相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS
本申请要求2015年1月29日提交的美国临时专利申请62/109,281的的优先权，通过引用将其并入本文。This application claims priority to U.S. Provisional Patent Application No. 62/109,281, filed January 29, 2015, which is incorporated herein by reference.
发明内容Summary of the Invention
在一个方面，本公开描述了用于在自然杀伤(NK)细胞中表达的嵌合抗原受体。通常，所述嵌合抗原受体包括包含抗原识别区的胞外结构域、连接至所述胞外结构域的跨膜结构域，以及连接至所述跨膜结构域的胞内结构域。所述胞内结构域可包含激活NK细胞的信号传导肽(signaling peptide)。In one aspect, the present disclosure describes a chimeric antigen receptor for expression in natural killer (NK) cells. Typically, the chimeric antigen receptor includes an extracellular domain comprising an antigen recognition region, a transmembrane domain connected to the extracellular domain, and an intracellular domain connected to the transmembrane domain. The intracellular domain may include a signaling peptide that activates NK cells.
在一些实施方式中，所述抗原识别结构域可特异性地结合与疾病相关的抗原。In some embodiments, the antigen recognition domain can specifically bind to an antigen associated with a disease.
在一些实施方式中，所述抗原识别结构域可特异性地结合肿瘤抗原。In some embodiments, the antigen recognition domain can specifically bind to a tumor antigen.
在一些实施方式中，所述胞外结构域还可包括信号肽(signal peptide)或前导序列(leader sequence)和/或间隔序列(spacer)。In some embodiments, the extracellular domain may further include a signal peptide or a leader sequence and/or a spacer sequence.
在一些实施方式中，所述胞内结构域可包括诸如2B4、DAP10、DAP12、IL21R、CD137(41BB)或CD3ζ的NK细胞膜结合信号接头蛋白(NK cell membrane-bound signalingadaptor protein)的信号传导结构域(signaling domain)。In some embodiments, the intracellular domain may include a signaling domain of a NK cell membrane-bound signaling adapter protein such as 2B4, DAP10, DAP12, IL21R, CD137 (41BB), or CD3ζ.
在一些实施方式中，所述跨膜结构域可包括诸如CD16、NKp44、NKp46或NKG2D的在NK细胞中表达的自然细胞毒性受体(natural cytotoxicity receptor)的跨膜区。In some embodiments, the transmembrane domain may include a transmembrane region of a natural cytotoxicity receptor expressed in NK cells, such as CD16, NKp44, NKp46, or NKG2D.
在另一方面，本公开描述了包括被修饰以便表达上文总结的嵌合抗原受体的任何实施方式的NK细胞(和/或iPSC)的药物组合物。In another aspect, the present disclosure describes pharmaceutical compositions comprising NK cells (and/or iPSCs) modified to express any of the embodiments of the chimeric antigen receptor summarized above.
在另一方面，本公开描述了向有病况(condition)的受试者提供免疫治疗的方法。通常，该方法包括将上文总结的治疗性组合物施用于该受试者，其中所述嵌合抗原受体的抗原识别区特异性地结合与该病况相关的抗原。In another aspect, the present disclosure describes a method of providing immunotherapy to a subject having a condition. Generally, the method comprises administering to the subject a therapeutic composition as summarized above, wherein the antigen recognition region of the chimeric antigen receptor specifically binds to an antigen associated with the condition.
以上的总结无意描述本发明的每个公开的实施方式或者每种实践。以下的说明书更具体地例举了阐释性实施方式。在该申请的若干处，通过列出实施例来提供指导，这些实施例可以以各种组合使用。每种情形下，所给出的列表仅用作代表性组，而不应解释为排他性列表。The above summary is not intended to describe every disclosed embodiment or every practice of the present invention. The following description more specifically exemplifies illustrative embodiments. In several places throughout this application, guidance is provided by listing examples, which can be used in various combinations. In each case, the list provided serves only as a representative group and should not be construed as an exclusive list.
附图简要说明BRIEF DESCRIPTION OF THE DRAWINGS
图1.(A)示例性自然细胞毒性受体以及这些受体与它们配体的结合对NK细胞脱颗粒和极化以及靶细胞杀伤的影响。(B)自然细胞毒性受体的代表性实例和它们相应的信号接头。(C)用在iPSC衍生的NK细胞中的第三代嵌合T细胞抗原受体构建体。Figure 1. (A) Exemplary natural cytotoxicity receptors and the effects of binding of these receptors to their ligands on NK cell degranulation and polarization, as well as target cell killing. (B) Representative examples of natural cytotoxicity receptors and their corresponding signaling adaptors. (C) Third-generation chimeric T cell antigen receptor constructs used in iPSC-derived NK cells.
图2.(A)NK激活CAR(NK-activating CAR)的一般化示意图。(B)新型嵌合抗原受体构建体的示意图。将嵌合抗原受体克隆进pkt2载体，该载体含有与SB100X转座酶一起使用的IR/DR、mCAGs启动子、嵌合抗原受体(CAR)序列、内部核糖体进入位点(IRES)，以及GFP:Zeo选择标记。嵌合抗原受体片段从UniProt获得，并通过gBlock合成和传统限制酶克隆(IDT)进行组装。Figure 2. (A) Generalized schematic diagram of NK-activating CAR. (B) Schematic diagram of the novel chimeric antigen receptor construct. The chimeric antigen receptor was cloned into the pkt2 vector, which contains IR/DR for use with SB100X transposase, mCAGs promoter, chimeric antigen receptor (CAR) sequence, internal ribosome entry site (IRES), and GFP:Zeo selection marker. The chimeric antigen receptor fragment was obtained from UniProt and assembled by gBlock synthesis and traditional restriction enzyme cloning (IDT).
图3.在NK92和iPS细胞中嵌合抗原受体的表面表达。采用SB100X使用SleepingBeaut转座子系统转染NK92细胞或iPS细胞。然后使用博莱霉素(Zeocin)筛选细胞，并进行流式细胞分析以评估各种嵌合抗原受体的细胞表面表达。采用生物素偶联的识别小鼠IgGF(ab’)2片段的多克隆山羊抗小鼠抗体来评估表达(Jackson ImmunoResearchLaboratories,Inc.,West Grove,PA,cat#115-065-072)。采用与荧光染料偶联的链霉亲和素来检测所结合的抗体。Figure 3. Surface expression of chimeric antigen receptors in NK92 and iPS cells. NK92 cells or iPS cells were transfected using the SleepingBeaut transposon system using SB100X. Cells were then screened with Zeocin and flow cytometry was performed to assess the cell surface expression of various chimeric antigen receptors. Expression was assessed using a biotin-coupled polyclonal goat anti-mouse antibody that recognizes mouse IgG F(ab')2 fragments (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, cat#115-065-072). Bound antibodies were detected using streptavidin coupled to a fluorescent dye.
图4.NK92细胞中CD107A的释放和IFN-γ的产生。NK92细胞脱颗粒和细胞因子生成采用流式细胞分析评价。将NK92细胞与间皮素(mesothelin)阴性(MA148)、间皮素阳性(A1847)卵巢癌靶细胞，或者与间皮素/Fc嵌合蛋白偶联或未偶联的蛋白A微珠以1:1混合。将细胞针对CD107a染色，针对IFN-γ的产生进行细胞内染色。Figure 4. CD107A release and IFN-γ production in NK92 cells. NK92 cell degranulation and cytokine production were assessed by flow cytometry. NK92 cells were mixed 1:1 with mesothelin-negative (MA148), mesothelin-positive (A1847) ovarian cancer target cells, or with protein A microbeads conjugated or unconjugated with a mesothelin/Fc chimeric protein. Cells were stained for CD107a, and intracellular staining was performed for IFN-γ production.
图5.采用NK92细胞的Cr-51释放测定。将NK92、NK92/28/41BB/CD3或NK92/CAR4细胞与K562、K562间皮素+、MA148或A1847细胞以标明的比例一起温育4小时。然后检测Cr-51的释放以评估细胞杀伤。本实验依照Woll等，2009,Blood113(24):6094-6101进行，除了用iPSC衍生的NK细胞替换hESC衍生的NK细胞。Figure 5. Cr-51 release assay using NK92 cells. NK92, NK92/28/41BB/CD3, or NK92/CAR4 cells were incubated with K562, K562 mesothelin+, MA148, or A1847 cells at the indicated ratios for 4 hours. Cr-51 release was then measured to assess cell killing. This experiment was performed according to Woll et al., 2009, Blood 113(24):6094-6101, except that iPSC-derived NK cells were used instead of hESC-derived NK cells.
图6.示例性的其他NK激活嵌合抗原受体。Figure 6. Exemplary additional NK-activating chimeric antigen receptors.
图7.比较第三代T细胞CAR和图2中反映的示例性NK CAR构建体的示意图。FIG7 . Schematic diagram comparing third generation T cell CARs and the exemplary NK CAR constructs reflected in FIG2 .
图8.示例性NK CAR构建体的示意图。Figure 8. Schematic diagram of exemplary NK CAR constructs.
图9.示例性NK CAR构建体的示意图。Figure 9. Schematic diagram of exemplary NK CAR constructs.
图10.显示NK CAR对K562细胞的细胞毒性的数据。Figure 10. Data showing the cytotoxicity of NK CAR on K562 cells.
图11.显示NK CAR对两种卵巢癌细胞系的细胞毒性的数据。Figure 11. Data showing the cytotoxicity of NK CAR against two ovarian cancer cell lines.
图12.显示通过诱导多能干细胞表达示例性NK CAR的数据。Figure 12. Data showing expression of exemplary NK CARs by induced pluripotent stem cells.
图13.显示通过iPSC衍生的NK细胞表达而进行的示例性CAR的表面表达的数据。Figure 13. Data showing surface expression of exemplary CARs by iPSC-derived NK cells.
图14.示例性的一般化的NK CAR载体构建体。FIG14. Exemplary generalized NK CAR vector constructs.
图15.示例性的一般化的NK CAR载体构建体。串联cHS4绝缘子可抑制CAR载体沉默，由此改善该CAR在NK细胞和iPSC中的表达。Figure 15. Exemplary generalized NK CAR vector constructs. Tandem cHS4 insulators inhibit CAR vector silencing, thereby improving the expression of the CAR in NK cells and iPSCs.
具体实施方式DETAILED DESCRIPTION
本公开描述了经设计特异性地并入NK细胞激活结构域的嵌合抗原受体。嵌合抗原受体可并入细胞内区和/或跨膜区，包括例如与来自例如2B4(CD244)、CD137(41BB)、IL21、DAP10、DAP12和/或CD3ζ的共刺激或信号传导结构域相连接的来自CD16、NKp44、NKp46和/或NKG2D的细胞内区和/或跨膜区。The present disclosure describes chimeric antigen receptors that are designed to specifically incorporate NK cell activation domains. The chimeric antigen receptors can be incorporated into intracellular and/or transmembrane regions, including, for example, intracellular and/or transmembrane regions from CD16, NKp44, NKp46, and/or NKG2D connected to costimulatory or signaling domains from, for example, 2B4 (CD244), CD137 (41BB), IL21, DAP10, DAP12, and/or CD3ζ.
嵌合抗原受体(CAR)为工程化人工受体，其可为表达该CAR的免疫细胞提供工程化特异性。通常，可从患有特定形式癌症的受试者收集免疫细胞群。所收集的免疫细胞可经修饰以表达与肿瘤细胞表达的抗原特异性地结合的嵌合抗原受体，然后回输进该受试者。表达嵌合抗原受体的经修饰的免疫细胞能够更好地识别并杀灭表达被该嵌合抗原受体特异性地识别的抗原的肿瘤细胞。Chimeric antigen receptors (CARs) are engineered artificial receptors that provide engineered specificity for immune cells expressing the CARs. Typically, immune cell populations can be collected from subjects with specific forms of cancer. The collected immune cells can be modified to express chimeric antigen receptors that specifically bind to antigens expressed by tumor cells and then infused back into the subject. Modified immune cells expressing chimeric antigen receptors are better able to recognize and kill tumor cells that express antigens specifically recognized by the chimeric antigen receptors.
嵌合抗原受体已被设计来激活T细胞，用于处理难治性ALL(靶向CD19)、胰腺癌(靶向间皮素)，以及其它恶性肿瘤。存在几种嵌合抗原受体构建体，但多数是设计来激活T细胞。Chimeric antigen receptors have been designed to activate T cells for the treatment of refractory ALL (targeting CD19), pancreatic cancer (targeting mesothelin), and other malignancies. Several chimeric antigen receptor constructs exist, but most are designed to activate T cells.
相对而言，本文描述的嵌合抗原受体经设计为在诱导性多功能干细胞(iPSC)中表达，这些细胞然后分化为NK细胞。它们也可直接表达进外周血(PB)-NK细胞、NK-92细胞，或另外的合适NK细胞系中。NK-92细胞或其它NK细胞系已经用在临床研究中用于抗癌治疗。表达嵌合抗原受体的NK细胞接着可用作免疫治疗用于处理多种癌症。本文描述的嵌合抗原受体可包括可与各种靶向抗体的抗原识别部分一起使用的信号传导结构域。具体地，本公开描述了反映靶向间皮素的嵌合抗原受体用于治疗卵巢癌的示例性实施方式。但是，由于间皮素在很多腺癌上表达，所描述的实施方式可具有更广泛的用途。此外，所描述的间皮素靶向结构域仅仅是示例性的；其它的单链可变片段(scFV)可被工程化进该NK特异性的嵌合抗原受体(NK-CAR)信号构建体中以靶向基本上任何恶性肿瘤。In contrast, the chimeric antigen receptors described herein are designed to be expressed in induced pluripotent stem cells (iPSCs), which are then differentiated into NK cells. They can also be directly expressed in peripheral blood (PB)-NK cells, NK-92 cells, or other suitable NK cell lines. NK-92 cells or other NK cell lines have been used in clinical studies for anti-cancer treatment. NK cells expressing chimeric antigen receptors can then be used as immunotherapy for treating a variety of cancers. The chimeric antigen receptors described herein may include a signaling domain that can be used with the antigen recognition portion of various targeting antibodies. Specifically, the present disclosure describes exemplary embodiments of chimeric antigen receptors that reflect targeting mesothelin for the treatment of ovarian cancer. However, since mesothelin is expressed on many adenocarcinomas, the described embodiments may have a wider range of uses. In addition, the mesothelin targeting domain described is merely exemplary; other single-chain variable fragments (scFV) can be engineered into the NK-specific chimeric antigen receptor (NK-CAR) signaling construct to target essentially any malignant tumor.
本文描述的NK细胞嵌合抗原受体的一个特征为可绕过自然细胞毒性受体(naturalcytotoxicity receptor)启动信号转导所需的接头分子/辅助蛋白(adaptormolecules/accessoryproteins)。另外一种方式或除此之外，包括有通常与接头分子/辅助蛋白结合的跨膜结构域使得辅助蛋白也能进行结合，让信号转导更可能启动。设计为包括例如CD3ζ的NK细胞嵌合抗原受体使得能够绕过其它的自然细胞毒性受体。并入跨膜结构域和其它的细胞内结构域可使得这些NK细胞嵌合抗原受体能够与接头蛋白结合并通过激活多种途径而提供比单独CD3ζ更佳的信号传导。One feature of the NK cell chimeric antigen receptor described herein is that it can bypass the natural cytotoxicity receptor (natural cytotoxicity receptor) to initiate the adapter molecules / accessory proteins required for signal transduction. Alternatively or in addition thereto, a transmembrane domain that is typically bound to an adapter molecule / accessory protein is included so that the accessory protein can also bind, making it more likely that signal transduction will be initiated. Designed to include, for example, an NK cell chimeric antigen receptor for CD3ζ enables bypassing other natural cytotoxicity receptors. Incorporation of a transmembrane domain and other intracellular domains enables these NK cell chimeric antigen receptors to bind to adapter proteins and provide better signal transduction than CD3ζ alone by activating multiple pathways.
尽管一些T细胞嵌合抗原受体构建体由于共享的信号传导结构域而可在一定程度上激活NK细胞，但是本文描述的嵌合抗原受体是特异性地设计为激活NK细胞。经设计为特异性地激活NK细胞的嵌合抗原受体可在NK细胞免疫疗法(例如，细胞介导的难治性肿瘤杀灭)中改善NK功能和受体用途。Although some T cell chimeric antigen receptor constructs can activate NK cells to a certain extent due to shared signaling domains, chimeric antigen receptors described herein are specifically designed to activate NK cells. Chimeric antigen receptors designed to specifically activate NK cells can improve NK function and receptor uses in NK cell immunotherapy (e.g., cell-mediated refractory tumor killing).
嵌合抗原受体典型地包括胞外结构域、跨膜结构域以及胞内结构域。所述胞内结构域通常驻留在细胞的细胞质中。一旦抗原被胞外结构域所识别，该胞内结构域就将激活信号传递至NK细胞，这诱导NK细胞破坏所靶向的肿瘤细胞。示例性信号传导胞内结构域包括例如膜结合信号接头蛋白(包括例如，2B4(CD244)、CD137(41BB)、IL21、DAP10、DAP12和/或CD3ζ)的信号传导结构域，或其部分，包括例如免疫受体酪氨酸活化基序(ITAM)、YxxM基序、TxYxxV/I基序、FcRγ、NKp80(通过非典型hemi-ITAM信号传导)和/或DNAM等。Chimeric antigen receptor typically includes an extracellular domain, a transmembrane domain and an intracellular domain. The intracellular domain usually resides in the cytoplasm of the cell. Once the antigen is recognized by the extracellular domain, the intracellular domain will transmit the activation signal to the NK cell, which induces the NK cell to destroy the targeted tumor cells. Exemplary signal transduction intracellular domain includes, for example, a membrane-bound signal adapter protein (including, for example, 2B4 (CD244), CD137 (41BB), IL21, DAP10, DAP12 and/or CD3ζ) signal transduction domain, or a portion thereof, including, for example, immunoreceptor tyrosine activation motif (ITAM), YxxM motif, TxYxxV/I motif, FcRγ, NKp80 (by atypical hemi-ITAM signal transduction) and/or DNAM etc.
跨膜结构域横跨质膜，将胞内结构域连接至胞外结构域。示例性跨膜结构域例如包括自然细胞毒性受体(NCR)(例如包括CD16、NKp44、NKp46、NKG2D、NKp30、NKp80和/或DNAM-1)的细胞内和/或跨膜结构域，或其包括例如一个或更多个带电荷氨基酸的部分。在一些实施方式中，该带电荷的氨基酸可以是赖氨酸和/或精氨酸残基。在一些情况下，跨膜区可以来自跨膜蛋白，意味着其天然具有细胞外C末端，而不是细胞外N末端。这种情况下，可反转跨膜区的方向，例如在图6中以“Rev TM”标明的，以便嵌合抗原受体在NK细胞膜中正确定向。The membrane spanning domain spans the plasma membrane, connecting the intracellular domain to the extracellular domain. Exemplary membrane spanning domains, for example, include the intracellular and/or membrane spaning domains of natural cytotoxicity receptors (NCRs) (for example, including CD16, NKp44, NKp46, NKG2D, NKp30, NKp80 and/or DNAM-1), or include, for example, the part of one or more charged amino acids. In some embodiments, the charged amino acids can be lysine and/or arginine residues. In some cases, the membrane spanning region can be from a transmembrane protein, meaning that it naturally has an extracellular C-terminal, rather than an extracellular N-terminal. In this case, the direction of the membrane spanning region can be reversed, such as indicated with "Rev TM" in Figure 6, so that the chimeric antigen receptor is correctly oriented in the NK cell membrane.
胞外结构域通常包括信号肽和抗原识别区。在很多实施方式中，胞外结构域还可包括间隔序列(spacer)。信号肽将新生多肽引导进内质网，以便其可被正确糖基化并锚定进质膜。一般来讲，可使用任何真核细胞信号肽，只要其引导蛋白至内质网。一个示例性信号肽包括CD8α前导序列，但其它的信号肽序列也可能是适合的。当间隔序列存在时，其将抗原识别结构域连接至跨膜结构域。该间隔序列通常提供柔韧性，从而抗原识别区可自由定向至不同方向，由此使得抗原识别区能够结合抗原靶。一个示例性间隔序列包括CD8α铰链序列，但其它的Ig铰链区也可能是适合的。抗原识别区可包括能够特异性地结合指定靶的任何肽序列。如本文所用，“特异性地结合”及其变型指对特定靶具有任何程度的差异化的或非一般的亲和力。因此，抗原识别区可包括特异性地结合特定抗原(例如，肿瘤抗原、病毒抗原、经修饰的自身抗原等)的抗体片段，例如scFv或Fab。在一些实施方式中，scFv可来自单克隆抗体。嵌合抗原受体可设计为包括可特异性地结合任何指定靶的抗原识别区。因此，尽管图2、图6、图7和图8显示了设计为特异性地结合间皮素的实施方式，但是NK激活嵌合抗原受体可被设计为特异性地结合并由此靶向与准备作为NK细胞介导杀伤标靶的细胞(例如致瘤细胞或病毒感染细胞)相关的任何抗原。例如，NK细胞和/或CAR已显示出针对各种各样的实体瘤和病毒感染细胞的活性，包括但不限于，HIV(人免疫缺陷病毒)、乙型肝炎、丙型肝炎、CMV(巨细胞病毒)、EBV(Epstein-Barr病毒)、HPV(人乳头瘤病毒)，等等。The extracellular domain generally includes a signal peptide and an antigen recognition region. In many embodiments, the extracellular domain may further include a spacer. The signal peptide guides the nascent polypeptide into the endoplasmic reticulum so that it can be correctly glycosylated and anchored into the plasma membrane. Generally speaking, any eukaryotic cell signal peptide can be used as long as it guides the protein to the endoplasmic reticulum. An exemplary signal peptide includes the CD8α leader sequence, but other signal peptide sequences may also be suitable. When the spacer is present, it connects the antigen recognition domain to the transmembrane domain. The spacer generally provides flexibility, so that the antigen recognition region can be freely oriented to different directions, thereby enabling the antigen recognition region to bind to the antigen target. An exemplary spacer includes the CD8α hinge sequence, but other Ig hinge regions may also be suitable. The antigen recognition region may include any peptide sequence that can specifically bind to a specified target. As used herein, "specifically bind" and its variants refer to having any degree of differentiated or non-general affinity for a specific target. Therefore, the antigen recognition region may include an antibody fragment, such as scFv or Fab, that specifically binds to a specific antigen (e.g., a tumor antigen, a viral antigen, a modified self-antigen, etc.). In some embodiments, the scFv may be derived from a monoclonal antibody. The chimeric antigen receptor can be designed to include an antigen recognition region that can specifically bind to any specified target. Therefore, although Figures 2, 6, 7, and 8 show embodiments designed to specifically bind to mesothelin, the NK activating chimeric antigen receptor can be designed to specifically bind to and thereby target any antigen associated with cells (e.g., tumorigenic cells or virus-infected cells) that are prepared as targets for NK cell-mediated killing. For example, NK cells and/or CARs have shown activity against a variety of solid tumors and virus-infected cells, including but not limited to HIV (human immunodeficiency virus), hepatitis B, hepatitis C, CMV (cytomegalovirus), EBV (Epstein-Barr virus), HPV (human papillomavirus), and the like.
于是，例如，为了更好地介导针对包括表达间皮素的细胞的肿瘤(例如，卵巢癌、胰腺癌、肺癌、结肠腺癌、间皮瘤，以及其它表达间皮素的腺癌)的NK细胞毒性，可以设计并在NK细胞中表达例如图2、图6、图7和图8中显示的嵌合抗原受体。所展示的嵌合抗原受体构建体含有NK细胞特异性跨膜结构域和激活结构域(activating domain)，并能够在NK细胞肿瘤系NK92中表达。跨膜区和细胞内区取自CD16、NKp44、NKp46和/或NKG2D，同时以试图最大化激活NK细胞的方式组合2B4、DAP10、DAP12和/或CD3ζ的激活结构域。图3显示NK92和iPS细胞表达图2中显示的嵌合抗原受体。Thus, for example, in order to better mediate NK cell toxicity against tumors including cells expressing mesothelin (e.g., ovarian cancer, pancreatic cancer, lung cancer, colon adenocarcinoma, mesothelioma, and other adenocarcinomas expressing mesothelin), chimeric antigen receptors such as those shown in Figures 2, 6, 7, and 8 can be designed and expressed in NK cells. The chimeric antigen receptor constructs shown contain NK cell-specific transmembrane domains and activation domains and can be expressed in the NK cell tumor line NK92. The transmembrane region and the intracellular region are taken from CD16, NKp44, NKp46, and/or NKG2D, while the activation domains of 2B4, DAP10, DAP12, and/or CD3ζ are combined in an attempt to maximize the activation of NK cells. Figure 3 shows that NK92 and iPS cells express the chimeric antigen receptor shown in Figure 2.
为了评估嵌合抗原受体的功能，针对抗原包覆微珠和间皮素表达细胞系测试了表达该嵌合抗原受体的NK细胞。图4显示，当表达嵌合抗原受体的NK细胞与间皮素阳性靶混合时，图2的嵌合抗原受体增强了NK细胞的脱颗粒和细胞因子生成。To evaluate the functionality of the chimeric antigen receptor, NK cells expressing this chimeric antigen receptor were tested against antigen-coated microbeads and a mesothelin-expressing cell line. Figure 4 shows that when chimeric antigen receptor-expressing NK cells were mixed with a mesothelin-positive target, the chimeric antigen receptor of Figure 2 enhanced NK cell degranulation and cytokine production.
图5显示，与第三代T细胞特异嵌合抗原受体(NK92/28/41BB/CD3ζ)或非转染NK92细胞相比，表达如本文描述的NK特异性嵌合抗原受体的NK92细胞改善了对间皮素阳性靶细胞的体外杀伤。Figure 5 shows that NK92 cells expressing the NK-specific chimeric antigen receptor described herein improved in vitro killing of mesothelin-positive target cells compared to third-generation T cell-specific chimeric antigen receptor (NK92/28/41BB/CD3ζ) or non-transfected NK92 cells.
图10显示了示例性NK CAR对K562细胞(左上插图)和作为该CAR的标靶的表达间皮素的K562细胞(右上插图)的细胞毒性。这些结果显示了以间皮素特异性方式明显改善了杀伤，以CAR7和CAR9最为显著。下方的插图为对以溶解单位表示的结果的总结(Bryant等,1992,J Immunol Methods 146(1):91-103)。CAR7和CAR9显示了比以前的研究中使用的第三代T细胞CAR(NK92meso 3rd)明显更高的细胞毒性。按之前描述的Cr-51释放测定法测量细胞毒性(Knorr等,2013,Stem Cells Transl Med 2(4):274-283；Woll等,2009,Blood 113(24):3094-6101；Woll等,2005,J Immunol 175(8):5095-5103)。图11显示了采用两种卵巢癌细胞系meso-高(A1497)和meso-低(MA148)的类似结果。具有不同的基于NK细胞的抗mesoCAR的NK92以meso特异性的方式杀伤。底部插图也反映了以溶解单位表示的总结。此外，当以如图10和图11中的标靶刺激时，NK CAR介导了增强的CD107a和/或IFN-γ表达(数据未示出)。Figure 10 shows the cytotoxicity of exemplary NK CAR to K562 cells (upper left illustration) and K562 cells expressing mesothelin as the target of the CAR (upper right illustration). These results show that killing was significantly improved in a mesothelin-specific manner, with CAR7 and CAR9 being the most significant. The illustration below is a summary of the results expressed in lysis units (Bryant et al., 1992, J Immunol Methods 146 (1): 91-103). CAR7 and CAR9 showed significantly higher cytotoxicity than the third generation T cell CAR (NK92meso3rd ) used in previous studies. Cytotoxicity was measured by the Cr-51 release assay described previously (Knorr et al., 2013, Stem Cells Transl Med 2(4):274-283; Woll et al., 2009, Blood 113(24):3094-6101; Woll et al., 2005, J Immunol 175(8):5095-5103). Figure 11 shows similar results using two ovarian cancer cell lines, meso-high (A1497) and meso-low (MA148). NK92 with different NK cell-based anti-mesoCARs killed in a meso-specific manner. The bottom inset also reflects the summary expressed in lytic units. In addition, when stimulated with targets as shown in Figures 10 and 11, NK CAR mediated enhanced CD107a and/or IFN-γ expression (data not shown).
在一些实施方式中，如本文描述的NK特异的嵌合抗原受体可在iPSC中表达，这些iPSC可随后分化成NK细胞。可以按Knorr等,2013,Stem Cells Transl Med.2(4):274-283或Ni等,2014,Stem Cells 32(4):1021-1031中所描述分化iPSC。图12显示通过诱导多能干细胞(iPSCs)表达示例性NK CAR，如通过CD45+CD56+细胞的产生所显示的(首行，第5插图)。图13显示通过iPSC衍生的NK细胞进行的CAR表面表达。图13的底部三行显示，与不表达CAR的PB-NK细胞和iPSC-NK细胞(未修饰的对照细胞，第4列，第4和第5行)相比，仅在iPSC-CAR4v2(第4列，底部三行)表面上的CAR表达。图12和13中的其它插图显示iPSC-CAR4v2上的其它典型NK细胞表面抗原/受体与存在于未修饰对照细胞上的那些相类似。这些结果表明，iPSC衍生的NK细胞可展示出与图10和图11中的靶向间皮素的CAR表达NK细胞(mesothelin-targeted-CAR-expressing NK cells)相同的靶特异性细胞毒性。In some embodiments, the chimeric antigen receptor of NK specific as described herein can be expressed in iPSC, and these iPSC can be subsequently differentiated into NK cells. Can be pressed Knorr et al., 2013, Stem Cells Transl Med.2 (4): 274-283 or Ni et al., 2014, Stem Cells 32 (4): 1021-1031 described in differentiation iPSC. Figure 12 shows that exemplary NK CAR is expressed by induced pluripotent stem cells (iPSCs), as shown by the generation of CD45+CD56+ cells (first row, the 5th illustration). Figure 13 shows the CAR surface expression carried out by NK cells derived from iPSC. The bottom three rows of Figure 13 show that, compared with PB-NK cells and iPSC-NK cells (unmodified control cells, the 4th column, the 4th and 5th rows) that do not express CAR, only CAR expression on the surface of iPSC-CAR4v2 (the 4th column, the bottom three rows) is expressed. Other illustrations in Figures 12 and 13 show that other typical NK cell surface antigens/receptors on iPSC-CAR4v2 are similar to those present on unmodified control cells. These results indicate that iPSC-derived NK cells can exhibit the same target-specific cytotoxicity as the mesothelin-targeted-CAR-expressing NK cells in Figures 10 and 11.
可以采用常规转染方法将编码NK CAR构建体的多核苷酸引入NK细胞或iPSC。由此，尽管本文是在采用Sleeping Beauty转座子系统将编码CAR的多核苷酸转染进细胞的示例性实施方式情形下描述，但可以采用任何适合的转染方法修饰NK细胞(和/或iPSC)。图14显示了可以用于修饰NK细胞(和/或iPSCs)以表达嵌合抗原受体的示例性载体构建体(vector construct)。图15显示了备选的示例性载体的构建，该载体还包括串联cHS4绝缘子(Aker等,2007,Hum Gene Ther 18(4):333-343)，其可抑制CAR载体沉默，由此改善该CAR在NK细胞和iPSC中的表达。Conventional transfection methods can be used to introduce polynucleotides encoding NK CAR constructs into NK cells or iPSC. Thus, although this article is described in the exemplary embodiment of the case where the polynucleotides encoding CAR are transfected into cells using the Sleeping Beauty transposon system, any suitable transfection method can be used to modify NK cells (and/or iPSC). Figure 14 shows exemplary vector constructs (vector constructs) that can be used to modify NK cells (and/or iPSCs) to express chimeric antigen receptors. Figure 15 shows the construction of an alternative exemplary vector, which also includes a series cHS4 insulator (Aker et al., 2007, Hum Gene Ther 18 (4): 333-343), which can suppress CAR vector silencing, thereby improving the expression of the CAR in NK cells and iPSC.
本文描述的经修饰以表达嵌合抗原受体的NK细胞和/或iPSs可以与“载体(carrier)”一起配制成药物组合物，用于递送至受试者，该受试者具有至少部分地以细胞可以是NK细胞毒性的标靶为特征的病况。如本文所用，“载体”包括任何溶剂、分散介质、媒介、包衣、稀释剂、抗菌剂和/或抗真菌剂、等渗剂、吸收延缓剂、缓冲剂、载体溶液、混悬剂、胶体等。将这些介质和/或试剂用于药学上有活性的物质在本领域内是公知的。除了任何常规媒介或试剂与该活性成分不相容之外，考虑将该活性成分用在治疗性组合物中。补充性活性成分也可掺入到该组合物中。The modified NK cells and/or iPSs described herein that express chimeric antigen receptors can be formulated into pharmaceutical compositions together with a "carrier" for delivery to a subject having a condition characterized at least in part by cells that can be targets of NK cell toxicity. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and/or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, etc. It is well known in the art to use these media and/or reagents for pharmaceutically active substances. Except that any conventional vehicle or reagent is incompatible with the active ingredient, it is considered that the active ingredient is used in a therapeutic composition. Supplementary active ingredients may also be incorporated into the composition.
“药学上可接受的”指并非生物学上或其它方面不利的物质，即，该物质可以与经修饰以表达嵌合抗原受体的NK细胞(和/或iPSC)一起施用于个体，而不引起任何不希望的生物效果或不与其所在的药物组合物中的任何其它组分以有害方式相互作用。"Pharmaceutically acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., the substance can be administered to an individual along with NK cells (and/or iPSCs) modified to express a chimeric antigen receptor without causing any undesirable biological effect or interacting in a deleterious manner with any other component of the pharmaceutical composition in which it is contained.
该药物组合物可以配制为适合于优选施用途径的各种形式。由此，组合物可以通过已知途径施用，例如包括胃肠外(例如，皮内、经皮、皮下、肌内、静脉内、腹膜内等等)或局部施用(例如，气管内、肺内等等)。组合物也可通过持续或延缓释放施用。The pharmaceutical composition can be formulated into various forms suitable for preferred routes of administration. Thus, the composition can be administered by known routes, including, for example, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.) or topical administration (e.g., intratracheal, intrapulmonary, etc.). The composition can also be administered by sustained or delayed release.
制剂可以方便地以单位剂型呈现，并且可以通过药学领域公知的方法制备。以药学上可接受的载体制备组合物的方法包括让经修饰以表达嵌合抗原受体的NK细胞(和/或iPSC)与构成一种或更多种辅助成分的载体结合的步骤。一般来讲，可以通过让NK细胞(和/或iPSC)均匀地和/或密切地与例如液体载体结合来制备制剂。The preparation can be conveniently presented in a unit dosage form and can be prepared by methods well known in the pharmaceutical field. The method for preparing a composition with a pharmaceutically acceptable carrier includes the step of combining NK cells (and/or iPSCs) modified to express a chimeric antigen receptor with a carrier constituting one or more auxiliary components. Generally speaking, the preparation can be prepared by combining NK cells (and/or iPSCs) uniformly and/or closely with, for example, a liquid carrier.
包括经修饰以表达嵌合抗原受体的NK细胞(和/或iPSC)的药物组合物可以以适合的形式提供，包括但不限于溶液、混悬液、乳剂、喷雾剂、气溶胶，或任何形式的混合物。该组合物可以在制剂中与任何药学上可接受的赋形剂、载体或媒介一起递送。The pharmaceutical composition comprising NK cells (and/or iPSCs) modified to express chimeric antigen receptors can be provided in a suitable form, including but not limited to a solution, a suspension, an emulsion, a spray, an aerosol, or a mixture of any form. The composition can be delivered in a formulation with any pharmaceutically acceptable excipient, carrier, or vehicle.
施用于受试者的经修饰以表达嵌合抗原受体的NK细胞(和/或iPSC)的量可随各种因素变动，包括但不限于，受试者的体重、身体状况，和/或年龄，是否施用一种或更多种嵌合抗原受体，和/或施用途径。因此，包括在给定单位剂型中的NK细胞(和/或iPSC)的绝对量可大范围变动，并取决于一些因素，例如受试者的物种、年龄、体重和身体状况，以及施用方法。相应地，普遍性地阐述对每个和/或所有可能的应用有效的经修饰以表达嵌合抗原受体的NK细胞(和/或iPSC)的量是不切实际的。然而，本领域普通技术人员通过适当考虑这些因素可容易地确定合适的量。The amount of NK cells (and/or iPSC) modified to express chimeric antigen receptors administered to a subject may vary with various factors, including but not limited to, the subject's weight, physical condition, and/or age, whether one or more chimeric antigen receptors are administered, and/or route of administration. Therefore, the absolute amount of NK cells (and/or iPSC) included in a given unit dosage form may vary widely and depends on factors such as the subject's species, age, weight, and physical condition, as well as the method of administration. Accordingly, it is impractical to generally describe the amount of NK cells (and/or iPSC) modified to express chimeric antigen receptors that are effective for each and/or all possible applications. However, one of ordinary skill in the art can easily determine a suitable amount by appropriately considering these factors.
在一些实施方式中，该方法可包括施用足够数量的经修饰以表达嵌合抗原受体的NK细胞(和/或iPSC)，以向受试者提供例如约105个细胞/kg至约1010个细胞/kg的剂量，尽管在一些实施方式中可以通过施用超出该范围的剂量的NK细胞(和/或iPSC)来实施该方法。在这些实施方式的一些中，该方法包括施用足够数量的经修饰以表达嵌合抗原受体的NK细胞(和/或iPSC)，以向受试者提供例如约107个细胞/kg至约108个细胞/kg的剂量，例如，约1×107个细胞/kg至约8×107个细胞/kg的剂量。In some embodiments, the method may include administering a sufficient number of NK cells (and/or iPSCs) modified to express a chimeric antigen receptor to provide, for example, a dose of about 105 cells/kg to about 1010 cells/kg to the subject, although in some embodiments the method may be implemented by administering a dose of NK cells (and/or iPSCs) outside this range. In some of these embodiments, the method includes administering a sufficient number of NK cells (and/or iPSCs) modified to express a chimeric antigen receptor to provide, for example, a dose of about 107 cells/kg to about 108 cells/kg to the subject, for example, a dose of about 1×107 cells/kg to about 8×107 cells/kg.
另外一种方式，可以采用在治疗过程开始前获得的实际体重来计算该剂量。对于以此方式计算的剂量，在治疗过程开始之前采用Dubois法计算身体表面积(m2)：m2＝(重量kg0.425×身高cm0.725)×0.007184。Alternatively, the dose can be calculated using actual body weight obtained before the start of the treatment course. For doses calculated in this manner, body surface area (m2 ) is calculated before the start of the treatment course using the Dubois method: m2 = (weight kg0.425 × height cm0.725 ) × 0.007184.
在一些实施方式中，包括经修饰以表达嵌合抗原受体的NK细胞(和/或iPSC)的药物组合物可以例如以每周单剂量至多剂量进行施用，尽管在一些实施方式中药物组合物可通过以超出该范围的频率施用来实施该方法。在某些实施方式中，该药物组合物可以每月施用约1次至每周施用约5次。In some embodiments, the pharmaceutical composition comprising NK cells (and/or iPSCs) modified to express a chimeric antigen receptor can be administered, for example, in a single dose to multiple doses per week, although in some embodiments the pharmaceutical composition can be administered at a frequency exceeding this range to implement the method. In certain embodiments, the pharmaceutical composition can be administered from about once a month to about five times a week.
通常，以病况的症状或临床迹象任何程度上的有效减少、限制其进展、缓解或消退的量和给药方案向受试者施用药物组合物。如本文所用，“缓解”指特定病况特征性的症状或临床征象在程度、严重性、频率，和/或可能性方面的任何减轻。“症状”指疾病或患者病况的任何主观证据(subjective evidence)。“征象”或“临床征象”指非患者本人发现的与具体状况相关的客观身体检查结果。Typically, the pharmaceutical composition is administered to a subject in an amount and dosage regimen effective to reduce, limit progression, alleviate, or resolve the symptoms or clinical signs of a condition to any extent. As used herein, "alleviation" refers to any reduction in the extent, severity, frequency, and/or likelihood of symptoms or clinical signs characteristic of a particular condition. "Symptoms" refers to any subjective evidence of a disease or patient's condition. "Signs" or "clinical signs" refer to objective physical findings related to a specific condition that are not observed by the patient himself.
在前面的描述中，为了清楚起见，可以孤立地描述特定实施方式。除非另有明确指明特定实施方式的特征与另一实施方式的特征不相容，否则某些实施方式可包括本文结合一个或更多个实施方式描述的相容特征的组合。In the foregoing description, particular embodiments may be described in isolation for clarity. Unless otherwise expressly stated that features of a particular embodiment are incompatible with features of another embodiment, certain embodiments may include a combination of compatible features described herein in conjunction with one or more embodiments.
对于本文描述的包括分开步骤的任何方法，这些方法可以以任何可行的顺序进行。视情况，两个或更多个步骤的任意组合可以同时进行。For any method described herein comprising separate steps, these methods can be performed in any feasible order. Optionally, any combination of two or more steps can be performed simultaneously.
本发明通过以上描述的示例性实施方式来阐释。应当理解，特定的实例、物质、量、以及操作应根据本文描述的发明的范围和精神在广义上解释。The present invention is illustrated by the exemplary embodiments described above. It should be understood that the specific examples, materials, amounts, and operations should be interpreted broadly in accordance with the scope and spirit of the invention described herein.
如本文所用，术语“和/或”指所列出的要素的一个或全部或所列出的要素的任意两个或更多个的组合；当出现在说明书和权利要求书中时，术语“包含”及其变型不具有限制性含义；除非另有指出，“a”、“an”、“the”以及“至少一个”可互换使用，指一个或多于一个；通过端点对数值范围的叙述包括归纳在该范围内的所有数字(例如，1至5包括1、1.5、2、2.75、3、3.80、4、5等等)。As used herein, the term "and/or" refers to one or all of the listed elements or any combination of two or more of the listed elements; the term "comprising" and its variations do not have a limiting meaning when appearing in the specification and claims; unless otherwise indicated, "a," "an," "the," and "at least one" are used interchangeably to mean one or more than one; the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
将本文提及的所有专利、专利申请和出版物以及以电子方式提供的材料(例如，包括例如GenBank和RefSeq中的核苷酸序列提交，例如SwissProt、PIR、PRF、PDB中氨基酸序列提交，以及GenBank和RefSeq中来自注释编码区域的翻译)的完整公开通过引用以其全文并入。如果本申请的公开与通过引用并入本文的任何文献的公开之间存在任何不一致，则适用本申请的公开。仅出于清楚理解的目的给出了前文的详细说明和实施例。不应从中理解不必要的限制。本发明不限于所示和所描述的精确细节，因为本领域技术人员显而易见的变化将被包括在由权利要求书所定义的发明内。The complete disclosures of all patents, patent applications, and publications mentioned herein, as well as electronically provided materials (e.g., including, for example, nucleotide sequence submissions in GenBank and RefSeq, amino acid sequence submissions in SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. If there is any inconsistency between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application applies. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be understood therefrom. The present invention is not limited to the precise details shown and described, as variations obvious to those skilled in the art will be included in the invention defined by the claims.
除非另有说明，在说明书和权利要求书中使用的表示组分数量、分子量等的所有数字应理解为在所有情况下都被术语“约”修饰。因此，除非有相反说明，在说明书和权利要求书中列出的数值参数为近似值，它们可以随本发明所寻求获得的期望性能而变化。最起码，并且并非试图将等同原则的应用限于权利要求的范围，每一数值参数应至少按照所报告的有效数以及应用普通舍入法来理解。Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and the like used in the specification and claims should be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximate and may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
尽管陈述本发明的广泛范围的数值范围和参数是近似值，但是具体实施例中列出的数值为尽可能精确地报告。然而，所有数值固有地包含由其各自测量中发现的标准偏差所必然产生的范围。Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain the range necessarily resulting from the standard deviation found in their respective measurements.
除非另有说明，所有的标题都是为了方便阅读者，而不应用于限制该标题后文本的含义。Unless otherwise indicated, all headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US62/109,281 | 2015-01-29 |
| Publication Number | Publication Date |
|---|---|
| HK1242705A1 HK1242705A1 (en) | 2018-06-29 |
| HK1242705Btrue HK1242705B (en) | 2022-03-18 |
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