本发明属于细胞治疗领域。更具体地，本发明涉及工程化的滋养细胞及其用途。The present invention belongs to the field of cell therapy. More specifically, the present invention relates to engineered trophoblastic cells and their uses.
自然杀伤(NK)细胞是体内重要的淋巴细胞，具有细胞毒性和免疫调节功能，参与体内天然免疫和适应性免疫，被认为是最有潜力的肿瘤杀伤效应细胞之一。NK细胞表达多种激活受体，其中的NKp46被认为是跨物种NK细胞的统一特征分子。Natural killer (NK) cells are important lymphocytes in the body, with cytotoxic and immunomodulatory functions, participating in innate immunity and adaptive immunity in the body, and are considered to be one of the most potential tumor-killing effector cells. NK cells express a variety of activation receptors, among which NKp46 is considered to be a unified characteristic molecule of NK cells across species.
NK细胞在细胞治疗领域有以下优势：1.NK细胞能自发地杀死肿瘤细胞的能力，无需提前敏化，无需抗原呈递，起效更迅速；2.NK细胞能通过多种途径杀死肿瘤细胞；3.NK细胞治疗不会产生严重不良反应，仅有较轻的移植物抗宿主病(GvHD)；4.NK细胞可以不用改造直接用于异体通用型的免疫细胞治疗等等。但是，由于NK细胞只占正常人外周血淋巴细胞的1％-32.6％，体外扩增难度较大，而且在体内存活寿命有限，NK细胞免疫治疗一直受限于无法获得足够数量的细胞等问题。NK cells have the following advantages in the field of cell therapy: 1. NK cells can spontaneously kill tumor cells without prior sensitization or antigen presentation, and take effect more quickly; 2. NK cells can kill tumor cells through multiple pathways; 3. NK cell therapy does not produce serious adverse reactions, only mild graft-versus-host disease (GvHD); 4. NK cells can be directly used for allogeneic universal immune cell therapy without modification, etc. However, since NK cells only account for 1%-32.6% of normal human peripheral blood lymphocytes, it is difficult to expand in vitro, and their survival lifespan in vivo is limited. NK cell immunotherapy has always been limited by the problem of not being able to obtain a sufficient number of cells.
已有研究报道，IL-2、IL-15等一些细胞因子可以用于扩增NK细胞，但作用有限。因此，仍有必要开发一种工程化滋养细胞，用于研究NK细胞的功能，并为细胞治疗奠定基础。Studies have reported that some cytokines such as IL-2 and IL-15 can be used to expand NK cells, but the effect is limited. Therefore, it is still necessary to develop an engineered trophoblast cell to study the function of NK cells and lay the foundation for cell therapy.
发明内容Summary of the invention
除非另有说明，否则本文中所使用的所有科学技术术语的含义与本发明所属领域的普通技术人员通常所了解的相同。Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
本发明的目的在于提供一种工程化滋养细胞，其表达特异性识别NKp46的细胞表面分子。与未经工程化的滋养细胞相比，将该工程化滋养细胞用于激活NK细胞，能够增强NK细胞扩增能力和肿瘤细胞杀伤活性。The purpose of the present invention is to provide an engineered trophoblast cell, which expresses a cell surface molecule that specifically recognizes NKp46. Compared with unengineered trophoblast cells, the engineered trophoblast cell is used to activate NK cells, which can enhance the NK cell proliferation ability and tumor cell killing activity.
工程化滋养细胞Engineered trophoblasts
在第一个方面，本发明提供一种工程化滋养细胞，其表达特异性识别NKp46的细胞表面分子，所述细胞表面分子至少包含靶向NKp46的抗体和跨膜结构域。In a first aspect, the present invention provides an engineered trophoblast cell expressing a cell surface molecule that specifically recognizes NKp46, wherein the cell surface molecule comprises at least an antibody targeting NKp46 and a transmembrane domain.
如本文所用，术语“滋养细胞”是指添加至靶细胞(例如NK细胞)培养物中以支持其存活和/或生长的细胞。滋养细胞提供完整和功能性的细胞外基质和基质相关的因子，并且将已知和未知的细胞因子分泌至条件培养基中。滋养细胞通常处于生长停滞状态，以防止其在培养物中增殖，但维持其存活。生长停滞可以通过有效剂量的辐照或用有效剂量的化学物质(如丝裂霉素C)处理来实现。适用于本发明的滋养细胞包括：PBMC、RPMI8866、HFWT、K562、EBV-LCL、721.221以及NK92细胞等。As used herein, the term "feeder cells" refers to cells that are added to target cell (e.g., NK cell) cultures to support Cells that maintain their survival and/or growth. Trophoblasts provide complete and functional extracellular matrix and matrix-related factors, and secrete known and unknown cytokines into conditioned medium. Trophoblasts are usually in a state of growth arrest to prevent them from proliferating in culture, but maintain their survival. Growth arrest can be achieved by irradiation of an effective dose or treatment with an effective dose of a chemical substance (such as mitomycin C). Trophoblasts suitable for the present invention include: PBMC, RPMI8866, HFWT, K562, EBV-LCL, 721.221 and NK92 cells, etc.
如本文所用，术语“细胞表面分子”是指在细胞表面表达的能够与NKp46特异性结合的分子。此类表面分子一般包含能够与NKp46特异性结合的抗体以及将表面分子锚定在细胞表面的跨膜结构域。任选地，表面分子还包含位于抗体和跨膜结构域之间的铰链区、负责信号传递的胞内结构域、外源的细胞活化分子等。As used herein, the term "cell surface molecule" refers to a molecule expressed on the cell surface that is capable of specifically binding to NKp46. Such surface molecules generally include antibodies that are capable of specifically binding to NKp46 and transmembrane domains that anchor the surface molecule to the cell surface. Optionally, the surface molecule also includes a hinge region between the antibody and the transmembrane domain, an intracellular domain responsible for signal transduction, an exogenous cell activation molecule, and the like.
如本文所用，“抗体”是指可以与抗原结合的任何结构或其功能性变体，包括但不限于单克隆抗体、多克隆抗体、重组抗体，可以选自人抗体、人源化抗体、鼠源抗体、嵌合抗体及其功能性片段。例如，抗体包括但不限于完整抗体、Fab、Fab'、F(ab')2、Fd、Fd′、Fv、scFv、sdFv、线性抗体、sdAb、纳米抗体、双体、anticalin、DARPIN等。在本发明中，抗体可以是单价或二价，且可以是单特异性、双特异性或多特异性的抗体。As used herein, "antibody" refers to any structure or functional variant thereof that can bind to an antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, and can be selected from human antibodies, humanized antibodies, mouse antibodies, chimeric antibodies, and functional fragments thereof. For example, antibodies include but are not limited to complete antibodies, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies, sdAb, nanobodies, diabodies, anticalins, DARPINs, etc. In the present invention, antibodies can be monovalent or divalent, and can be monospecific, bispecific, or multispecific antibodies.
本领域已知的靶向NKp46的抗体均可用于本发明。在一些实施方案中，所述靶向NKp46的抗体选自完整抗体、Fab、Fab’、F(ab’)2、Fd、Fd′、Fv、scFv、sdFv、线性抗体、双体、sdAb和纳米抗体，优选选自Fab、scFv、单域抗体和纳米抗体。优选地，所述所述靶向NKp46的抗体包含轻链可变区和重链可变区，其中所述轻链可变区包含的CDR1-L、CDR2-L和CDR3-L与SEQ ID NO:7所包含的CDR1-L、CDR2-L和CDR3-L相同；其中所述重链可变区包含的CDR1-H、CDR2-H和CDR3-H与SEQ ID NO:8所包含的CDR1-H、CDR2-H和CDR3-H相同。优选地，所述CDR1-L如SEQ ID NO:1所示，CDR2-L如SEQ ID NO:2所示，CDR3-L如SEQ ID NO:3所示，CDR1-H如SEQ ID NO:4所示，CDR2-H如SEQ ID NO:5所示，CDR3-H如SEQ ID NO:6所示。Antibodies targeting NKp46 known in the art can be used in the present invention. In some embodiments, the antibody targeting NKp46 is selected from a complete antibody, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibody, diabody, sdAb and nanobody, preferably selected from Fab, scFv, single domain antibody and nanobody. Preferably, the antibody targeting NKp46 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are the same as the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO:7; wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are the same as the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO:8. Preferably, the CDR1-L is shown as SEQ ID NO: 1, CDR2-L is shown as SEQ ID NO: 2, CDR3-L is shown as SEQ ID NO: 3, CDR1-H is shown as SEQ ID NO: 4, CDR2-H is shown as SEQ ID NO: 5, and CDR3-H is shown as SEQ ID NO: 6.
在一些实施方案中，所述轻链可变区与SEQ ID NO:7具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性；所述重链可变区与SEQ ID NO:8具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。优选地，其中所述靶向NKp4的抗体与SEQ ID NO:10所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性In some embodiments, the light chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with SEQ ID NO:7; the heavy chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with SEQ ID NO:8. Preferably, the antibody targeting NKp4 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:10. or 100% sequence identity
术语“重链”是指抗体分子中天然存在的构象中存在的两种类型多肽链中较大的一种，并且通常决定抗体所属的类别。术语“轻链”是指抗体分子中天然存在的构象中存在的两种类型多肽链中较小的一种。Kappa(κ)和lambda(λ)轻链是指两种主要的抗体轻链同种型。The term "heavy chain" refers to the larger of the two types of polypeptide chains that occur in naturally occurring conformations in antibody molecules and generally determines the class to which the antibody belongs. The term "light chain" refers to the smaller of the two types of polypeptide chains that occur in naturally occurring conformations in antibody molecules. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.
术语“互补决定区”或“CDR”是指赋予抗原特异性和结合亲和力的抗体可变区内的氨基酸序列。例如，一般来说，每个重链可变区中存在三个CDR(例如CDR1-H、CDR2-H、和CDR3-H)，并且每个轻链可变区中存在三个CDR(CDR1-L、CDR2-L、和CDR3-L)。CDR的精确氨基酸序列边界可以使用许多熟知的方案中的任一种来确定，包括：Kabat编号方案、Chothia编号方案、IMGT编号方案、AHo编号方案、AbM编号方案。给定CDR或FR的精确氨基酸序列可能由于选择的编号方案不同而不同，应理解，给定抗体或其区域(如其可变区)的“CDR”或“FR”涵盖由任何上述方案或其他已知方案所定义的CDR或FR，在指定的CDR或FR含有给定氨基酸序列的情况下，应理解，这样的CDR或FR还可以具有由任何上述方案或其他已知方案所定义的相应CDR或FR的序列。在本文中用于界定CDR和FR的边界的编号方案为Chothia方案。The term "complementarity determining region" or "CDR" refers to the amino acid sequence in the variable region of an antibody that imparts antigen specificity and binding affinity. For example, in general, there are three CDRs (e.g., CDR1-H, CDR2-H, and CDR3-H) in each heavy chain variable region, and there are three CDRs (CDR1-L, CDR2-L, and CDR3-L) in each light chain variable region. The precise amino acid sequence boundaries of CDRs can be determined using any of many well-known schemes, including: Kabat numbering scheme, Chothia numbering scheme, IMGT numbering scheme, AHo numbering scheme, AbM numbering scheme. The precise amino acid sequence of a given CDR or FR may be different due to the different numbering schemes selected, and it should be understood that a "CDR" or "FR" of a given antibody or its region (such as its variable region) covers a CDR or FR defined by any of the above schemes or other known schemes, and in the case where a specified CDR or FR contains a given amino acid sequence, it should be understood that such CDR or FR can also have a sequence of a corresponding CDR or FR defined by any of the above schemes or other known schemes. The numbering scheme used herein to define the boundaries of CDRs and FRs is the Chothia scheme.
术语“单链抗体”或“scFv”是指包含至少一个轻链可变区(VL)和至少一个重链可变区(VH)的融合蛋白，其中所述轻链可变区和重链可变区是邻接的(例如经由接头连接)，并且能够以单链多肽形式表达，且其中所述scFv保留其来源的完整抗体的特异性。除非另有说明，否则本文中的scFv可以以任何顺序(例如相对于多肽的N-末端和C末端)具有所述的VL和VH，scFv从N端到C端可以包含VL-接头-VH，也可以包含VH-接头-VL。术语“接头”是指连接两个分子或同一分子上的两个序列的分子序列。在一些实施方案中，接头是肽接头。优选地，接头不会不利地影响多肽的表达、分泌或生物活性。此外，接头优选地不是抗原性的并且不引发免疫应答。在一些实施方案中，接头可以是内源氨基酸序列、外源氨基酸序列(例如，富含GS的序列)或非肽化学接头。在一种实施方案中，本发明所述的接头与SEQ ID NO:28或29所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。The term "single-chain antibody" or "scFv" refers to a fusion protein comprising at least one light chain variable region (VL) and at least one heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region are adjacent (e.g., connected via a linker) and can be expressed in the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, the scFv herein may have the VL and VH in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may include VL-linker-VH or VH-linker-VL from the N-terminus to the C-terminus. The term "linker" refers to a molecular sequence connecting two molecules or two sequences on the same molecule. In some embodiments, the linker is a peptide linker. Preferably, the linker does not adversely affect the expression, secretion or biological activity of the polypeptide. In addition, the linker is preferably not antigenic and does not induce an immune response. In some embodiments, the linker may be an endogenous amino acid sequence, an exogenous amino acid sequence (e.g., a sequence rich in GS) or a non-peptide chemical linker. In one embodiment, the linker described in the present invention has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:28 or 29.
如本文所用，术语“跨膜结构域”是指能够使细胞表面分子在细胞表面上表达的多肽结构。跨膜结构域可以是天然或合成的，也可以源自任何膜结合蛋白或跨膜蛋白。当抗体与靶抗原结合时，跨膜结构域能够进行信号传导。特别适用于本发明中的跨膜结构域可以源自例如TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137和CD154。优选地，所述跨膜结构域选自CD8α、CD4、CD28或CD278的跨膜结构域。更为优选地，所述跨膜结构域为CD8α或CD28跨膜结构域，与SEQ ID NO:12-14任一项所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables cell surface molecules to be expressed on the cell surface. The transmembrane domain may be natural or synthetic, and may also be derived from any membrane-bound protein or transmembrane protein. When the antibody binds to the target antigen, the transmembrane domain is capable of signal transduction. Transmembrane domains particularly suitable for use in the present invention can be derived from, for example, TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 zeta subunit, CD3 epsilon subunit, CD3 gamma subunit, CD3 delta subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. Preferably, the transmembrane domain is selected from the transmembrane domain of CD8 alpha, CD4, CD28 or CD278. More preferably, the transmembrane domain is a CD8 alpha or CD28 transmembrane domain, having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 12-14.
在一些实施方案中，本发明的工程化滋养细胞还可以表达位于抗体和跨膜结构域之间的铰链区。如本文所用，术语“铰链区”一般是指作用为连接跨膜结构域至抗体的任何寡肽或多肽。具体地，铰链区用来为抗体提供更大的灵活性和可及性。铰链区可以包含最多达300个氨基酸，优选10至100个氨基酸并且最优选25至50个氨基酸。铰链区可以源自全部或部分的天然分子，如源自全部或部分的CD8、CD4或CD28的胞外区，或源自全部或部分的抗体恒定区。或者，铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。优选地，所述铰链区选自以下蛋白的铰链区：CD8α、CD28、FcγRIIIα受体、IgG4和IgG1，更优选与SEQ ID NO:21-24所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。In some embodiments, the engineered trophoblast of the present invention can also express a hinge region between the antibody and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the antibody. Specifically, the hinge region is used to provide greater flexibility and accessibility for the antibody. The hinge region can contain up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region can be derived from all or part of a natural molecule, such as from the extracellular region of all or part of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a fully synthetic hinge sequence. Preferably, the hinge region is selected from the hinge regions of the following proteins: CD8α, CD28, FcγRIIIα receptor, IgG4 and IgG1, and more preferably has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:21-24.
在一些实施方案中，本发明的细胞表面分子还包含胞内结构域。优选地，所述胞内结构域包含共刺激结构域和/或初级信号传导结构域。In some embodiments, the cell surface molecule of the present invention further comprises an intracellular domain. Preferably, the intracellular domain comprises a co-stimulatory domain and/or a primary signaling domain.
本发明的初级信号传导结构域可以是T细胞受体和共受体的细胞质序列，其在抗原受体结合以后一同起作用以引发信号传导，以及这些序列的任何衍生物或变体和具有相同或相似功能的任何合成序列。初级信号传导结构域包含两种不同类型的细胞质信号序列：引发抗原依赖性初级活化的那些，以及以不依赖抗原的方式起作用以提供次级或共刺激信号的那些。初级细胞质信号序列可以包含许多免疫受体酪氨酸激活基序(ITAM)。本发明的初级信号传导结构域包括但不限于源自FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b、CD66d及其任意组合。优选地，所述初级信号传导结构域包含CD3ζ的信号传导结构域。更为优选地，所述初级信号传导结构域与SEQ ID NO:18-20任一所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。The primary signaling domain of the present invention can be a cytoplasmic sequence of a T cell receptor and a co-receptor that works together to initiate signaling after antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same or similar functions. The primary signaling domain comprises two different types of cytoplasmic signal sequences: those that induce antigen-dependent primary activation, and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals. The primary cytoplasmic signal sequence may comprise a number of immunoreceptor tyrosine activation motifs (ITAMs). The primary signaling domain of the present invention includes, but is not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, and any combination thereof. Preferably, the primary signaling domain comprises the signaling domain of CD3ζ. More preferably, the primary signaling domain has at least 70%, preferably at least 80%, with the amino acid sequence shown in any one of SEQ ID NOs: 18-20, More preferred is a sequence identity of at least 90%, 95%, 97% or 99% or 100%.
本发明的共刺激结构域可以是来自共刺激分子的细胞内功能性信号传导结构域，其可以包含所述共刺激分子的整个细胞内部分，或其功能片段。“共刺激分子”是指在T细胞上与共刺激配体特异性结合，由此介导T细胞的共刺激反应(例如增殖)的同源结合配偶体。共刺激分子包括但不限于1类MHC分子、BTLA和Toll配体受体。本发明的共刺激结构域的非限制性施例包括但不限于源自以下蛋白质的共刺激信号传导结构域：LTB、CD94、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18、CD27、CD28、CD30、CD40、CD54、CD83、CD134、CD137、CD270、CD272、CD276、CD278、CD357、DAP10、DAP12、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM、ZAP70及其任意组合。优选地，所述共刺激结构域选自4-1BB、CD28、CD27、OX40、CD278或其任意组合。更为优选地，所述共刺激结构域为4-1BB或CD28，与SEQ ID NO:15-17任一所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。The costimulatory domain of the present invention can be an intracellular functional signaling domain from a costimulatory molecule, which can include the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof. "Costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell. Costimulatory molecules include, but are not limited to, class 1 MHC molecules, BTLA, and Toll ligand receptors. Non-limiting examples of the costimulatory domains of the present invention include, but are not limited to, costimulatory signaling domains derived from the following proteins: LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD137, CD270, CD272, CD276, CD278, CD357, DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and any combination thereof. Preferably, the costimulatory domain is selected from 4-1BB, CD28, CD27, OX40, CD278, or any combination thereof. More preferably, the co-stimulatory domain is 4-1BB or CD28, having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:15-17.
在一些实施方案中，本发明的细胞表面分子还包含信号肽，所述信号肽选自以下蛋白的信号肽：CD8α、IgG1、GM-CSFRα、IgG4或其任意组合。优选地，所述信号肽包含CD8α或B2M的信号肽。更为优选地，所述信号肽与SEQ ID NO:25-27任一所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。In some embodiments, the cell surface molecule of the present invention further comprises a signal peptide, and the signal peptide is selected from the signal peptides of the following proteins: CD8α, IgG1, GM-CSFRα, IgG4 or any combination thereof. Preferably, the signal peptide comprises a signal peptide of CD8α or B2M. More preferably, the signal peptide has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:25-27.
在一些实施方案中，本发明的细胞表面分子还包含接头，其用于隔开任何本文中所述的结构域/区。例如，接头可位于信号肽与抗体之间、抗体的VH与VL之间、抗体与铰链区之间、铰链区与跨膜结构域之间、侧接共刺激结构域或在共刺激结构域的N-或C-区上、和/或在跨膜结构域与初级信号传导结构域之间。接头可以是长度为约6至约40个氨基酸、或长度为约6至约25个氨基酸的肽。In some embodiments, the cell surface molecules of the present invention further comprise a linker for separating any domain/region described herein. For example, the linker may be located between the signal peptide and the antibody, between the VH and VL of the antibody, between the antibody and the hinge region, between the hinge region and the transmembrane domain, on the side of the co-stimulatory domain or on the N- or C- region of the co-stimulatory domain, and/or between the transmembrane domain and the primary signaling domain. The linker may be a peptide of about 6 to about 40 amino acids in length, or about 6 to about 25 amino acids in length.
如本文所用，术语“同一性”或“同源性”定义为在比对序列及必要时引入空位以实现最大序列同一性百分比后且不考虑任何保守取代作为序列同一性的一部分下，候选序列中与特定肽或多肽序列中的氨基酸残基相同的氨基酸残基的百分比。可根据所属领域技能内的多种方式，使用公众可获得的计算器软件，诸如BLAST、BLAST-2、ALIGN或MEGALIGNTM(DNASTAR)软件，实现比对，以确定氨基酸序列同一性百分比。所属领域技术人员可确定用于量测比对的适当参数，包括实现所比较的全长序列上的最大比对所需的任何算法。As used herein, the term "identity" or "homology" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity and without considering any conservative substitutions as part of the sequence identity. Alignment to determine the percentage of amino acid sequence identity can be achieved in a variety of ways within the skill of the art using publicly available calculator software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
如本文所用，术语“变体”是指具有与参考氨基酸序列基本上相同的氨基酸序列，或由基本上相同的核苷酸序列编码的多肽。在一些实施方案中，变体是功能性变体。术语“功能性变体”是指具有与参考氨基酸序列基本上相同的氨基酸序列，或由基本上相同的核苷酸序列编码，并且能够具有参考氨基酸序列的一种或多种活性的多肽。As used herein, the term "variant" refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant. The term "functional variant" refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or encoded by a substantially identical nucleotide sequence, and capable of having one or more activities of the reference amino acid sequence.
在一些实施方案中，本发明的工程化滋养细胞还表达至少一种细胞活化分子，所述细胞活化分子选自分泌型或膜结合型细胞因子、共刺激配体、激活性受体激动剂及其他具备细胞活化功能的分子。在一些实施方案中，所述细胞因子选自分泌型或膜结合型的IL-2、IL-5、IL-7、IL-12、IL-15、IL-18、IL-21及其任意组合；所述共刺激配体选自4-1BBL、OX40L、B7-H6、CD58、CD112、CD155、MIC-A/B、ULBPs及其任意组合；所述激活性受体激动剂选自NKG2D激动剂、NKp44激动剂、NKp30激动剂、CD16激动剂及其任意组合；所述其他具备细胞活化功能的分子包括但不限于DNER、Jedi、SOM-11、CCN3、MAGP2、MAGP1、TSP2、YB-1、EGFL7、CCR7、DAP12和DAP10、Notch配体等。优选地，所述细胞活化分子选自IL-21、4-1BBL、IL-15、IL-21和4-1BBL、IL-21和IL-15、以及IL-15和4-1BBL，所述IL-21、IL-15或IL-21以分泌型或膜结合型的形式存在，更优选IL-21和4-1BBL。In some embodiments, the engineered trophoblast cells of the present invention further express at least one cell activation molecule selected from secreted or membrane-bound cytokines, co-stimulatory ligands, activating receptor agonists and other molecules with cell activation function. In some embodiments, the cytokine is selected from secreted or membrane-bound IL-2, IL-5, IL-7, IL-12, IL-15, IL-18, IL-21 and any combination thereof; the co-stimulatory ligand is selected from 4-1BBL, OX40L, B7-H6, CD58, CD112, CD155, MIC-A/B, ULBPs and any combination thereof; the activating receptor agonist is selected from NKG2D agonist, NKp44 agonist, NKp30 agonist, CD16 agonist and any combination thereof; the other molecules with cell activation function include but are not limited to DNER, Jedi, SOM-11, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12 and DAP10, Notch ligand, etc. Preferably, the cell activation molecule is selected from IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, and IL-15 and 4-1BBL, and the IL-21, IL-15 or IL-21 exists in a secretory or membrane-bound form, more preferably IL-21 and 4-1BBL.
培养基Culture medium
在第二个方面，本发明提供一种细胞培养基，其包含如上所述的工程化滋养细胞。In a second aspect, the present invention provides a cell culture medium comprising the engineered trophoblast cells as described above.
在一些实施方案中，所述NK细胞可以是CD56dim NK细胞和/或CD56bright NK细胞。在一些实施方案中，所述NK细胞可以来源于原代NK细胞、干细胞衍生的NK细胞、NK细胞系等。其中，所述原代NK细胞可以来源于受试者(如人、小鼠或者大鼠、猫、狗、奶牛、马、绵羊、山羊或其他牲畜等)，选自外周血NK细胞、脐带血NK细胞、脾脏NK细胞等；所述干细胞衍生的NK细胞可以选自多能干细胞(IPSC)衍生的NK细胞、胚胎干细胞(ESC)衍生的NK细胞、造血干细胞(HSPC)衍生的NK细胞等；所述NK细胞系选自NK92、NK92.26.5、NK92.MI、NK92Ci、NK92Fc、NK3.3、NKL、NKG、NK-YT、NK-YTS、KHYG-1、HATAK细胞等。在一些实施方案中，所述NK细胞为NK92细胞。In some embodiments, the NK cells may be CD56dim NK cells and/or CD56bright NK cells. In some embodiments, the NK cells may be derived from primary NK cells, stem cell-derived NK cells, NK cell lines, etc. Among them, the primary NK cells may be derived from subjects (such as humans, mice or rats, cats, dogs, cows, horses, sheep, goats or other livestock, etc.), selected from peripheral blood NK cells, umbilical cord blood NK cells, spleen NK cells, etc.; the stem cell-derived NK cells may be selected from NK cells derived from pluripotent stem cells (IPSC), NK cells derived from embryonic stem cells (ESC), NK cells derived from hematopoietic stem cells (HSPC), etc.; the NK cell line is selected from NK92, NK92.26.5, NK92.MI, NK92Ci, NK92Fc, NK3.3, NKL, NKG, NK-YT, NK-YTS, KHYG-1, HATAK cells, etc. In some embodiments, the NK cells are NK92 cells.
在一些实施方案中，所述细胞选自天然NK细胞或工程化的NK细胞。In some embodiments, the cell is selected from a natural NK cell or an engineered NK cell.
在一些实施方案中，所述工程化的NK细胞选自表达嵌合抗原受体(CAR)、T细胞受体(TCR)、T细胞受体融合蛋白(TFP)、T细胞抗原耦合器(TAC)、免疫动员单克隆T细胞受体(ImmTAC)的NK细胞。In some embodiments, the engineered NK cells are selected from NK cells expressing a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell receptor fusion protein (TFP), a T cell antigen coupler (TAC), and an immune mobilizing monoclonal T cell receptor (ImmTAC).
术语“嵌合抗原受体”或“CAR”是指人工构建的杂合多肽，该杂合多肽一般包括抗原(例如肿瘤抗原)结合结构域(例如抗体或抗原的配体)、跨膜结构域、任选的共刺激结构域和初级信号传导结构域，各个结构域之间通过接头连接。CAR能够以非MHC限制性的方式将T细胞和其它免疫细胞的特异性和反应性重定向至所选择的靶标。在一些实施方案中，本发明的功能性外源受体是嵌合抗原受体，其包含抗原结合结构域、跨膜结构域、一个或多个共刺激结构域和初级信号传导结构域。任选地，所述嵌合抗原受体还包含以下结构中的一个或多个：信号肽、铰链区、自杀基因、开关结构等。适用于本发明中的“跨膜结构域”、“共刺激结构域”、“初级信号传导结构域”、“铰链区”、“信号肽”等已在上文中定义。The term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide, which generally includes an antigen (e.g., tumor antigen) binding domain (e.g., a ligand of an antibody or antigen), a transmembrane domain, an optional co-stimulatory domain, and a primary signaling domain, and each domain is connected by a linker. CAR can redirect the specificity and reactivity of T cells and other immune cells to the selected target in a non-MHC restricted manner. In some embodiments, the functional exogenous receptor of the present invention is a chimeric antigen receptor, which comprises an antigen binding domain, a transmembrane domain, one or more co-stimulatory domains, and a primary signaling domain. Optionally, the chimeric antigen receptor also includes one or more of the following structures: a signal peptide, a hinge region, a suicide gene, a switch structure, etc. "Transmembrane domain", "co-stimulatory domain", "primary signaling domain", "hinge region", "signal peptide", etc. applicable to the present invention have been defined above.
如本文所用，术语“T细胞受体”或“TCR”是指响应于抗原呈递并参与T细胞活化的膜蛋白复合体。TCR的刺激由抗原呈递细胞上的主要组织相容性复合体分子(MHC)触发，所述抗原呈递细胞将抗原肽呈递至T细胞并且结合至TCR复合体以诱发一系列胞内信号传导。TCR由分别形成异二聚体的六条肽链组成，其一般分为αβ型和γδ型。每条肽链包括恒定区和可变区，其中可变区负责结合特异性的特定的抗原和MHC分子。TCR的可变区可以包含抗原结合结构域或与抗原结合结构域可操作连接。As used herein, the term "T cell receptor" or "TCR" refers to a membrane protein complex that responds to antigen presentation and participates in T cell activation. The stimulation of TCR is triggered by the major histocompatibility complex molecule (MHC) on the antigen presenting cell, which presents the antigen peptide to the T cell and binds to the TCR complex to induce a series of intracellular signal transductions. TCR consists of six peptide chains that form heterodimers, which are generally divided into αβ type and γδ type. Each peptide chain includes a constant region and a variable region, wherein the variable region is responsible for binding to a specific antigen and MHC molecule of specificity. The variable region of TCR may include an antigen binding domain or be operably connected to an antigen binding domain.
术语“T细胞抗原耦合器”或“TAC”包括三个功能结构域：(1)肿瘤靶向结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；(2)细胞外结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；(3)跨膜结构域和CD4共受体的细胞内结构域，其中，细胞内结构域连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAM)磷酸化作为T细胞活化的初始步骤。The term “T cell antigen coupler” or “TAC” includes three functional domains: (1) a tumor targeting domain, which may include a single-chain antibody, a designed ankyrin repeat protein (DARPin), or other targeting moieties; (2) an extracellular domain, which is a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; and (3) a transmembrane domain and an intracellular domain of the CD4 co-receptor, wherein the intracellular domain is linked to the protein kinase LCK, which catalyzes the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of the TCR complex as the initial step in T cell activation.
术语“T细胞受体融合蛋白”或“TFP”是指由TCR各组分衍生的重组多肽，通常由TCR亚基和与其连接的抗原结合区组成并在细胞表面表达。其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内信号结构域。The term "T cell receptor fusion protein" or "TFP" refers to a recombinant polypeptide derived from various components of TCR, which is usually composed of a TCR subunit and an antigen binding region connected thereto and expressed on the cell surface. Among them, the TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular signaling domain.
术语“免疫动员单克隆T细胞受体”或“ImmTAC”是由工程化改造的T细胞受体(TCR)以及抗CD3的scFv组成，其中：改造后的TCR能以显著提高的亲和力特异性识别并结合肿瘤细胞表面的HLA-肽复合物，并通过scFv抗体片段与CD3的相互作用促进T细胞介导的效应器功能。The term "immune mobilizing monoclonal T cell receptor" or "ImmTAC" is composed of an engineered T cell receptor (TCR) and an anti-CD3 scFv, wherein: the engineered TCR can specifically bind to the target cell with significantly improved affinity. It specifically recognizes and binds to HLA-peptide complexes on the surface of tumor cells and promotes T cell-mediated effector functions through the interaction of scFv antibody fragments with CD3.
方法method
在第三个方面，本发明还提供一种扩增NK细胞的方法，其包括：将NK细胞与如上所述的工程化滋养细胞共培养。共培养的时间及条件可以根据需要以及本领域内常见的培养方法进行选择。In a third aspect, the present invention also provides a method for expanding NK cells, comprising: co-culturing NK cells with the engineered trophoblast cells as described above. The time and conditions of co-culture can be selected according to needs and common culture methods in the art.
在一些实施方案中，在所述共培养前还包括将所述工程化滋养细胞进行辐照处理或丝裂霉素处理。In some embodiments, the engineered trophoblast cells are further subjected to irradiation or mitomycin treatment before the co-culturing.
下面结合具体实施例和附图，进一步阐述本发明。应理解，这些实施例和附图仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。The present invention will be further described below in conjunction with specific examples and accompanying drawings. It should be understood that these examples and accompanying drawings are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.
图1：经过工程化K562细胞处理后，NK92细胞的激活与扩增效果。Figure 1: Activation and expansion of NK92 cells after treatment with engineered K562 cells.
图2：经过工程化K562细胞处理后，分选后的PBNK细胞的激活与扩增效果。Figure 2: Activation and expansion of sorted PBNK cells after treatment with engineered K562 cells.
图3：经过工程化K562细胞处理后，未分选的PBNK细胞的激活与扩增效果。Figure 3: Activation and expansion of unsorted PBNK cells after treatment with engineered K562 cells.
图4：工程化K562细胞激活的NK92细胞对K562细胞的杀伤作用。Figure 4: Killing effect of NK92 cells activated by engineered K562 cells on K562 cells.
图5：工程化K562细胞激活的NK92细胞的CD107a阳性率。Figure 5: CD107a positivity rate of NK92 cells activated by engineered K562 cells.
实施例1工程化K562细胞的构建Example 1 Construction of engineered K562 cells
合成编码4-1BBL(氨基酸序列如SEQ ID NO:10所示)的DNA，将其克隆至慢病毒载体pLv-EF1a(即启动子替换为EF1a的pLVX载体(PPL，Cat No.:PPL00157-4a))中，构建得到pLv-EF1a-4-1BBL质粒。DNA encoding 4-1BBL (amino acid sequence as shown in SEQ ID NO: 10) was synthesized and cloned into the lentiviral vector pLv-EF1a (i.e., pLVX vector (PPL, Cat No.: PPL00157-4a) in which the promoter is replaced with EF1a) to construct the pLv-EF1a-4-1BBL plasmid.
合成编码mbIL21(氨基酸序列如SEQ ID NO:11所示)的DNA，将其克隆至慢病毒载体pLv-CMV(即启动子替换为CMV的pLVX载体(PPL，Cat No.:PPL00157-4a))中，构建得到pLv-CMV-mbIL21质粒。DNA encoding mbIL21 (amino acid sequence as shown in SEQ ID NO: 11) was synthesized and cloned into the lentiviral vector pLv-CMV (i.e., pLVX vector (PPL, Cat No.: PPL00157-4a) in which the promoter is replaced with CMV) to construct the pLv-CMV-mbIL21 plasmid.
分别合成编码抗NKp46 scFv(氨基酸序列如SEQ ID NO:9所示)、CD8α铰链区(氨基酸序列如SEQ ID NO:22所示)、CD8α跨膜结构域(氨基酸序列如SEQ ID NO:13所示)、4-1BB共刺激结构域(氨基酸序列如SEQ ID NO:16所示)、CD3ζ初级信号传导结构域(氨基酸序列如SEQ ID NO:19所示)的DNA，将其克隆至慢病毒载体pLv-EF1a中，构建得到pLv-EF1a-NKp46 CAR质粒。The anti-NKp46 scFv (amino acid sequence as shown in SEQ ID NO: 9), CD8α hinge region (amino acid sequence as shown in SEQ ID NO: 22), CD8α transmembrane domain (amino acid sequence as shown in SEQ ID NO: 13) were synthesized. The DNA of 147 amino acid sequences was cloned into the lentiviral vector pLv-EF1a to construct the pLv-EF1a-NKp46 CAR plasmid.
分别合成编码抗NKp46 scFv(氨基酸序列如SEQ ID NO:9所示)、CD8α铰链区(氨基酸序列如SEQ ID NO:22所示)、CD8α跨膜结构域(氨基酸序列如SEQ ID NO:13所示)的DNA，将其克隆至慢病毒载体pLv-EF1a中，构建得到pLv-EF1a-NKp46 CAR-SHT质粒。DNAs encoding anti-NKp46 scFv (amino acid sequence as shown in SEQ ID NO:9), CD8α hinge region (amino acid sequence as shown in SEQ ID NO:22), and CD8α transmembrane domain (amino acid sequence as shown in SEQ ID NO:13) were synthesized respectively and cloned into the lentiviral vector pLv-EF1a to construct the pLv-EF1a-NKp46 CAR-SHT plasmid.
分别合成编码4-1BBL(氨基酸序列如SEQ ID NO:10所示)、T2A(氨基酸序列如SEQ ID NO:30所示)、抗NKp46 scFv(氨基酸序列如SEQ ID NO:9所示)、CD8α铰链区(氨基酸序列如SEQ ID NO:22所示)、CD8α跨膜结构域(氨基酸序列如SEQ ID NO:13所示)、4-1BB共刺激结构域(氨基酸序列如SEQ ID NO:16所示)、CD3ζ初级信号传导结构域(氨基酸序列如SEQ ID NO:19所示)的DNA，将其克隆至慢病毒载体pLv-EF1a中，构建得到pLv-EF1a-4-1BBL-T2A-NKp46 CAR质粒。DNA encoding 4-1BBL (amino acid sequence as shown in SEQ ID NO:10), T2A (amino acid sequence as shown in SEQ ID NO:30), anti-NKp46 scFv (amino acid sequence as shown in SEQ ID NO:9), CD8α hinge region (amino acid sequence as shown in SEQ ID NO:22), CD8α transmembrane domain (amino acid sequence as shown in SEQ ID NO:13), 4-1BB co-stimulatory domain (amino acid sequence as shown in SEQ ID NO:16), and CD3ζ primary signaling domain (amino acid sequence as shown in SEQ ID NO:19) were synthesized respectively and cloned into the lentiviral vector pLv-EF1a to construct the pLv-EF1a-4-1BBL-T2A-NKp46 CAR plasmid.
将上述质粒包装入慢病毒，并进一步转染K562细胞，获得相应的工程化K562细胞，质粒及包含其的工程化K562细胞如表1所示。流式检测阳性细胞比例，确定各个慢病毒有效转染至K562细胞。The above plasmids were packaged into lentiviruses, and further transfected into K562 cells to obtain corresponding engineered K562 cells. The plasmids and engineered K562 cells containing them are shown in Table 1. The proportion of positive cells was detected by flow cytometry to determine that each lentivirus was effectively transfected into K562 cells.
表1构建的各工程化K562细胞及所需要的病毒
Table 1 Constructed engineered K562 cells and required viruses
实施例2 NK92细胞的激活与扩增Example 2 Activation and expansion of NK92 cells
2.1丝裂霉素处理工程化K562细胞2.1 Treatment of engineered K562 cells with mitomycin
细胞激活当天(Day0)，分别转移实施例1中制备的Mock K562(Mock)、NKp46 CAR K562(46)、NKp46 CAR-SHT K562(46SHT)细胞至离心管中，室温下300g离心5分钟，弃上清。加入25μg/ml丝裂霉素(PBS稀释)，37℃处理45分钟。处理后，用PBS重悬细胞，室温下300g离心5分钟，弃上清。按此步骤重复洗细胞4次，得到丝裂霉素处理后的工程化K562细胞。On the day of cell activation (Day 0), transfer the Mock K562 (Mock), NKp46 CAR K562 (46), and NKp46 CAR-SHT K562 (46SHT) cells prepared in Example 1 to centrifuge tubes, centrifuge at 300g for 5 minutes at room temperature, and discard the supernatant. Add 25μg/ml mitomycin (diluted with PBS) and treat at 37°C for 45 minutes. After treatment, resuspend the cells with PBS, centrifuge at 300g for 5 minutes at room temperature, and discard the supernatant. Repeat this step to wash the cells 4 times. The engineered K562 cells were obtained after being treated with mitomycin.
2.2激活NK92细胞2.2 Activation of NK92 cells
对NK92细胞和丝裂霉素处理后的工程化K562细胞进行计数。以NK92细胞：K562细胞＝1：0.65的数量比，取出NK92细胞和K562细胞，分别转移至离心管中，室温下300g离心5分钟，弃上清。向NK92细胞和K562细胞分别加入NK92培养基(XVIVO-15+12％FBS+900IU/ml IL2)重悬，将两者混合一起加入细胞培养板中至NK92细胞密度为2.5×105个/ml。将细胞放置于细胞培养箱，37℃培养。每隔2～3天，用NK92培养基半换液。培养至第4天、第8天、第12天进行细胞计数。结果如图1所示。NK92 cells and engineered K562 cells treated with mitomycin were counted. With a ratio of NK92 cells: K562 cells = 1: 0.65, NK92 cells and K562 cells were taken out and transferred to centrifuge tubes respectively, centrifuged at 300g for 5 minutes at room temperature, and the supernatant was discarded. NK92 cells and K562 cells were resuspended in NK92 culture medium (XVIVO-15 + 12% FBS + 900IU / ml IL2), respectively, and the two were mixed and added to the cell culture plate until the NK92 cell density was 2.5 × 105 / ml. The cells were placed in a cell culture incubator and cultured at 37 ° C. Every 2 to 3 days, half of the medium was replaced with NK92 culture medium. Cell counts were performed on the 4th, 8th, and 12th days of culture. The results are shown in Figure 1.
可以看出，相比于Mock K562，用NKp46 CAR K562和NKp46 CAR-SHT K562细胞激活NK92细胞，均能够显著促进NK92细胞增殖，而且两者没有明显差异，表明NKp46 CAR K562和NKp46 CAR-SHT K562对NK细胞扩增均有显著的促进作用，这种作用不依赖于胞内结构域。It can be seen that compared with Mock K562, activating NK92 cells with NKp46 CAR K562 and NKp46 CAR-SHT K562 cells can significantly promote the proliferation of NK92 cells, and there is no significant difference between the two, indicating that NKp46 CAR K562 and NKp46 CAR-SHT K562 both have a significant promoting effect on NK cell expansion, and this effect is not dependent on the intracellular domain.
实施例3 PBNK细胞(经过分选)的激活与扩增Example 3 Activation and expansion of PBNK cells (after sorting)
细胞激活前一天(Day-1)，复苏PBMC细胞，加入10ml PBMC培养基(XVIVO-15+5％FBS)。室温下300g离心5分钟，弃上清。加入PBMC培养基，使细胞密度调整至1×106个/ml。放置于细胞培养箱，37℃过夜培养。Day0，激活当天，将过夜培养PBMC中的悬浮细胞转移至离心管里，室温下400g离心5分钟，弃上清，加入磁珠分选缓冲液。使用NK细胞分选试剂盒(美天旎，130-092-657)分出PBNK。One day before cell activation (Day-1), PBMC cells were revived and 10 ml PBMC culture medium (XVIVO-15 + 5% FBS) was added. Centrifuge at 300g for 5 minutes at room temperature and discard the supernatant. Add PBMC culture medium to adjust the cell density to 1×106 cells/ml. Place in a cell culture incubator and culture overnight at 37°C. Day 0, the day of activation, transfer the suspended cells in the overnight cultured PBMC to a centrifuge tube, centrifuge at 400g for 5 minutes at room temperature, discard the supernatant, and add magnetic bead sorting buffer. Use the NK cell sorting kit (Miltenyi Biotec, 130-092-657) to separate PBNK.
对PBNK细胞和实施例2.1中经过丝裂霉素处理后的工程化K562细胞进行计数。以PBNK细胞：K562细胞＝1：4的数量比取出相应的细胞，分别转移至离心管中，室温下300g离心5分钟，弃上清。加入PBNK培养基(XVIVO-15+10％FBS+900IU/ml IL2)，调整PBNK密度至2.5×105个/ml，转移到细胞培养板。放置于细胞培养箱，37℃培养。每隔2～3天，用PBNK培养基半换液。培养至第8天进行细胞计数。结果如图2所示。Count the PBNK cells and the engineered K562 cells treated with mitomycin in Example 2.1. Take out the corresponding cells at a ratio of PBNK cells: K562 cells = 1:4, transfer them to centrifuge tubes respectively, centrifuge at 300g for 5 minutes at room temperature, and discard the supernatant. Add PBNK culture medium (XVIVO-15+10% FBS+900IU/ml IL2), adjust the PBNK density to 2.5×105 cells/ml, and transfer to a cell culture plate. Place in a cell culture incubator and culture at 37°C. Replace half of the medium with PBNK culture medium every 2 to 3 days. Count the cells on the 8th day of culture. The results are shown in Figure 2.
可以看出，相比于Mock K562，用NKp46 CAR K562和NKp46 CAR-SHT K562细胞激活分选过的PBNK细胞，其细胞增殖倍数均有明显增加，而且两者之间没有明显差异，表明NKp46 CAR K562和NKp46 CAR-SHT K562对分选过的PBNK细胞扩增有显著的促进作用，这种作用不依赖于胞内结构域。It can be seen that compared with Mock K562, the proliferation times of the sorted PBNK cells activated by NKp46 CAR K562 and NKp46 CAR-SHT K562 cells were significantly increased, and there was no significant difference between the two, indicating that NKp46 CAR K562 and NKp46 CAR-SHT K562 have a significant effect on the proliferation of sorted PBNK cells. The increase was significantly promoted, and this effect was independent of the intracellular domain.
实施例4 PBNK细胞(未经分选)的激活与扩增Example 4 Activation and expansion of PBNK cells (unsorted)
细胞激活前一天(Day-1)，复苏PBMC细胞，加入10ml PBMC培养基(XVIVO-15+5％FBS)。室温下300g离心5分钟，弃上清。加入PBMC培养基，使细胞密度调整至1×106个/ml。放置于细胞培养箱，37℃过夜培养。细胞激活当天(Day0)，将过夜培养PBMC中的悬浮细胞转移至离心管里，室温下400g离心5分钟，弃上清。取一部分进行流式分析，对PBNK细胞进行绝对计数，计数的结果作为PBNK扩增的起始值。One day before cell activation (Day-1), PBMC cells were revived and 10 ml PBMC culture medium (XVIVO-15 + 5% FBS) was added. Centrifuge at 300g for 5 minutes at room temperature and discard the supernatant. Add PBMC culture medium to adjust the cell density to 1×106 cells/ml. Place in a cell culture incubator and culture overnight at 37°C. On the day of cell activation (Day0), the suspended cells in the overnight cultured PBMC were transferred to a centrifuge tube, centrifuged at 400g for 5 minutes at room temperature, and the supernatant was discarded. Take a portion for flow cytometry analysis and perform absolute counting of PBNK cells. The counting result is used as the starting value of PBNK amplification.
对PBMC细胞和按照实施例2.1中的丝裂霉素处理方法处理后的Mock K562(Mock)，4-1BBL+NKp46 CAR K562(bbl46)、NKp46 CAR+mbIL21 K562(4621)、4-1BBL+NKp46 CAR+mbIL21 K562(bbl4621)进行计数。以未分选的PBMC细胞：K562细胞＝1：0.8的数量比取出相应细胞，转移至离心管中，室温下300g离心5分钟，弃上清。加入PBNK培养基(XVIVO-15+10％FBS+900IU/ml IL2)，调整PBMC密度至6×105个/ml，转移到细胞培养板。放置于细胞培养箱，37℃培养。每隔2～3天，用PBNK培养基半换液。培养至第6天进行细胞计数。结果如图3所示。PBMC cells and Mock K562 (Mock) treated with mitomycin in Example 2.1, 4-1BBL + NKp46 CAR K562 (bbl46), NKp46 CAR + mbIL21 K562 (4621), 4-1BBL + NKp46 CAR + mbIL21 K562 (bbl4621) were counted. The corresponding cells were taken out at a ratio of unsorted PBMC cells: K562 cells = 1:0.8, transferred to a centrifuge tube, centrifuged at 300g for 5 minutes at room temperature, and the supernatant was discarded. PBNK culture medium (XVIVO-15 + 10% FBS + 900IU / ml IL2) was added, the PBMC density was adjusted to 6 × 105 / ml, and transferred to a cell culture plate. Place in a cell culture incubator and culture at 37 ° C. Every 2 to 3 days, half of the medium was replaced with PBNK culture medium. Cell counts were performed on the 6th day of culture. The results are shown in Figure 3.
可以看出，相比于Mock K562，4-1BBL+NKp46 CAR K562、NKp46 CAR+mbIL21 K562、4-1BBL+NKp46 CAR+mbIL21 K562细胞对PBNK细胞的激活效果均显著增强。值得注意的是，相比于表达anti-NKp46 CAR和mbIL21、以及anti-NKp46 CAR和4-1BBL的K562细胞，同时表达anti-NKp46 CAR、mbIL21和4-1BBL的K562细胞进一步提高了PBNK细胞的扩增倍数。It can be seen that compared with Mock K562, 4-1BBL+NKp46 CAR K562, NKp46 CAR+mbIL21 K562, and 4-1BBL+NKp46 CAR+mbIL21 K562 cells significantly enhanced the activation effect of PBNK cells. It is worth noting that compared with K562 cells expressing anti-NKp46 CAR and mbIL21, and anti-NKp46 CAR and 4-1BBL, K562 cells expressing anti-NKp46 CAR, mbIL21, and 4-1BBL further increased the expansion multiple of PBNK cells.
实施例5不同工程化K562细胞激活的NK92细胞对K562细胞的杀伤Example 5 Killing of K562 cells by NK92 cells activated by different engineered K562 cells
5.1利用工程化K562细胞激活NK92细胞5.1 Activation of NK92 cells using engineered K562 cells
在实验前4天(Day-4)，按照实施例2.1中的丝裂霉素处理方法处理工程化K562细胞。将细胞分为两组：Four days before the experiment (Day-4), the engineered K562 cells were treated with mitomycin according to the method of Example 2.1. The cells were divided into two groups:
第一组：未转染K562细胞(NT)、NKp46 CAR K562(46)、NKp46 CAR-SHT K562(46SHT)；Group 1: non-transfected K562 cells (NT), NKp46 CAR K562 (46), NKp46 CAR-SHT K562 (46SHT);
第二组：Mock K562(Mock)、NKp46 CAR K562(46)、NKp46 CAR+mbIL21 K562(4621)、4-1BBL+NKp46 CAR K562(bbl46)、4-1BBL+NKp46 CAR+mbIL21 K562(bbl4621)。Group 2: Mock K562 (Mock), NKp46 CAR K562 (46), NKp46 CAR + mbIL21 K562 (4621), 4-1BBL+NKp46 CAR K562(bbl46), 4-1BBL+NKp46 CAR+mbIL21 K562(bbl4621).
按照实施例2.2中的激活方式，用这些丝裂霉素处理过的工程化K562分别激活NK92。加入NK92培养基(XVIVO-15+12％FBS+900IU/ml IL2)，将细胞放置于细胞培养箱，37℃培养。每隔2～3天，用NK92培养基半换液。According to the activation method in Example 2.2, the engineered K562 treated with these mitomycins were used to activate NK92. NK92 culture medium (XVIVO-15 + 12% FBS + 900 IU/ml IL2) was added, and the cells were placed in a cell culture incubator and cultured at 37°C. Half of the medium was replaced with NK92 culture medium every 2 to 3 days.
5.2利用激活后的NK92细胞杀伤K562细胞5.2 Using activated NK92 cells to kill K562 cells
实验当天(Day0)，配置R10培养基50ml(45ml RPMI 1640+5ml FBS)。对各NK92细胞以及稳定表达荧光素酶的K562细胞进行计数。On the day of the experiment (Day 0), prepare 50 ml of R10 culture medium (45 ml RPMI 1640 + 5 ml FBS). Count the NK92 cells and K562 cells stably expressing luciferase.
取稳定表达荧光素酶的K562细胞转移至离心管中，室温下300g离心5分钟，弃上清。加入R10培养基调整细胞密度至1×105个/ml。取各组NK92细胞分别转移至离心管中，室温下300g离心5分钟，弃上清。加入R10培养基调整细胞密度至5×105个/ml。将NK92细胞与稳定表达荧光素酶的K562细胞分别按100μl/孔吸取细胞悬液，转移至同一块96孔板对应的孔中。37℃孵育4小时。4小时后，每孔取100μl转移到全白96孔板中。加入荧光素酶底物。使用酶标仪读取荧光值。结果如图4所示。Transfer the K562 cells stably expressing luciferase to a centrifuge tube, centrifuge at 300g for 5 minutes at room temperature, and discard the supernatant. Add R10 medium to adjust the cell density to 1×105 cells/ml. Transfer the NK92 cells of each group to a centrifuge tube, centrifuge at 300g for 5 minutes at room temperature, and discard the supernatant. Add R10 medium to adjust the cell density to 5×105 cells/ml. Pipette the cell suspension of NK92 cells and K562 cells stably expressing luciferase at 100μl/well, respectively, and transfer them to the corresponding wells of the same 96-well plate. Incubate at 37°C for 4 hours. After 4 hours, transfer 100μl from each well to a full-white 96-well plate. Add luciferase substrate. Use an enzyme reader to read the fluorescence value. The results are shown in Figure 4.
可以看出，相比于NT细胞，NKp46 CAR K562和NKp46 CAR-SHT K562细胞激活的NK92细胞对K562细胞的杀伤作用均显著增强，而且它们之间没有明显差异，表明利用NKp46 CAR K562和NKp46 CAR-SHT K562细胞激活NK细胞均可以增强NK细胞对肿瘤细胞的杀伤作用，此作用不依赖于胞内结构域。另外，相比于Mock K562细胞，各工程化K562细胞激活的NK92细胞的杀伤作用均显著增强；相比于仅表达anti-NKp46 CAR的K562细胞，额外表达mbIL21、或4-1BBL和mbIL21能够进一步增强NK92细胞对K562的杀伤作用，表明在anti-NKp46 CAR的基础上额外表达一些外源细胞活化分子，如mbIL21、或4-1BBL和mbIL21，可进一步增强对NK细胞的激活作用，增强NK细胞的肿瘤细胞杀伤活性。It can be seen that compared with NT cells, the killing effect of NK92 cells activated by NKp46 CAR K562 and NKp46 CAR-SHT K562 cells on K562 cells was significantly enhanced, and there was no obvious difference between them, indicating that activating NK cells using NKp46 CAR K562 and NKp46 CAR-SHT K562 cells can enhance the killing effect of NK cells on tumor cells, and this effect is not dependent on the intracellular domain. In addition, compared with Mock K562 cells, the killing effect of NK92 cells activated by each engineered K562 cell was significantly enhanced; compared with K562 cells expressing only anti-NKp46 CAR, the additional expression of mbIL21, or 4-1BBL and mbIL21 can further enhance the killing effect of NK92 cells on K562, indicating that the additional expression of some exogenous cell activation molecules on the basis of anti-NKp46 CAR, such as mbIL21, or 4-1BBL and mbIL21, can further enhance the activation effect of NK cells and enhance the tumor cell killing activity of NK cells.
实施例6不同工程化K562细胞激活的NK92细胞与K562细胞共孵育后的CD107a阳性率检测Example 6 Detection of CD107a positive rate after co-incubation of NK92 cells activated by different engineered K562 cells with K562 cells
6.1利用工程化K562细胞激活NK92细胞6.1 Activation of NK92 cells using engineered K562 cells
在实验前4天(Day-4)，按照实施例2.1中的丝裂霉素处理方法处理工程化K562细胞。将细胞分为两组：Four days before the experiment (Day-4), the engineered K562 cells were treated with mitomycin according to the method of Example 2.1. The cells were divided into two groups:
第一组：未转染K562细胞(NT)、NKp46 CAR K562(46)、NKp46 CAR-SHT K562(46SHT)；Group 1: non-transfected K562 cells (NT), NKp46 CAR K562 (46), NKp46 CAR-SHT K562 (46SHT);
第二组：Mock K562(Mock)、NKp46 CAR K562(46)、NKp46 CAR+mbIL21 K562(4621)、4-1BBL+mbIL21 K562(bbl21)、4-1BBL+NKp46 CAR K562(bbl46)、4-1BBL+NKp46 CAR+mbIL21 K562(bbl4621)。Group 2: Mock K562 (Mock), NKp46 CAR K562 (46), NKp46 CAR + mbIL21 K562 (4621), 4-1BBL + mbIL21 K562 (bbl21), 4-1BBL + NKp46 CAR K562 (bbl46), 4-1BBL + NKp46 CAR + mbIL21 K562 (bbl4621).
按照实施例2.2中的激活方式，用这些丝裂霉素处理过的工程化K562分别激活NK92。加入NK92培养基(XVIVO-15+12％FBS+900IU/ml IL2)，将细胞放置于细胞培养箱，37℃培养。每隔2～3天，用NK92培养基半换液。According to the activation method in Example 2.2, the engineered K562 treated with these mitomycins were used to activate NK92. NK92 culture medium (XVIVO-15 + 12% FBS + 900 IU/ml IL2) was added, and the cells were placed in a cell culture incubator and cultured at 37°C. Half of the medium was replaced with NK92 culture medium every 2 to 3 days.
6.2检测激活后的NK92细胞与K562细胞共孵育后的CD107a阳性率6.2 Detection of CD107a positive rate after co-incubation of activated NK92 cells and K562 cells
提前一天(Day-1)将所有NK92细胞的培养基换成低浓度IL-2培养基(90IU/ml IL2)。实验当天(Day0)，配置X10培养基50ml(45ml X-VIVO 15+5ml FBS)；配置Golgi Stop稀释液：2μl Golgi Stop加入3ml X10培养液中。对各NK92细胞以及野生型K562细胞进行计数。One day in advance (Day-1), the culture medium of all NK92 cells was replaced with low-concentration IL-2 culture medium (90 IU/ml IL2). On the day of the experiment (Day0), 50 ml of X10 culture medium (45 ml X-VIVO 15 + 5 ml FBS) was prepared; Golgi Stop diluent was prepared: 2 μl Golgi Stop was added to 3 ml X10 culture medium. Count the NK92 cells and wild-type K562 cells.
向96孔板中每孔加入1×105个NK92细胞和1×105个野生型K562细胞，以及CD107a荧光抗体，每孔细胞悬液总体积为170μl，37℃孵育1小时。每孔加入20μl Golgi Stop稀释液，37℃孵育2.5小时。最后加入5μl CD56荧光抗体，37℃孵育30分钟。用FACS缓冲液洗涤样品。用流式细胞仪对样品的CD56+细胞群进行分析，计算CD107+细胞占比。结果如图5所示。1×105 NK92 cells and 1×105 wild-type K562 cells were added to each well of a 96-well plate, as well as CD107a fluorescent antibodies. The total volume of the cell suspension in each well was 170 μl, and the cells were incubated at 37°C for 1 hour. 20 μl of Golgi Stop diluent was added to each well and incubated at 37°C for 2.5 hours. Finally, 5 μl of CD56 fluorescent antibodies were added and incubated at 37°C for 30 minutes. The samples were washed with FACS buffer. The CD56+ cell population of the samples was analyzed by flow cytometry, and the proportion of CD107+ cells was calculated. The results are shown in Figure 5.
可以看出，与Mock K562和4-1BBL+mbIL21 K562相比，本发明中制备的各工程化K562细胞激活的NK细胞在与野生型K562共孵育后，CD107a阳性率均显著提高，且各工程化K562细胞激活的NK细胞之间相差不大，表明用本发明制备的表达包含靶向NKp46的抗体和跨膜结构域的细胞表面分子的各工程化K562细胞激活的NK细胞，细胞毒性均显著增强。It can be seen that compared with Mock K562 and 4-1BBL+mbIL21 K562, the CD107a positivity rate of NK cells activated by each engineered K562 cell prepared in the present invention was significantly increased after co-incubation with wild-type K562, and there was little difference between the NK cells activated by each engineered K562 cell, indicating that the cytotoxicity of NK cells activated by each engineered K562 cell expressing a cell surface molecule comprising an antibody targeting NKp46 and a transmembrane domain prepared in the present invention was significantly enhanced.
以上结果表明，利用表达包含靶向NKp46的抗体、跨膜结构域以及任选的胞内结构域的细胞表面分子，以及任选的外源细胞活化分子(如4-1BBL和/或mbIL21等)的工程化滋养细胞激活NK细胞，能够显著增强NK细胞扩增能力和肿瘤细胞杀伤活性。The above results indicate that activating NK cells using engineered trophoblasts expressing antibodies targeting NKp46, cell surface molecules comprising a transmembrane domain and an optional intracellular domain, as well as optional exogenous cell activation molecules (such as 4-1BBL and/or mbIL21, etc.) can significantly enhance the NK cell expansion capacity and tumor cell killing activity.
需要说明的是，以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。本领域技术人员理解的是，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。It should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. It is clear to those skilled in the art that the present invention may be modified and varied in various ways. It is understood by those skilled in the art that any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2023/100058WO2024254772A1 (en) | 2023-06-14 | 2023-06-14 | Engineered trophoblast and use thereof |
| Application Number | Priority Date | Filing Date | Title |
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| PCT/CN2023/100058WO2024254772A1 (en) | 2023-06-14 | 2023-06-14 | Engineered trophoblast and use thereof |
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| WO2024254772A1true WO2024254772A1 (en) | 2024-12-19 |
| Application Number | Title | Priority Date | Filing Date |
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| PCT/CN2023/100058PendingWO2024254772A1 (en) | 2023-06-14 | 2023-06-14 | Engineered trophoblast and use thereof |
| Country | Link |
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| WO (1) | WO2024254772A1 (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011110657A1 (en)* | 2010-03-11 | 2011-09-15 | Universite Paris Descartes | Specific detection of human chorionic gonadotropin beta subunit type ii produced by trophoblastic and neoplastic cells |
| CA3007898A1 (en)* | 2015-12-28 | 2017-07-06 | Innate Pharma | Variable regions for nkp46 binding proteins |
| CN109666640A (en)* | 2019-01-14 | 2019-04-23 | 武汉睿健医药科技有限公司 | The method of the external pure culture of natural killer cells |
| CN110300765A (en)* | 2017-01-24 | 2019-10-01 | 依奈特制药公司 | Nkp46 binders |
| CN111206052A (en)* | 2020-02-21 | 2020-05-29 | 秦皇岛中邦干细胞医学科技有限公司 | Trophoblast, preparation method thereof and application thereof in large-scale rapid amplification of gamma delta T cells |
| CN112029001A (en)* | 2020-09-02 | 2020-12-04 | 南京北恒生物科技有限公司 | Chimeric antigen receptors targeting NK activating receptors |
| CN115595310A (en)* | 2021-07-08 | 2023-01-13 | 中国医学科学院血液病医院（中国医学科学院血液学研究所）（Cn） | NK cell trophoblast, preparation method and application thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011110657A1 (en)* | 2010-03-11 | 2011-09-15 | Universite Paris Descartes | Specific detection of human chorionic gonadotropin beta subunit type ii produced by trophoblastic and neoplastic cells |
| CA3007898A1 (en)* | 2015-12-28 | 2017-07-06 | Innate Pharma | Variable regions for nkp46 binding proteins |
| CN108779175A (en)* | 2015-12-28 | 2018-11-09 | 依奈特制药公司 | Variable regions of NKp46-binding proteins |
| CN110300765A (en)* | 2017-01-24 | 2019-10-01 | 依奈特制药公司 | Nkp46 binders |
| CN109666640A (en)* | 2019-01-14 | 2019-04-23 | 武汉睿健医药科技有限公司 | The method of the external pure culture of natural killer cells |
| CN111206052A (en)* | 2020-02-21 | 2020-05-29 | 秦皇岛中邦干细胞医学科技有限公司 | Trophoblast, preparation method thereof and application thereof in large-scale rapid amplification of gamma delta T cells |
| CN112029001A (en)* | 2020-09-02 | 2020-12-04 | 南京北恒生物科技有限公司 | Chimeric antigen receptors targeting NK activating receptors |
| CN115595310A (en)* | 2021-07-08 | 2023-01-13 | 中国医学科学院血液病医院（中国医学科学院血液学研究所）（Cn） | NK cell trophoblast, preparation method and application thereof |
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