gly Gly Tyr Leu
100 105 110
Asn Glu Leu Gly Ala Phe Gly Leu Asp Arg Ser Tyr Met Gly Lys Ser
115 120 125
Leu Lys Asp Leu Gly Lys Asn Ser Glu Tyr Asn Ser Ser Asn Cys Ser
130 135 140
Leu Ser Ser Ser Glu Asn Pro Tyr Ala Thr Ile Lys Asp Pro Pro Val
145 150 155 160
Leu Ile Pro Lys Ser Ser Glu Cys Gly Tyr Val Glu Met Lys Ser Pro
165 170 175
Ala Arg Arg Asp Ser Pro Tyr Ala Glu Ile Asn Asn Ser Thr Ser Ala
180 185 190
Asn Arg Asn Val Tyr Glu Val Glu Pro Thr Val Ser Val Val Gln Gly
195 200 205
Val Phe Ser Asn Asn Gly Arg Leu Ser Gln Asp Pro Tyr Asp Leu Pro
210 215 220
Lys Asn Ser His Ile Pro Cys His Tyr Asp Leu Leu Pro Val Arg Asp
225 230 235 240
Ser Ser Ser Ser Pro Lys Gln Glu Asp Ser Gly Gly Ser Ser Ser Asn
245 250 255
Ser Ser Ser Ser Ser Glu
260
<210> 50
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of human Megf10
<400> 50
Ala Ile Ala Gly Ile Ile Ile Leu Val Leu Val Val Leu Phe Leu Leu
1 5 10 15
Ala Leu Phe Ile Ile
20
<210> 51
<211> 832
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of human Megf10
<400> 51
Leu Asn Leu Glu Asp Pro Asn Val Cys Ser His Trp Glu Ser Tyr Ser
1 5 10 15
Val Thr Val Gln Glu Ser Tyr Pro His Pro Phe Asp Gln Ile Tyr Tyr
20 25 30
Thr Ser Cys Thr Asp Ile Leu Asn Trp Phe Lys Cys Thr Arg His Arg
35 40 45
Val Ser Tyr Arg Thr Ala Tyr Arg His Gly Glu Lys Thr Met Tyr Arg
50 55 60
Arg Lys Ser Gln Cys Cys Pro Gly Phe Tyr Glu Ser Gly Glu Met Cys
65 70 75 80
Val Pro His Cys Ala Asp Lys Cys Val His Gly Arg Cys Ile Ala Pro
85 90 95
Asn Thr Cys Gln Cys Glu Pro Gly Trp Gly Gly Thr Asn Cys Ser Ser
100 105 110
Ala Cys Asp Gly Asp His Trp Gly Pro His Cys Thr Ser Arg Cys Gln
115 120 125
Cys Lys Asn Gly Ala Leu Cys Asn Pro Ile Thr Gly Ala Cys His Cys
130 135 140
Ala Ala Gly Phe Arg Gly Trp Arg Cys Glu Asp Arg Cys Glu Gln Gly
145 150 155 160
Thr Tyr Gly Asn Asp Cys His Gln Arg Cys Gln Cys Gln Asn Gly Ala
165 170 175
Thr Cys Asp His Val Thr Gly Glu Cys Arg Cys Pro Pro Gly Tyr Thr
180 185 190
Gly Ala Phe Cys Glu Asp Leu Cys Pro Pro Gly Lys His Gly Pro Gln
195 200 205
Cys Glu Gln Arg Cys Pro Cys Gln Asn Gly Gly Val Cys His His Val
210 215 220
Thr Gly Glu Cys Ser Cys Pro Ser Gly Trp Met Gly Thr Val Cys Gly
225 230 235 240
Gln Pro Cys Pro Glu Gly Arg Phe Gly Lys Asn Cys Ser Gln Glu Cys
245 250 255
Gln Cys His Asn Gly Gly Thr Cys Asp Ala Ala Thr Gly Gln Cys His
260 265 270
Cys Ser Pro Gly Tyr Thr Gly Glu Arg Cys Gln Asp Glu Cys Pro Val
275 280 285
Gly Thr Tyr Gly Val Leu Cys Ala Glu Thr Cys Gln Cys Val Asn Gly
290 295 300
Gly Lys Cys Tyr His Val Ser Gly Ala Cys Leu Cys Glu Ala Gly Phe
305 310 315 320
Ala Gly Glu Arg Cys Glu Ala Arg Leu Cys Pro Glu Gly Leu Tyr Gly
325 330 335
Ile Lys Cys Asp Lys Arg Cys Pro Cys His Leu Glu Asn Thr His Ser
340 345 350
Cys His Pro Met Ser Gly Glu Cys Ala Cys Lys Pro Gly Trp Ser Gly
355 360 365
Leu Tyr Cys Asn Glu Thr Cys Ser Pro Gly Phe Tyr Gly Glu Ala Cys
370 375 380
Gln Gln Ile Cys Ser Cys Gln Asn Gly Ala Asp Cys Asp Ser Val Thr
385 390 395 400
Gly Lys Cys Thr Cys Ala Pro Gly Phe Lys Gly Ile Asp Cys Ser Thr
405 410 415
Pro Cys Pro Leu Gly Thr Tyr Gly Ile Asn Cys Ser Ser Arg Cys Gly
420 425 430
Cys Lys Asn Asp Ala Val Cys Ser Pro Val Asp Gly Ser Cys Thr Cys
435 440 445
Lys Ala Gly Trp His Gly Val Asp Cys Ser Ile Arg Cys Pro Ser Gly
450 455 460
Thr Trp Gly Phe Gly Cys Asn Leu Thr Cys Gln Cys Leu Asn Gly Gly
465 470 475 480
Ala Cys Asn Thr Leu Asp Gly Thr Cys Thr Cys Ala Pro Gly Trp Arg
485 490 495
Gly Glu Lys Cys Glu Leu Pro Cys Gln Asp Gly Thr Tyr Gly Leu Asn
500 505 510
Cys Ala Glu Arg Cys Asp Cys Ser His Ala Asp Gly Cys His Pro Thr
515 520 525
Thr Gly His Cys Arg Cys Leu Pro Gly Trp Ser Gly Val His Cys Asp
530 535 540
Ser Val Cys Ala Glu Gly Arg Trp Gly Pro Asn Cys Ser Leu Pro Cys
545 550 555 560
Tyr Cys Lys Asn Gly Ala Ser Cys Ser Pro Asp Asp Gly Ile Cys Glu
565 570 575
Cys Ala Pro Gly Phe Arg Gly Thr Thr Cys Gln Arg Ile Cys Ser Pro
580 585 590
Gly Phe Tyr Gly His Arg Cys Ser Gln Thr Cys Pro Gln Cys Val His
595 600 605
Ser Ser Gly Pro Cys His His Ile Thr Gly Leu Cys Asp Cys Leu Pro
610 615 620
Gly Phe Thr Gly Ala Leu Cys Asn Glu Val Cys Pro Ser Gly Arg Phe
625 630 635 640
Gly Lys Asn Cys Ala Gly Ile Cys Thr Cys Thr Asn Asn Gly Thr Cys
645 650 655
Asn Pro Ile Asp Arg Ser Cys Gln Cys Tyr Pro Gly Trp Ile Gly Ser
660 665 670
Asp Cys Ser Gln Pro Cys Pro Pro Ala His Trp Gly Pro Asn Cys Ile
675 680 685
His Thr Cys Asn Cys His Asn Gly Ala Phe Cys Ser Ala Tyr Asp Gly
690 695 700
Glu Cys Lys Cys Thr Pro Gly Trp Thr Gly Leu Tyr Cys Thr Gln Arg
705 710 715 720
Cys Pro Leu Gly Phe Tyr Gly Lys Asp Cys Ala Leu Ile Cys Gln Cys
725 730 735
Gln Asn Gly Ala Asp Cys Asp His Ile Ser Gly Gln Cys Thr Cys Arg
740 745 750
Thr Gly Phe Met Gly Arg His Cys Glu Gln Lys Cys Pro Ser Gly Thr
755 760 765
Tyr Gly Tyr Gly Cys Arg Gln Ile Cys Asp Cys Leu Asn Asn Ser Thr
770 775 780
Cys Asp His Ile Thr Gly Thr Cys Tyr Cys Ser Pro Gly Trp Lys Gly
785 790 795 800
Ala Arg Cys Asp Gln Ala Gly Val Ile Ile Val Gly Asn Leu Asn Ser
805 810 815
Leu Ser Arg Thr Ser Thr Ala Leu Pro Ala Asp Ser Tyr Gln Ile Gly
820 825 830
<210> 52
<211> 1147
<212> PRT
<213> Mus musculus
<220>
<221> misc_feature
<223> Mouse Megf10
<400> 52
Met Ala Ile Ser Ser Ser Ser Cys Leu Gly Leu Ile Cys Ser Leu Leu
1 5 10 15
Cys His Trp Val Gly Thr Ala Ser Ser Leu Asn Leu Glu Asp Pro Asn
20 25 30
Val Cys Ser His Trp Glu Ser Tyr Ser Val Thr Val Gln Glu Ser Tyr
35 40 45
Pro His Pro Phe Asp Gln Ile Tyr Tyr Thr Ser Cys Thr Asp Ile Leu
50 55 60
Asn Trp Phe Lys Cys Thr Arg His Arg Ile Ser Tyr Arg Thr Ala Tyr
65 70 75 80
Arg His Gly Glu Lys Thr Met Tyr Arg Arg Lys Ser Gln Cys Cys Pro
85 90 95
Gly Phe Tyr Glu Ser Arg Asp Met Cys Val Pro His Cys Ala Asp Lys
100 105 110
Cys Val His Gly Arg Cys Ile Ala Pro Asn Thr Cys Gln Cys Glu Pro
115 120 125
Gly Trp Gly Gly Thr Asn Cys Ser Ser Ala Cys Asp Gly Asp His Trp
130 135 140
Gly Pro His Cys Ser Ser Arg Cys Gln Cys Lys Asn Arg Ala Leu Cys
145 150 155 160
Asn Pro Ile Thr Gly Ala Cys His Cys Ala Ala Gly Tyr Arg Gly Trp
165 170 175
Arg Cys Glu Asp Arg Cys Glu Gln Gly Thr Tyr Gly Asn Asp Cys His
180 185 190
Gln Arg Cys Gln Cys Gln Asn Gly Ala Thr Cys Asp His Ile Thr Gly
195 200 205
Glu Cys Arg Cys Ser Pro Gly Tyr Thr Gly Ala Phe Cys Glu Asp Leu
210 215 220
Cys Pro Pro Gly Lys His Gly Pro His Cys Glu Gln Arg Cys Pro Cys
225 230 235 240
Gln Asn Gly Gly Val Cys His His Val Thr Gly Glu Cys Ser Cys Pro
245 250 255
Ser Gly Trp Met Gly Thr Val Cys Gly Gln Pro Cys Pro Glu Gly Arg
260 265 270
Phe Gly Lys Asn Cys Ser Gln Glu Cys Gln Cys His Asn Gly Gly Thr
275 280 285
Cys Asp Ala Ala Thr Gly Gln Cys His Cys Ser Pro Gly Tyr Thr Gly
290 295 300
Glu Arg Cys Gln Asp Glu Cys Pro Val Gly Ser Tyr Gly Val Arg Cys
305 310 315 320
Ala Glu Ala Cys Arg Cys Val Asn Gly Gly Lys Cys Tyr His Val Ser
325 330 335
Gly Thr Cys Leu Cys Glu Ala Gly Phe Ser Gly Glu Leu Cys Glu Ala
340 345 350
Arg Leu Cys Pro Glu Gly Leu Tyr Gly Ile Lys Cys Asp Lys Arg Cys
355 360 365
Pro Cys His Leu Asp Asn Thr His Ser Cys His Pro Met Ser Gly Glu
370 375 380
Cys Gly Cys Lys Pro Gly Trp Ser Gly Leu Tyr Cys Asn Glu Thr Cys
385 390 395 400
Ser Pro Gly Phe Tyr Gly Glu Ala Cys Gln Gln Ile Cys Ser Cys Gln
405 410 415
Asn Gly Ala Asp Cys Asp Ser Val Thr Gly Arg Cys Ala Cys Ala Pro
420 425 430
Gly Phe Lys Gly Thr Asp Cys Ser Thr Pro Cys Pro Leu Gly Arg Tyr
435 440 445
Gly Ile Asn Cys Ser Ser Arg Cys Gly Cys Lys Asn Asp Ala Val Cys
450 455 460
Ser Pro Val Asp Gly Ser Cys Ile Cys Lys Ala Gly Trp His Gly Val
465 470 475 480
Asp Cys Ser Ile Arg Cys Pro Ser Gly Thr Trp Gly Phe Gly Cys Asn
485 490 495
Leu Thr Cys Gln Cys Leu Asn Gly Gly Ala Cys Asn Thr Leu Asp Gly
500 505 510
Thr Cys Thr Cys Ala Pro Gly Trp Arg Gly Ala Lys Cys Glu Phe Pro
515 520 525
Cys Gln Asp Gly Thr Tyr Gly Leu Asn Cys Ala Glu Arg Cys Asp Cys
530 535 540
Ser His Ala Asp Gly Cys His Pro Thr Thr Gly His Cys Arg Cys Leu
545 550 555 560
Pro Gly Trp Ser Gly Val His Cys Asp Ser Val Cys Ala Glu Gly Arg
565 570 575
Trp Gly Pro Asn Cys Ser Leu Pro Cys Tyr Cys Lys Asn Gly Ala Ser
580 585 590
Cys Ser Pro Asp Asp Gly Ile Cys Glu Cys Ala Pro Gly Phe Arg Gly
595 600 605
Thr Thr Cys Gln Arg Ile Cys Ser Pro Gly Phe Tyr Gly His Arg Cys
610 615 620
Ser Gln Thr Cys Pro Gln Cys Val His Ser Ser Gly Pro Cys His His
625 630 635 640
Ile Thr Gly Leu Cys Asp Cys Leu Pro Gly Phe Thr Gly Ala Leu Cys
645 650 655
Asn Glu Val Cys Pro Ser Gly Arg Phe Gly Lys Asn Cys Ala Gly Val
660 665 670
Cys Thr Cys Thr Asn Asn Gly Thr Cys Asn Pro Ile Asp Arg Ser Cys
675 680 685
Gln Cys Tyr Pro Gly Trp Ile Gly Ser Asp Cys Ser Gln Pro Cys Pro
690 695 700
Pro Ala His Trp Gly Pro Asn Cys Ile His Thr Cys Asn Cys His Asn
705 710 715 720
Gly Ala Phe Cys Ser Ala Tyr Asp Gly Glu Cys Lys Cys Thr Pro Gly
725 730 735
Trp Thr Gly Leu Tyr Cys Thr Gln Arg Cys Pro Leu Gly Phe Tyr Gly
740 745 750
Lys Asp Cys Ala Leu Ile Cys Gln Cys Gln Asn Gly Ala Asp Cys Asp
755 760 765
His Ile Ser Gly Gln Cys Thr Cys Arg Thr Gly Phe Met Gly Arg His
770 775 780
Cys Glu Gln Lys Cys Pro Ala Gly Thr Tyr Gly Tyr Gly Cys Arg Gln
785 790 795 800
Ile Cys Asp Cys Leu Asn Asn Ser Thr Cys Asp His Ile Thr Gly Thr
805 810 815
Cys Tyr Cys Ser Pro Gly Trp Lys Gly Ala Arg Cys Asp Gln Ala Gly
820 825 830
Val Ile Ile Val Gly Asn Leu Asn Ser Leu Ser Arg Thr Ser Thr Ala
835 840 845
Leu Pro Ala Asp Ser Tyr Gln Ile Gly Ala Ile Ala Gly Ile Val Val
850 855 860
Leu Val Leu Val Val Leu Phe Leu Leu Ala Leu Phe Ile Ile Tyr Arg
865 870 875 880
His Lys Gln Lys Arg Lys Glu Ser Ser Met Pro Ala Val Thr Tyr Thr
885 890 895
Pro Ala Met Arg Val Ile Asn Ala Asp Tyr Thr Ile Ala Glu Thr Leu
900 905 910
Pro His Ser Asn Gly Gly Asn Ala Asn Ser His Tyr Phe Thr Asn Pro
915 920 925
Ser Tyr His Thr Leu Ser Gln Cys Ala Thr Ser Pro His Val Asn Asn
930 935 940
Arg Asp Arg Met Thr Ile Ala Lys Ser Lys Asn Asn Gln Leu Phe Val
945 950 955 960
Asn Leu Lys Asn Val Asn Pro Gly Lys Arg Gly Thr Leu Val Asp Cys
965 970 975
Thr Gly Thr Leu Pro Ala Asp Trp Lys Gln Gly Gly Tyr Leu Asn Glu
980 985 990
Leu Gly Ala Phe Gly Leu Asp Arg Ser Tyr Met Gly Lys Ser Leu Lys
995 1000 1005
Asp Leu Gly Lys Asn Ser Glu Tyr Asn Ser Ser Thr Cys Ser Leu
1010 1015 1020
Ser Ser Ser Glu Asn Pro Tyr Ala Thr Ile Lys Asp Pro Pro Ala
1025 1030 1035
Leu Leu Pro Lys Ser Ser Glu Cys Gly Tyr Val Glu Met Lys Ser
1040 1045 1050
Pro Ala Arg Arg Asp Ser Pro Tyr Ala Glu Ile Asn Asn Ser Thr
1055 1060 1065
Pro Ala Asn Arg Asn Val Tyr Glu Val Glu Pro Thr Val Ser Val
1070 1075 1080
Val Gln Gly Val Phe Ser Asn Ser Gly His Val Thr Gln Asp Pro
1085 1090 1095
Tyr Asp Leu Pro Lys Asn Ser His Ile Pro Cys His Tyr Asp Leu
1100 1105 1110
Leu Pro Val Arg Asp Ser Ser Ser Ser Pro Lys Arg Glu Asp Gly
1115 1120 1125
Gly Gly Ser Asn Ser Thr Ser Ser Asn Ser Thr Ser Ser Ser Ser
1130 1135 1140
Ser Ser Ser Glu
1145
<210> 53
<211> 269
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of mouse Megf10
<400> 53
Tyr Arg His Lys Gln Lys Arg Lys Glu Ser Ser Met Pro Ala Val Thr
1 5 10 15
Tyr Thr Pro Ala Met Arg Val Ile Asn Ala Asp Tyr Thr Ile Ala Glu
20 25 30
Thr Leu Pro His Ser Asn Gly Gly Asn Ala Asn Ser His Tyr Phe Thr
35 40 45
Asn Pro Ser Tyr His Thr Leu Ser Gln Cys Ala Thr Ser Pro His Val
50 55 60
Asn Asn Arg Asp Arg Met Thr Ile Ala Lys Ser Lys Asn Asn Gln Leu
65 70 75 80
Phe Val Asn Leu Lys Asn Val Asn Pro Gly Lys Arg Gly Thr Leu Val
85 90 95
Asp Cys Thr Gly Thr Leu Pro Ala Asp Trp Lys Gln Gly Gly Tyr Leu
100 105 110
Asn Glu Leu Gly Ala Phe Gly Leu Asp Arg Ser Tyr Met Gly Lys Ser
115 120 125
Leu Lys Asp Leu Gly Lys Asn Ser Glu Tyr Asn Ser Ser Thr Cys Ser
130 135 140
Leu Ser Ser Ser Glu Asn Pro Tyr Ala Thr Ile Lys Asp Pro Pro Ala
145 150 155 160
Leu Leu Pro Lys Ser Ser Glu Cys Gly Tyr Val Glu Met Lys Ser Pro
165 170 175
Ala Arg Arg Asp Ser Pro Tyr Ala Glu Ile Asn Asn Ser Thr Pro Ala
180 185 190
Asn Arg Asn Val Tyr Glu Val Glu Pro Thr Val Ser Val Val Gln Gly
195 200 205
Val Phe Ser Asn Ser Gly His Val Thr Gln Asp Pro Tyr Asp Leu Pro
210 215 220
Lys Asn Ser His Ile Pro Cys His Tyr Asp Leu Leu Pro Val Arg Asp
225 230 235 240
Ser Ser Ser Ser Pro Lys Arg Glu Asp Gly Gly Gly Ser Asn Ser Thr
245 250 255
Ser Ser Asn Ser Thr Ser Ser Ser Ser Ser Ser Ser Glu
260 265
<210> 54
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of mouse Megf10
<400> 54
Ala Ile Ala Gly Ile Val Val Leu Val Leu Val Val Leu Phe Leu Leu
1 5 10 15
Ala Leu Phe Ile Ile
20
<210> 55
<211> 832
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of mouse Megf10
<400> 55
Leu Asn Leu Glu Asp Pro Asn Val Cys Ser His Trp Glu Ser Tyr Ser
1 5 10 15
Val Thr Val Gln Glu Ser Tyr Pro His Pro Phe Asp Gln Ile Tyr Tyr
20 25 30
Thr Ser Cys Thr Asp Ile Leu Asn Trp Phe Lys Cys Thr Arg His Arg
35 40 45
Ile Ser Tyr Arg Thr Ala Tyr Arg His Gly Glu Lys Thr Met Tyr Arg
50 55 60
Arg Lys Ser Gln Cys Cys Pro Gly Phe Tyr Glu Ser Arg Asp Met Cys
65 70 75 80
Val Pro His Cys Ala Asp Lys Cys Val His Gly Arg Cys Ile Ala Pro
85 90 95
Asn Thr Cys Gln Cys Glu Pro Gly Trp Gly Gly Thr Asn Cys Ser Ser
100 105 110
Ala Cys Asp Gly Asp His Trp Gly Pro His Cys Ser Ser Arg Cys Gln
115 120 125
Cys Lys Asn Arg Ala Leu Cys Asn Pro Ile Thr Gly Ala Cys His Cys
130 135 140
Ala Ala Gly Tyr Arg Gly Trp Arg Cys Glu Asp Arg Cys Glu Gln Gly
145 150 155 160
Thr Tyr Gly Asn Asp Cys His Gln Arg Cys Gln Cys Gln Asn Gly Ala
165 170 175
Thr Cys Asp His Ile Thr Gly Glu Cys Arg Cys Ser Pro Gly Tyr Thr
180 185 190
Gly Ala Phe Cys Glu Asp Leu Cys Pro Pro Gly Lys His Gly Pro His
195 200 205
Cys Glu Gln Arg Cys Pro Cys Gln Asn Gly Gly Val Cys His His Val
210 215 220
Thr Gly Glu Cys Ser Cys Pro Ser Gly Trp Met Gly Thr Val Cys Gly
225 230 235 240
Gln Pro Cys Pro Glu Gly Arg Phe Gly Lys Asn Cys Ser Gln Glu Cys
245 250 255
Gln Cys His Asn Gly Gly Thr Cys Asp Ala Ala Thr Gly Gln Cys His
260 265 270
Cys Ser Pro Gly Tyr Thr Gly Glu Arg Cys Gln Asp Glu Cys Pro Val
275 280 285
Gly Ser Tyr Gly Val Arg Cys Ala Glu Ala Cys Arg Cys Val Asn Gly
290 295 300
Gly Lys Cys Tyr His Val Ser Gly Thr Cys Leu Cys Glu Ala Gly Phe
305 310 315 320
Ser Gly Glu Leu Cys Glu Ala Arg Leu Cys Pro Glu Gly Leu Tyr Gly
325 330 335
Ile Lys Cys Asp Lys Arg Cys Pro Cys His Leu Asp Asn Thr His Ser
340 345 350
Cys His Pro Met Ser Gly Glu Cys Gly Cys Lys Pro Gly Trp Ser Gly
355 360 365
Leu Tyr Cys Asn Glu Thr Cys Ser Pro Gly Phe Tyr Gly Glu Ala Cys
370 375 380
Gln Gln Ile Cys Ser Cys Gln Asn Gly Ala Asp Cys Asp Ser Val Thr
385 390 395 400
Gly Arg Cys Ala Cys Ala Pro Gly Phe Lys Gly Thr Asp Cys Ser Thr
405 410 415
Pro Cys Pro Leu Gly Arg Tyr Gly Ile Asn Cys Ser Ser Arg Cys Gly
420 425 430
Cys Lys Asn Asp Ala Val Cys Ser Pro Val Asp Gly Ser Cys Ile Cys
435 440 445
Lys Ala Gly Trp His Gly Val Asp Cys Ser Ile Arg Cys Pro Ser Gly
450 455 460
Thr Trp Gly Phe Gly Cys Asn Leu Thr Cys Gln Cys Leu Asn Gly Gly
465 470 475 480
Ala Cys Asn Thr Leu Asp Gly Thr Cys Thr Cys Ala Pro Gly Trp Arg
485 490 495
Gly Ala Lys Cys Glu Phe Pro Cys Gln Asp Gly Thr Tyr Gly Leu Asn
500 505 510
Cys Ala Glu Arg Cys Asp Cys Ser His Ala Asp Gly Cys His Pro Thr
515 520 525
Thr Gly His Cys Arg Cys Leu Pro Gly Trp Ser Gly Val His Cys Asp
530 535 540
Ser Val Cys Ala Glu Gly Arg Trp Gly Pro Asn Cys Ser Leu Pro Cys
545 550 555 560
Tyr Cys Lys Asn Gly Ala Ser Cys Ser Pro Asp Asp Gly Ile Cys Glu
565 570 575
Cys Ala Pro Gly Phe Arg Gly Thr Thr Cys Gln Arg Ile Cys Ser Pro
580 585 590
Gly Phe Tyr Gly His Arg Cys Ser Gln Thr Cys Pro Gln Cys Val His
595 600 605
Ser Ser Gly Pro Cys His His Ile Thr Gly Leu Cys Asp Cys Leu Pro
610 615 620
Gly Phe Thr Gly Ala Leu Cys Asn Glu Val Cys Pro Ser Gly Arg Phe
625 630 635 640
Gly Lys Asn Cys Ala Gly Val Cys Thr Cys Thr Asn Asn Gly Thr Cys
645 650 655
Asn Pro Ile Asp Arg Ser Cys Gln Cys Tyr Pro Gly Trp Ile Gly Ser
660 665 670
Asp Cys Ser Gln Pro Cys Pro Pro Ala His Trp Gly Pro Asn Cys Ile
675 680 685
His Thr Cys Asn Cys His Asn Gly Ala Phe Cys Ser Ala Tyr Asp Gly
690 695 700
Glu Cys Lys Cys Thr Pro Gly Trp Thr Gly Leu Tyr Cys Thr Gln Arg
705 710 715 720
Cys Pro Leu Gly Phe Tyr Gly Lys Asp Cys Ala Leu Ile Cys Gln Cys
725 730 735
Gln Asn Gly Ala Asp Cys Asp His Ile Ser Gly Gln Cys Thr Cys Arg
740 745 750
Thr Gly Phe Met Gly Arg His Cys Glu Gln Lys Cys Pro Ala Gly Thr
755 760 765
Tyr Gly Tyr Gly Cys Arg Gln Ile Cys Asp Cys Leu Asn Asn Ser Thr
770 775 780
Cys Asp His Ile Thr Gly Thr Cys Tyr Cys Ser Pro Gly Trp Lys Gly
785 790 795 800
Ala Arg Cys Asp Gln Ala Gly Val Ile Ile Val Gly Asn Leu Asn Ser
805 810 815
Leu Ser Arg Thr Ser Thr Ala Leu Pro Ala Asp Ser Tyr Gln Ile Gly
820 825 830
<210> 56
<211> 164
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Human CD3 zeta chain
<400> 56
Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu
1 5 10 15
Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys
20 25 30
Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala
35 40 45
Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
50 55 60
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
65 70 75 80
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
85 90 95
Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
100 105 110
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
115 120 125
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
130 135 140
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
145 150 155 160
Leu Pro Pro Arg
<210> 57
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of human CD3 zeta chain
<400> 57
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
50 55 60
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
65 70 75 80
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
85 90 95
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
100 105 110
Arg
<210> 58
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of human CD3 zeta chain
<400> 58
Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu
1 5 10 15
Thr Ala Leu Phe Leu
20
<210> 59
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of human CD3 zeta chain
<400> 59
Gln Ser Phe Gly Leu Leu Asp Pro Lys
1 5
<210> 60
<211> 164
<212> PRT
<213> Mus musculus
<220>
<221> misc_feature
<223> Mouse CD3 zeta chain
<400> 60
Met Lys Trp Lys Val Ser Val Leu Ala Cys Ile Leu His Val Arg Phe
1 5 10 15
Pro Gly Ala Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys
20 25 30
Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Ile Thr Ala
35 40 45
Leu Tyr Leu Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn
50 55 60
Leu Gln Asp Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
65 70 75 80
Glu Glu Tyr Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu Met
85 90 95
Gly Gly Lys Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr Asn
100 105 110
Ala Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Thr
115 120 125
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
130 135 140
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Thr
145 150 155 160
Leu Ala Pro Arg
<210> 61
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of mouse CD3 zeta chain
<400> 61
Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn Leu Gln Asp
1 5 10 15
Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr Asn Ala Leu Gln
50 55 60
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Thr Lys Gly Glu
65 70 75 80
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
85 90 95
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Thr Leu Ala Pro
100 105 110
Arg
<210> 62
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of mouse CD3 zeta chain
<400> 62
Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Ile
1 5 10 15
Thr Ala Leu Tyr Leu
20
<210> 63
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of mouse CD3 zeta chain
<400> 63
Gln Ser Phe Gly Leu Leu Asp Pro Lys
1 5
<210> 64
<211> 760
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Human transferrin receptor
<400> 64
Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu
1 5 10 15
Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp
20 25 30
Asn Ser His Val Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn Ala
35 40 45
Asp Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser Gly
50 55 60
Ser Ile Cys Tyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile Gly
65 70 75 80
Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr
85 90 95
Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu Pro
100 105 110
Gly Glu Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu Lys
115 120 125
Arg Lys Leu Ser Glu Lys Leu Asp Ser Thr Asp Phe Thr Gly Thr Ile
130 135 140
Lys Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly Ser Gln
145 150 155 160
Lys Asp Glu Asn Leu Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu Phe
165 170 175
Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val
180 185 190
Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg
195 200 205
Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys
210 215 220
Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys
225 230 235 240
Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile
245 250 255
Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu
260 265 270
Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe
275 280 285
Pro Ile Val Asn Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly
290 295 300
Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln
305 310 315 320
Phe Pro Pro Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr
325 330 335
Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp
340 345 350
Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser
355 360 365
Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile
370 375 380
Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp
385 390 395 400
His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala
405 410 415
Ala Lys Ser Gly Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met
420 425 430
Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile
435 440 445
Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr
450 455 460
Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr
465 470 475 480
Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
485 490 495
Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asn
500 505 510
Val Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn Trp
515 520 525
Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe
530 535 540
Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp
545 550 555 560
Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu
565 570 575
Ile Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu
580 585 590
Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn
595 600 605
Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp
610 615 620
Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln
625 630 635 640
Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu
645 650 655
Thr Thr Asp Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys
660 665 670
Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu Ser Pro
675 680 685
Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser
690 695 700
Gly Ser His Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys
705 710 715 720
Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
725 730 735
Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp
740 745 750
Val Trp Asp Ile Asp Asn Glu Phe
755 760
<210> 65
<211> 67
<212> PRT
<213> Artificial Sequence
<220>
<223>
The cytoplasmic domain of human transferrin receptor
<400> 65
Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu
1 5 10 15
Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp
20 25 30
Asn Ser His Val Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn Ala
35 40 45
Asp Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser Gly
50 55 60
Ser Ile Cys
65
<210> 66
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of human transferrin receptor
<400> 66
Tyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile Gly Phe Met Ile
1 5 10 15
Gly Tyr Leu Gly Tyr
20
<210> 67
<211> 672
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of human transferrin receptor
<400> 67
Cys Lys Gly Val Glu Pro Lys Thr Glu Cys Glu Arg Leu Ala Gly Thr
1 5 10 15
Glu Ser Pro Val Arg Glu Glu Pro Gly Glu Asp Phe Pro Ala Ala Arg
20 25 30
Arg Leu Tyr Trp Asp Asp Leu Lys Arg Lys Leu Ser Glu Lys Leu Asp
35 40 45
Ser Thr Asp Phe Thr Gly Thr Ile Lys Leu Leu Asn Glu Asn Ser Tyr
50 55 60
Val Pro Arg Glu Ala Gly Ser Gln Lys Asp Glu Asn Leu Ala Leu Tyr
65 70 75 80
Val Glu Asn Gln Phe Arg Glu Phe Lys Leu Ser Lys Val Trp Arg Asp
85 90 95
Gln His Phe Val Lys Ile Gln Val Lys Asp Ser Ala Gln Asn Ser Val
100 105 110
Ile Ile Val Asp Lys Asn Gly Arg Leu Val Tyr Leu Val Glu Asn Pro
115 120 125
Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly Lys Leu
130 135 140
Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Asp Leu Tyr Thr
145 150 155 160
Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly Lys Ile Thr Phe
165 170 175
Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly Val Leu
180 185 190
Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Asn Ala Glu Leu Ser
195 200 205
Phe Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly
210 215 220
Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser Ser Gly
225 230 235 240
Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys
245 250 255
Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser Asp Trp Lys Thr Asp
260 265 270
Ser Thr Cys Arg Met Val Thr Ser Glu Ser Lys Asn Val Lys Leu Thr
275 280 285
Val Ser Asn Val Leu Lys Glu Ile Lys Ile Leu Asn Ile Phe Gly Val
290 295 300
Ile Lys Gly Phe Val Glu Pro Asp His Tyr Val Val Val Gly Ala Gln
305 310 315 320
Arg Asp Ala Trp Gly Pro Gly Ala Ala Lys Ser Gly Val Gly Thr Ala
325 330 335
Leu Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met Val Leu Lys Asp
340 345 350
Gly Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser Trp Ser Ala Gly
355 360 365
Asp Phe Gly Ser Val Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser
370 375 380
Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu Asp Lys Ala Val
385 390 395 400
Leu Gly Thr Ser Asn Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr
405 410 415
Leu Ile Glu Lys Thr Met Gln Asn Val Lys His Pro Val Thr Gly Gln
420 425 430
Phe Leu Tyr Gln Asp Ser Asn Trp Ala Ser Lys Val Glu Lys Leu Thr
435 440 445
Leu Asp Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala
450 455 460
Val Ser Phe Cys Phe Cys Glu Asp Thr Asp Tyr Pro Tyr Leu Gly Thr
465 470 475 480
Thr Met Asp Thr Tyr Lys Glu Leu Ile Glu Arg Ile Pro Glu Leu Asn
485 490 495
Lys Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln Phe Val Ile Lys
500 505 510
Leu Thr His Asp Val Glu Leu Asn Leu Asp Tyr Glu Arg Tyr Asn Ser
515 520 525
Gln Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr Arg Ala Asp Ile
530 535 540
Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp
545 550 555 560
Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe Gly Asn Ala Glu
565 570 575
Lys Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp Arg Val Met Arg
580 585 590
Val Glu Tyr His Phe Leu Ser Pro Tyr Val Ser Pro Lys Glu Ser Pro
595 600 605
Phe Arg His Val Phe Trp Gly Ser Gly Ser His Thr Leu Pro Ala Leu
610 615 620
Leu Glu Asn Leu Lys Leu Arg Lys Gln Asn Asn Gly Ala Phe Asn Glu
625 630 635 640
Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly
645 650 655
Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile Asp Asn Glu Phe
660 665 670
<210> 68
<211> 202
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of human transferrin receptor
<400> 68
Glu Asp Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys
1 5 10 15
Glu Leu Ile Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala
20 25 30
Ala Glu Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu
35 40 45
Leu Asn Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val
50 55 60
Arg Asp Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser
65 70 75 80
Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser
85 90 95
Arg Leu Thr Thr Asp Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val
100 105 110
Met Lys Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu
115 120 125
Ser Pro Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp
130 135 140
Gly Ser Gly Ser His Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu
145 150 155 160
Arg Lys Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln
165 170 175
Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser
180 185 190
Gly Asp Val Trp Asp Ile Asp Asn Glu Phe
195 200
<210> 69
<211> 229
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Human Clec-2 receptor
<400> 69
Met Gln Asp Glu Asp Gly Tyr Ile Thr Leu Asn Ile Lys Thr Arg Lys
1 5 10 15
Pro Ala Leu Ile Ser Val Gly Ser Ala Ser Ser Ser Trp Trp Arg Val
20 25 30
Met Ala Leu Ile Leu Leu Ile Leu Cys Val Gly Met Val Val Gly Leu
35 40 45
Val Ala Leu Gly Ile Trp Ser Val Met Gln Arg Asn Tyr Leu Gln Gly
50 55 60
Glu Asn Glu Asn Arg Thr Gly Thr Leu Gln Gln Leu Ala Lys Arg Phe
65 70 75 80
Cys Gln Tyr Val Val Lys Gln Ser Glu Leu Lys Gly Thr Phe Lys Gly
85 90 95
His Lys Cys Ser Pro Cys Asp Thr Asn Trp Arg Tyr Tyr Gly Asp Ser
100 105 110
Cys Tyr Gly Phe Phe Arg His Asn Leu Thr Trp Glu Glu Ser Lys Gln
115 120 125
Tyr Cys Thr Asp Met Asn Ala Thr Leu Leu Lys Ile Asp Asn Arg Asn
130 135 140
Ile Val Glu Tyr Ile Lys Ala Arg Thr His Leu Ile Arg Trp Val Gly
145 150 155 160
Leu Ser Arg Gln Lys Ser Asn Glu Val Trp Lys Trp Glu Asp Gly Ser
165 170 175
Val Ile Ser Glu Asn Met Phe Glu Phe Leu Glu Asp Gly Lys Gly Asn
180 185 190
Met Asn Cys Ala Tyr Phe His Asn Gly Lys Met His Pro Thr Phe Cys
195 200 205
Glu Asn Lys His Tyr Leu Met Cys Glu Arg Lys Ala Gly Met Thr Lys
210 215 220
Val Asp Gln Leu Pro
225
<210> 70
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of human Clec-2 receptor
<400> 70
Met Gln Asp Glu Asp Gly Tyr Ile Thr Leu Asn Ile Lys Thr Arg Lys
1 5 10 15
Pro Ala Leu Ile Ser Val Gly Ser Ala Ser Ser Ser Trp Trp Arg Val
20 25 30
Met
<210> 71
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of human Clec-2 receptor
<400> 71
Ala Leu Ile Leu Leu Ile Leu Cys Val Gly Met Val Val Gly Leu Val
1 5 10 15
Ala Leu Gly Ile Trp
20
<210> 72
<211> 175
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of human Clec-2 receptor
<400> 72
Ser Val Met Gln Arg Asn Tyr Leu Gln Gly Glu Asn Glu Asn Arg Thr
1 5 10 15
Gly Thr Leu Gln Gln Leu Ala Lys Arg Phe Cys Gln Tyr Val Val Lys
20 25 30
Gln Ser Glu Leu Lys Gly Thr Phe Lys Gly His Lys Cys Ser Pro Cys
35 40 45
Asp Thr Asn Trp Arg Tyr Tyr Gly Asp Ser Cys Tyr Gly Phe Phe Arg
50 55 60
His Asn Leu Thr Trp Glu Glu Ser Lys Gln Tyr Cys Thr Asp Met Asn
65 70 75 80
Ala Thr Leu Leu Lys Ile Asp Asn Arg Asn Ile Val Glu Tyr Ile Lys
85 90 95
Ala Arg Thr His Leu Ile Arg Trp Val Gly Leu Ser Arg Gln Lys Ser
100 105 110
Asn Glu Val Trp Lys Trp Glu Asp Gly Ser Val Ile Ser Glu Asn Met
115 120 125
Phe Glu Phe Leu Glu Asp Gly Lys Gly Asn Met Asn Cys Ala Tyr Phe
130 135 140
His Asn Gly Lys Met His Pro Thr Phe Cys Glu Asn Lys His Tyr Leu
145 150 155 160
Met Cys Glu Arg Lys Ala Gly Met Thr Lys Val Asp Gln Leu Pro
165 170 175
<210> 73
<211> 231
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Human KLRF1 receptor
<400> 73
Met Gln Asp Glu Glu Arg Tyr Met Thr Leu Asn Val Gln Ser Lys Lys
1 5 10 15
Arg Ser Ser Ala Gln Thr Ser Gln Leu Thr Phe Lys Asp Tyr Ser Val
20 25 30
Thr Leu His Trp Tyr Lys Ile Leu Leu Gly Ile Ser Gly Thr Val Asn
35 40 45
Gly Ile Leu Thr Leu Thr Leu Ile Ser Leu Ile Leu Leu Val Ser Gln
50 55 60
Gly Val Leu Leu Lys Cys Gln Lys Gly Ser Cys Ser Asn Ala Thr Gln
65 70 75 80
Tyr Glu Asp Thr Gly Asp Leu Lys Val Asn Asn Gly Thr Arg Arg Asn
85 90 95
Ile Ser Asn Lys Asp Leu Cys Ala Ser Arg Ser Ala Asp Gln Thr Val
100 105 110
Leu Cys Gln Ser Glu Trp Leu Lys Tyr Gln Gly Lys Cys Tyr Trp Phe
115 120 125
Ser Asn Glu Met Lys Ser Trp Ser Asp Ser Tyr Val Tyr Cys Leu Glu
130 135 140
Arg Lys Ser His Leu Leu Ile Ile His Asp Gln Leu Glu Met Ala Phe
145 150 155 160
Ile Gln Lys Asn Leu Arg Gln Leu Asn Tyr Val Trp Ile Gly Leu Asn
165 170 175
Phe Thr Ser Leu Lys Met Thr Trp Thr Trp Val Asp Gly Ser Pro Ile
180 185 190
Asp Ser Lys Ile Phe Phe Ile Lys Gly Pro Ala Lys Glu Asn Ser Cys
195 200 205
Ala Ala Ile Lys Glu Ser Lys Ile Phe Ser Glu Thr Cys Ser Ser Val
210 215 220
Phe Lys Trp Ile Cys Gln Tyr
225 230
<210> 74
<211> 38
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of human KLRF1 receptor
<400> 74
Met Gln Asp Glu Glu Arg Tyr Met Thr Leu Asn Val Gln Ser Lys Lys
1 5 10 15
Arg Ser Ser Ala Gln Thr Ser Gln Leu Thr Phe Lys Asp Tyr Ser Val
20 25 30
Thr Leu His Trp Tyr Lys
35
<210> 75
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of human KLRF1 receptor
<400> 75
Ile Leu Leu Gly Ile Ser Gly Thr Val Asn Gly Ile Leu Thr Leu Thr
1 5 10 15
Leu Ile Ser Leu Ile
20
<210> 76
<211> 172
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of human KLRF1 receptor
<400> 76
Leu Leu Val Ser Gln Gly Val Leu Leu Lys Cys Gln Lys Gly Ser Cys
1 5 10 15
Ser Asn Ala Thr Gln Tyr Glu Asp Thr Gly Asp Leu Lys Val Asn Asn
20 25 30
Gly Thr Arg Arg Asn Ile Ser Asn Lys Asp Leu Cys Ala Ser Arg Ser
35 40 45
Ala Asp Gln Thr Val Leu Cys Gln Ser Glu Trp Leu Lys Tyr Gln Gly
50 55 60
Lys Cys Tyr Trp Phe Ser Asn Glu Met Lys Ser Trp Ser Asp Ser Tyr
65 70 75 80
Val Tyr Cys Leu Glu Arg Lys Ser His Leu Leu Ile Ile His Asp Gln
85 90 95
Leu Glu Met Ala Phe Ile Gln Lys Asn Leu Arg Gln Leu Asn Tyr Val
100 105 110
Trp Ile Gly Leu Asn Phe Thr Ser Leu Lys Met Thr Trp Thr Trp Val
115 120 125
Asp Gly Ser Pro Ile Asp Ser Lys Ile Phe Phe Ile Lys Gly Pro Ala
130 135 140
Lys Glu Asn Ser Cys Ala Ala Ile Lys Glu Ser Lys Ile Phe Ser Glu
145 150 155 160
Thr Cys Ser Ser Val Phe Lys Trp Ile Cys Gln Tyr
165 170
<210> 77
<211> 247
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Human Dectin-1 receptor
<400> 77
Met Glu Tyr His Pro Asp Leu Glu Asn Leu Asp Glu Asp Gly Tyr Thr
1 5 10 15
Gln Leu His Phe Asp Ser Gln Ser Asn Thr Arg Ile Ala Val Val Ser
20 25 30
Glu Lys Gly Ser Cys Ala Ala Ser Pro Pro Trp Arg Leu Ile Ala Val
35 40 45
Ile Leu Gly Ile Leu Cys Leu Val Ile Leu Val Ile Ala Val Val Leu
50 55 60
Gly Thr Met Ala Ile Trp Arg Ser Asn Ser Gly Ser Asn Thr Leu Glu
65 70 75 80
Asn Gly Tyr Phe Leu Ser Arg Asn Lys Glu Asn His Ser Gln Pro Thr
85 90 95
Gln Ser Ser Leu Glu Asp Ser Val Thr Pro Thr Lys Ala Val Lys Thr
100 105 110
Thr Gly Val Leu Ser Ser Pro Cys Pro Pro Asn Trp Ile Ile Tyr Glu
115 120 125
Lys Ser Cys Tyr Leu Phe Ser Met Ser Leu Asn Ser Trp Asp Gly Ser
130 135 140
Lys Arg Gln Cys Trp Gln Leu Gly Ser Asn Leu Leu Lys Ile Asp Ser
145 150 155 160
Ser Asn Glu Leu Gly Phe Ile Val Lys Gln Val Ser Ser Gln Pro Asp
165 170 175
Asn Ser Phe Trp Ile Gly Leu Ser Arg Pro Gln Thr Glu Val Pro Trp
180 185 190
Leu Trp Glu Asp Gly Ser Thr Phe Ser Ser Asn Leu Phe Gln Ile Arg
195 200 205
Thr Thr Ala Thr Gln Glu Asn Pro Ser Pro Asn Cys Val Trp Ile His
210 215 220
Val Ser Val Ile Tyr Asp Gln Leu Cys Ser Val Pro Ser Tyr Ser Ile
225 230 235 240
Cys Glu Lys Lys Phe Ser Met
245
<210> 78
<211> 44
<212> PRT
<213> Artificial Sequence
<220>
<223> The cytoplasmic domain of human Dectin-1 receptor
<400> 78
Met Glu Tyr His Pro Asp Leu Glu Asn Leu Asp Glu Asp Gly Tyr Thr
1 5 10 15
Gln Leu His Phe Asp Ser Gln Ser Asn Thr Arg Ile Ala Val Val Ser
20 25 30
Glu Lys Gly Ser Cys Ala Ala Ser Pro Pro Trp Arg
35 40
<210> 79
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of human Dectin-1 receptor
<400> 79
Leu Ile Ala Val Ile Leu Gly Ile Leu Cys Leu Val Ile Leu Val Ile
1 5 10 15
Ala Val Val Leu Gly
20
<210> 80
<211> 182
<212> PRT
<213> Artificial Sequence
<220>
<223> The extracellular domain of human Dectin-1 receptor
<400> 80
Thr Met Ala Ile Trp Arg Ser Asn Ser Gly Ser Asn Thr Leu Glu Asn
1 5 10 15
Gly Tyr Phe Leu Ser Arg Asn Lys Glu Asn His Ser Gln Pro Thr Gln
20 25 30
Ser Ser Leu Glu Asp Ser Val Thr Pro Thr Lys Ala Val Lys Thr Thr
35 40 45
Gly Val Leu Ser Ser Pro Cys Pro Pro Asn Trp Ile Ile Tyr Glu Lys
50 55 60
Ser Cys Tyr Leu Phe Ser Met Ser Leu Asn Ser Trp Asp Gly Ser Lys
65 70 75 80
Arg Gln Cys Trp Gln Leu Gly Ser Asn Leu Leu Lys Ile Asp Ser Ser
85 90 95
Asn Glu Leu Gly Phe Ile Val Lys Gln Val Ser Ser Gln Pro Asp Asn
100 105 110
Ser Phe Trp Ile Gly Leu Ser Arg Pro Gln Thr Glu Val Pro Trp Leu
115 120 125
Trp Glu Asp Gly Ser Thr Phe Ser Ser Asn Leu Phe Gln Ile Arg Thr
130 135 140
Thr Ala Thr Gln Glu Asn Pro Ser Pro Asn Cys Val Trp Ile His Val
145 150 155 160
Ser Val Ile Tyr Asp Gln Leu Cys Ser Val Pro Ser Tyr Ser Ile Cys
165 170 175
Glu Lys Lys Phe Ser Met
180
<210> 81
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> chimeric protein
<220>
<221> misc_feature
<222> (4)..(5)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (7)..(8)
<223> Xaa can be any naturally occurring amino acid
<400> 81
His Ala Glu Xaa Xaa Tyr Xaa Xaa Leu Gln Trp Asp
1 5 10
<210> 82
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> chimeric protein
<220>
<221> misc_feature
<222> (4)..(5)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (7)..(8)
<223> Xaa can be any naturally occurring amino acid
<400> 82
His Glu Glu Xaa Xaa Tyr Xaa Xaa Leu Gln Trp Asp
1 5 10
<210> 83
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> chimeric protein
<220>
<221> misc_feature
<222> (5)..(6)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (8)..(9)
<223> Xaa can be any naturally occurring amino acid
<400> 83
Met His Ala Glu Xaa Xaa Tyr Xaa Xaa Leu
1 5 10
<210> 84
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> chimeric protein
<220>
<221> misc_feature
<222> (5)..(6)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (8)..(9)
<223> Xaa can be any naturally occurring amino acid
<400> 84
Met His Glu Glu Xaa Xaa Tyr Xaa Xaa Leu
1 5 10
<210> 85
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> chimeric protein
<220>
<221> misc_feature
<222> (4)..(5)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (7)..(8)
<223> Xaa can be any naturally occurring amino acid
<400> 85
His Ala Glu Xaa Xaa Tyr Xaa Xaa Leu
1 5
<210> 86
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> chimeric protein
<220>
<221> misc_feature
<222> (4)..(5)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (7)..(8)
<223> Xaa can be any naturally occurring amino acid
<400> 86
His Glu Glu Xaa Xaa Tyr Xaa Xaa Leu
1 5
<210> 87
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> chimeric protein
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (4)..(5)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (7)..(8)
<223> Xaa can be any naturally occurring amino acid
<400> 87
His Xaa Glu Xaa Xaa Tyr Xaa Xaa Leu
1 5
<210> 88
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> The transmembrane domain of mouse TIMD4
<400> 88
Ile Leu Ile Ile Ala Cys Cys Val Gly Phe Val Leu Met Val Leu Leu
1 5 10 15
Phe Leu Ala Phe Leu
20
<210> 89
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> The signalling portion of human DNGR-1 cytoplasmic domain
<400> 89
Met His Glu Glu Glu Ile Tyr Thr Ser Leu Gln Trp Asp
1 5 10
<210> 90
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Syk binding sequence
<220>
<221> misc_feature
<222> (5)..(6)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (8)..(9)
<223> Xaa can be any naturally occurring amino acid
<400> 90
Met His Glu Glu Xaa Xaa Tyr Xaa Xaa Leu Gln Trp Asp
1 5 10
<210> 91
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Syk-binding sequence
<220>
<221> misc_feature
<222> (3)..(3)
<223> Xaa can be any naturally occurring amino acid
<400> 91
Met His Xaa Glu Glu Ile Tyr Thr Ser Leu Gln Trp Asp
1 5 10
<210> 92
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> linker sequence
<400> 92
Gly Gly Gly Ser Gly Gly Gly
1 5
<210> 93
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> linker sequence
<400> 93
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 94
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> linker sequence
<400> 94
Pro Gly Pro Gly
1
<210> 95
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> linker sequence
<400> 95
Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe
1 5 10
<210> 96
<211> 226
<212> PRT
<213> Artificial Sequence
<220>
<223> chimeric receptor
<400> 96
Met His Ala Glu Glu Ile Tyr Thr Ser Leu Gln Trp Asp Ile Pro Thr
1 5 10 15
Ser Glu Ala Ser Gln Lys Cys Gln Ser Pro Ser Lys Cys Ser Gly Ala
20 25 30
Val Gly Leu Gly Ile Leu Cys Phe Val Val Val Val Val Ala Ala Val
35 40 45
Leu Gly Ala Leu Ala Phe Trp Arg His Asn Ser Gly Arg Asn Pro Glu
50 55 60
Glu Lys Asp Ser Phe Leu Ser Arg Asn Lys Glu Asn His Lys Pro Thr
65 70 75 80
Glu Ser Ser Leu Asp Glu Lys Val Ala Pro Ser Lys Ala Ser Gln Thr
85 90 95
Thr Gly Gly Phe Ser Gln Ser Cys Leu Pro Asn Trp Ile Met His Gly
100 105 110
Lys Ser Cys Tyr Leu Phe Ser Phe Ser Gly Asn Ser Trp Tyr Gly Ser
115 120 125
Lys Arg His Cys Ser Gln Leu Gly Ala His Leu Leu Lys Ile Asp Asn
130 135 140
Ser Lys Glu Phe Glu Phe Ile Glu Ser Gln Thr Ser Ser His Arg Ile
145 150 155 160
Asn Ala Phe Trp Ile Gly Leu Ser Arg Asn Gln Ser Glu Gly Pro Trp
165 170 175
Phe Trp Glu Asp Gly Ser Ala Phe Phe Pro Asn Ser Phe Gln Val Arg
180 185 190
Asn Ala Val Pro Gln Glu Ser Leu Leu His Asn Cys Val Trp Ile His
195 200 205
Gly Ser Glu Val Tyr Asn Gln Ile Cys Asn Thr Ser Ser Tyr Ser Ile
210 215 220
Cys Glu
225
<210> 97
<211> 728
<212> PRT
<213> Artificial Sequence
<220>
<223> chimeric receptor
<400> 97
Met His Glu Glu Glu Ile Tyr Thr Ser Leu Gln Trp Asp Ser Pro Ala
1 5 10 15
Pro Asp Thr Tyr Gln Lys Cys Leu Ser Ser Asn Lys Cys Ser Gly Ala
20 25 30
Cys Cys Leu Val Met Val Ile Ser Cys Val Phe Cys Met Gly Leu Leu
35 40 45
Thr Ala Ser Ile Phe Leu Gly Val Cys Lys Gly Val Glu Pro Lys Thr
50 55 60
Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu Pro
65 70 75 80
Gly Glu Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu Lys
85 90 95
Arg Lys Leu Ser Glu Lys Leu Asp Ser Thr Asp Phe Thr Gly Thr Ile
100 105 110
Lys Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly Ser Gln
115 120 125
Lys Asp Glu Asn Leu Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu Phe
130 135 140
Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val
145 150 155 160
Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg
165 170 175
Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys
180 185 190
Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys
195 200 205
Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile
210 215 220
Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu
225 230 235 240
Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe
245 250 255
Pro Ile Val Asn Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly
260 265 270
Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln
275 280 285
Phe Pro Pro Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr
290 295 300
Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp
305 310 315 320
Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser
325 330 335
Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile
340 345 350
Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp
355 360 365
His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala
370 375 380
Ala Lys Ser Gly Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met
385 390 395 400
Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile
405 410 415
Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr
420 425 430
Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr
435 440 445
Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
450 455 460
Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asn
465 470 475 480
Val Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn Trp
485 490 495
Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe
500 505 510
Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp
515 520 525
Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu
530 535 540
Ile Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu
545 550 555 560
Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn
565 570 575
Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp
580 585 590
Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln
595 600 605
Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu
610 615 620
Thr Thr Asp Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys
625 630 635 640
Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu Ser Pro
645 650 655
Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser
660 665 670
Gly Ser His Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys
675 680 685
Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
690 695 700
Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp
705 710 715 720
Val Trp Asp Ile Asp Asn Glu Phe
725
<210> 98
<211> 340
<212> PRT
<213> Artificial Sequence
<220>
<223> chimeric receptor
<400> 98
Met Ile Leu Thr Ser Phe Gly Asp Asp Met Trp Leu Leu Thr Thr Leu
1 5 10 15
Leu Leu Trp Val Pro Val Gly Gly Glu Val Val Asn Ala Thr Lys Ala
20 25 30
Val Ile Thr Leu Gln Pro Pro Trp Val Ser Ile Phe Gln Lys Glu Asn
35 40 45
Val Thr Leu Trp Cys Glu Gly Pro His Leu Pro Gly Asp Ser Ser Thr
50 55 60
Gln Trp Phe Ile Asn Gly Thr Ala Val Gln Ile Ser Thr Pro Ser Tyr
65 70 75 80
Ser Ile Pro Glu Ala Ser Phe Gln Asp Ser Gly Glu Tyr Arg Cys Gln
85 90 95
Ile Gly Ser Ser Met Pro Ser Asp Pro Val Gln Leu Gln Ile His Asn
100 105 110
Asp Trp Leu Leu Leu Gln Ala Ser Arg Arg Val Leu Thr Glu Gly Glu
115 120 125
Pro Leu Ala Leu Arg Cys His Gly Trp Lys Asn Lys Leu Val Tyr Asn
130 135 140
Val Val Phe Tyr Arg Asn Gly Lys Ser Phe Gln Phe Ser Ser Asp Ser
145 150 155 160
Glu Val Ala Ile Leu Lys Thr Asn Leu Ser His Ser Gly Ile Tyr His
165 170 175
Cys Ser Gly Thr Gly Arg His Arg Tyr Thr Ser Ala Gly Val Ser Ile
180 185 190
Thr Val Lys Glu Leu Phe Thr Thr Pro Val Leu Arg Ala Ser Val Ser
195 200 205
Ser Pro Phe Pro Glu Gly Ser Leu Val Thr Leu Asn Cys Glu Thr Asn
210 215 220
Leu Leu Leu Gln Arg Pro Gly Leu Gln Leu His Phe Ser Phe Tyr Val
225 230 235 240
Gly Ser Lys Ile Leu Glu Tyr Arg Asn Thr Ser Ser Glu Tyr His Ile
245 250 255
Ala Arg Ala Glu Arg Glu Asp Ala Gly Phe Tyr Trp Cys Glu Val Ala
260 265 270
Thr Glu Asp Ser Ser Val Leu Lys Arg Ser Pro Glu Leu Glu Leu Gln
275 280 285
Val Leu Gly Pro Gln Ser Ser Ala Pro Val Trp Phe His Ile Leu Phe
290 295 300
Tyr Leu Ser Val Gly Ile Met Phe Ser Leu Asn Thr Val Leu Tyr Val
305 310 315 320
Gly Gly Gly Ser Gly Gly Gly Met His Glu Glu Glu Ile Tyr Thr Ser
325 330 335
Leu Gln Trp Asp
340
Claims (26)
1.一种嵌合受体，所述嵌合受体包含细胞外靶结合结构域、跨膜结构域和细胞内结构域，所述细胞内结构域包含来源于DNGR-1的胞质尾区的信号传导结构域的Syk结合序列，其中所述Syk结合序列含有酪氨酸残基。
2.根据权利要求1所述的嵌合受体，其中所述Syk结合序列包含如SEQ ID NO:15(MHAEXXYXXLQWD)中所示或如SEQ ID NO:90(MHEEXXYXXLQWD)中所示的氨基酸序列。
3.根据权利要求2所述的嵌合受体，其中所述Syk结合序列包含如SEQ ID NO:11(MHAEEIYTSLQWD)中所示的氨基酸序列或如SEQ ID NO:89(MHEEEIYTSLQWD)中所示的氨基酸序列。
4.根据前述权利要求中任一项所述的嵌合受体，其中所述靶结合结构域来源于非DNGR-1凝集素、转铁蛋白受体，或者其中所述靶结合结构域包含抗体可变区重链(VH)和/或轻链(VL)。
5.一种细胞，所述细胞包含根据权利要求1-4中任一项所述的嵌合受体。
6.根据权利要求5所述的细胞，其中所述细胞是专职抗原呈递细胞(APC)。
7.根据权利要求6所述的细胞，其中所述专职APC是巨噬细胞。
8.根据权利要求5所述的细胞，其中所述细胞不是专职抗原呈递细胞(APC)。
9.一种将生物聚合物递送至细胞的胞质溶胶的方法，其中所述细胞表达包含细胞内结构域的跨膜蛋白，所述细胞内结构域包含来源于DNGR-1的胞质尾区的信号传导结构域的Syk结合序列，其中所述生物聚合物包含可以特异性地结合所述跨膜蛋白的细胞外部分的结合结构域，其中所述方法包括使所述细胞与所述生物聚合物接触以允许所述结合结构域与所述跨膜蛋白的细胞外部分结合，使得所述生物聚合物被内化并易位到所述胞质溶胶中而不在吞噬体中降解，并且其中所述生物聚合物进一步包含编码基因产物的核酸。
10.根据权利要求9所述的方法，其中所述基因产物是促凋亡蛋白、酶、细胞毒性肽或抗原。
11.根据权利要求10所述的方法，其中所述生物聚合物的结合结构域是包含抗体可变区重链(VH)和/或可变区轻链(VL)的多肽。
12.根据权利要求9或权利要求10所述的方法，其中所述第二结构域经由接头与所述结合结构域共价连接，所述接头可以被所述细胞的胞质溶胶中存在的蛋白酶切割。
13.根据权利要求9至12中任一项所述的方法，其中所述跨膜蛋白是DNGR-1，并且其中所述生物聚合物的结合结构域特异性地结合DNGR-1的细胞外部分。
14.一种生物聚合物，所述生物聚合物包含结合结构域和第二结构域，其中所述结合结构域可以特异性地结合DNGR-1的细胞外部分，并且其中所述第二结构域是编码基因产物的核酸。
15.根据权利要求14所述的生物聚合物，其中所述基因产物是促凋亡蛋白、酶、细胞毒性肽或抗原。
16.一种核酸，所述核酸编码根据权利要求1至4中任一项所述的嵌合受体，或编码根据权利要求14或15所述的生物聚合物。
17.一种载体，所述载体包含根据权利要求16所述的核酸。
18.一种细胞，所述细胞包含根据权利要求16所述的核酸或根据权利要求17所述的载体。
19.一种药物组合物，所述药物组合物包含根据权利要求17所述的载体或根据权利要求18所述的细胞。
20.根据权利要求19所述的药物组合物，其用于药物中。
21.根据权利要求19所述的药物组合物，其用于治疗癌症的方法中，所述方法包括向所述患者施用所述药物组合物。
22.根据权利要求20所述的用于所述用途的药物组合物，其中所述治疗引发所述患者中的抗癌Th1反应。
23.根据权利要求18所述的药物组合物，其用于治疗感染性疾病的方法中，所述方法包括向所述受感染的患者施用所述药物组合物。
24.根据权利要求18所述的药物组合物，其用作疫苗。
25.根据权利要求18-22中任一项所述的用于所述用途的药物组合物，其中所述方法将抗原表达到患者细胞的胞质溶胶中。
26.根据权利要求18-23中任一项所述的用于所述用途的药物组合物，其中所述方法激活STING途径、RIG-I途径和/或MDA5途径。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB2011859.2A GB202011859D0 (en) | 2020-07-30 | 2020-07-30 | Cytosolic delivery |
| GB2011859.2 | 2020-07-30 | ||
| PCT/EP2021/071399 WO2022023528A1 (en) | 2020-07-30 | 2021-07-30 | Cytosolic delivery |
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| Publication Number | Publication Date |
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| CN116419761A true CN116419761A (zh) | 2023-07-11 |
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| CN202180065007.XA Pending CN116419761A (zh) | 2020-07-30 | 2021-07-30 | 胞质溶胶递送 |
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| Country | Link |
|---|---|
| US (1) | US20240100161A1 (zh) |
| EP (1) | EP4188946A1 (zh) |
| JP (1) | JP2023536441A (zh) |
| CN (1) | CN116419761A (zh) |
| AU (1) | AU2021319013A1 (zh) |
| CA (1) | CA3187062A1 (zh) |
| GB (1) | GB202011859D0 (zh) |
| WO (1) | WO2022023528A1 (zh) |
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| WO2023203198A1 (en) * | 2022-04-21 | 2023-10-26 | The Francis Crick Institute Limited | Bispecific binding agent |
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| GB0805159D0 (en) | 2008-03-19 | 2008-04-23 | Sancho Madrid David | Immune modulation via C-type lectin |
-
2020
- 2020-07-30 GB GBGB2011859.2A patent/GB202011859D0/en not_active Ceased
-
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- 2021-07-30 EP EP21762354.5A patent/EP4188946A1/en active Pending
- 2021-07-30 WO PCT/EP2021/071399 patent/WO2022023528A1/en unknown
- 2021-07-30 CN CN202180065007.XA patent/CN116419761A/zh active Pending
- 2021-07-30 CA CA3187062A patent/CA3187062A1/en active Pending
- 2021-07-30 US US18/018,685 patent/US20240100161A1/en active Pending
- 2021-07-30 JP JP2023505425A patent/JP2023536441A/ja active Pending
- 2021-07-30 AU AU2021319013A patent/AU2021319013A1/en active Pending
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| Publication number | Publication date |
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| AU2021319013A1 (en) | 2023-03-02 |
| JP2023536441A (ja) | 2023-08-25 |
| CA3187062A1 (en) | 2022-02-03 |
| GB202011859D0 (en) | 2020-09-16 |
| US20240100161A1 (en) | 2024-03-28 |
| WO2022023528A1 (en) | 2022-02-03 |
| EP4188946A1 (en) | 2023-06-07 |
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