Engineered trophoblast and use thereof

Abstract

An engineered trophoblast, which expresses a cell surface molecule specifically recognizing NKp46, wherein the cell surface molecule at least contains an antibody targeting NKp46 and a transmembrane domain. A cell culture medium and a method for expanding NK cells. Compared with non-engineered trophoblasts, the engineered trophoblast is used for activating NK cells, and can enhance the NK cell expansion capability and the tumor cell killing activity.

Description

Engineered trophoblast cells and their uses Technical Field

[0001] The present invention belongs to the field of cell therapy. More specifically, the present invention relates to engineered trophoblast cells and their uses. Background Art

[0002] Natural killer (NK) cells are important lymphocytes in the body, possessing cytotoxic and immunomodulatory functions. They participate in both innate and adaptive immunity and are considered one of the most promising tumor-killing effector cells. NK cells express multiple activating receptors, among which NKp46 is considered a unifying signature molecule for NK cells across species.

[0003] NK cells offer the following advantages in cell therapy: 1. NK cells can spontaneously kill tumor cells, eliminating the need for prior sensitization or antigen presentation, resulting in a more rapid onset of action; 2. NK cells can kill tumor cells through multiple pathways; 3. NK cell therapy produces no serious adverse reactions, with only mild graft-versus-host disease (GvHD); and 4. NK cells can be used directly in allogeneic universal immune cell therapies without modification. However, because NK cells only account for 1%-32.6% of normal human peripheral blood lymphocytes, they are difficult to expand in vitro and have a limited lifespan in vivo. Consequently, NK cell immunotherapy has been hampered by the inability to obtain sufficient numbers of cells.

[0004] Studies have reported that some cytokines, such as IL-2 and IL-15, can be used to expand NK cells, but their effects are limited. Therefore, there is still a need to develop engineered trophoblasts to study the function of NK cells and lay the foundation for cell therapy.

[0005] Summary of the Invention

[0006] 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.

[0007] The present invention aims to provide engineered trophoblasts that express a cell surface molecule that specifically recognizes NKp46. Compared to unengineered trophoblasts, these engineered trophoblasts can be used to activate NK cells and enhance their proliferation and tumor cell-killing activity.

[0008] Engineered trophoblasts

[0009] 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.

[0010] As used herein, the term "trophoblast" refers to cells that are added to a target cell (e.g., NK cell) culture to support its 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 typically 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.

[0011] 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 an antibody capable of specifically binding to NKp46 and a transmembrane domain that anchors the surface molecule to the cell surface. Optionally, the surface molecule further includes a hinge region between the antibody and the transmembrane domain, an intracellular domain responsible for signaling, an exogenous cell activation molecule, and the like.

[0012] 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, and recombinant antibodies, and can be selected from human antibodies, humanized antibodies, murine antibodies, chimeric antibodies, and functional fragments thereof. For example, antibodies include but are not limited to whole antibodies, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies, sdAb, nanobodies, diabodies, anticalins, DARPINs, and the like. In the present invention, antibodies can be monovalent or divalent, and can be monospecific, bispecific, or multispecific antibodies.

[0013] Any antibody targeting NKp46 known in the art can be used in the present invention. In some embodiments, the antibody targeting NKp46 is selected from a whole 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.

[0014] 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%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 10.

[0015] The term "heavy chain" refers to the larger of the two types of polypeptide chains found 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 found in naturally occurring conformations in antibody molecules. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0016] The term "complementarity determining region" or "CDR" refers to the amino acid sequence in the antibody variable region 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 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. It should be understood that "CDR" or "FR" of a given antibody or its region (e.g., its variable region) encompasses CDRs or FRs defined by any of the above-mentioned schemes or other known schemes. 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 the sequence of the corresponding CDR or FR defined by any of the above-mentioned schemes or other known schemes. The numbering scheme used herein to define the boundaries of CDRs and FRs is the Chothia scheme.

[0017] 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 indicated, 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 comprise VL-linker-VH or VH-linker-VL from the N-terminus to the C-terminus. The term "linker" refers to a molecular sequence that connects 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 elicit an immune response. In some embodiments, the linker may be an endogenous amino acid sequence, an exogenous amino acid sequence (e.g., a GS-rich sequence), or a non-peptide chemical linker. In one embodiment, the linker of the present invention 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: 28 or 29.

[0018] 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 can be natural or synthetic, and can also be derived from any membrane-bound protein or transmembrane protein. When the antibody binds to the target antigen, the transmembrane domain can carry out signal transduction. Particularly suitable transmembrane domains for use in the present invention can be derived from, for example, TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. Preferably, the transmembrane domain is selected from the transmembrane domain of CD8α, CD4, CD28 or CD278. More preferably, the transmembrane domain is a CD8α or CD28 transmembrane domain, which 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 NOs: 12-14.

[0019] In some embodiments, the engineered trophoblasts of the present invention may 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 may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region may 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 may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may 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.

[0020] In some embodiments, the cell surface molecule of the present invention further comprises an intracellular domain. Preferably, the intracellular domain comprises a costimulatory domain and / or a primary signaling domain.

[0021] The primary signaling domain of the present invention can be a cytoplasmic sequence of a T cell receptor and a co-receptor that acts together to initiate signaling after antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences with the same or similar functions. The primary signaling domain comprises two different types of cytoplasmic signaling 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 signaling sequence can 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 a signaling domain of CD3ζ. More preferably, the primary signaling domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 18-20.

[0022] 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. A "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 costimulatory 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 NOs: 15-17.

[0023] In some embodiments, the cell surface molecule of the present invention further comprises a signal peptide selected from the group consisting of 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%, and more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 25-27.

[0024] In some embodiments, the cell surface molecules of the present invention further comprise a linker for separating any domain / region described herein. For example, a linker may be located between a signal peptide and an antibody, between the VH and VL of an antibody, between an antibody and a hinge region, between a hinge region and a transmembrane domain, on the side of a costimulatory domain or on the N- or C- region of a costimulatory domain, and / or between a transmembrane domain and a primary signaling domain. The linker may be a peptide having a length of about 6 to about 40 amino acids, or a length of about 6 to about 25 amino acids.

[0025] 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, if necessary, introducing gaps to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment 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, to determine the percentage of amino acid sequence identity. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full-length sequences being compared.

[0026] 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.

[0027] 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 functions. 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.

[0028] culture medium

[0029] In a second aspect, the present invention provides a cell culture medium comprising the engineered feeder cells as described above.

[0030] 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. Wherein, the primary NK cells may be derived from a subject (such as a human, mouse or rat, cat, dog, cow, horse, sheep, goat 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.

[0031] In some embodiments, the cell is selected from a natural NK cell or an engineered NK cell.

[0032] 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), or an immune mobilizing monoclonal T cell receptor (ImmTAC).

[0033] The term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes an antigen (e.g., tumor antigen) binding domain (e.g., an antibody or antigen ligand), a transmembrane domain, an optional costimulatory domain, and a primary signaling domain, with each domain connected by a linker. CAR is capable of redirecting 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 costimulatory domains, and a primary signaling domain. Optionally, the chimeric antigen receptor further comprises one or more of the following structures: a signal peptide, a hinge region, a suicide gene, a switch structure, etc. "Transmembrane domain," "costimulatory domain," "primary signaling domain," "hinge region," "signal peptide," etc. applicable to the present invention have been defined above.

[0034] 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 molecules (MHC) on antigen-presenting cells, which present antigenic peptides to T cells and bind to the TCR complex to induce a series of intracellular signaling. 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 specific specific antigens and MHC molecules. The variable region of TCR can include an antigen binding domain or be operably connected to an antigen binding domain.

[0035] The term "T cell antigen coupler" or "TAC" includes three functional domains: (1) a tumor targeting domain, which can be 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, which is linked to the protein kinase LCK and catalyzes the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of the TCR complex as the initial step in T cell activation.

[0036] The term "T cell receptor fusion protein" or "TFP" refers to a recombinant polypeptide derived from various components of a TCR, typically consisting of a TCR subunit and an antigen-binding region attached thereto, and expressed on the cell surface. The TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular signaling domain.

[0037] 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 recognize and bind to the HLA-peptide complex on the surface of tumor cells with significantly improved affinity, and promote T cell-mediated effector function through the interaction of the scFv antibody fragment with CD3.

[0038] method

[0039] In a third aspect, the present invention further provides a method for expanding NK cells, comprising: co-culturing NK cells with the engineered trophoblast cells described above. The co-culturing time and conditions can be selected according to needs and common culture methods in the art.

[0040] In some embodiments, the engineered feeder cells are further subjected to irradiation or mitomycin treatment before the co-culturing.

[0041] The present invention will be further described below with reference to specific examples and accompanying drawings. It should be understood that these examples and accompanying drawings are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, where specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1: Activation and expansion of NK92 cells after treatment with engineered K562 cells.

[0043] Figure 2: Activation and expansion of sorted PBNK cells after treatment with engineered K562 cells.

[0044] Figure 3: Activation and expansion of unsorted PBNK cells after treatment with engineered K562 cells.

[0045] Figure 4: Killing effect of NK92 cells activated by engineered K562 cells on K562 cells.

[0046] Figure 5: CD107a positivity rate of NK92 cells activated by engineered K562 cells. DETAILED DESCRIPTION

[0047] Example 1 Construction of engineered K562 cells

[0048] DNA encoding 4-1BBL (amino acid sequence 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.

[0049] DNA encoding mbIL21 (amino acid sequence 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.

[0050] DNA encoding 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), 4-1BB costimulatory 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 and cloned into the lentiviral vector pLv-EF1a to construct the pLv-EF1a-NKp46 CAR plasmid.

[0051] DNA encoding the anti-NKp46 scFv (amino acid sequence shown in SEQ ID NO: 9), CD8α hinge region (amino acid sequence shown in SEQ ID NO: 22), and CD8α transmembrane domain (amino acid sequence shown in SEQ ID NO: 13) were synthesized and cloned into the lentiviral vector pLv-EF1a to construct the pLv-EF1a-NKp46 CAR-SHT plasmid.

[0052] 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 costimulatory 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 and cloned into the lentiviral vector pLv-EF1a to construct the pLv-EF1a-4-1BBL-T2A-NKp46 CAR plasmid.

[0053] The above plasmids were packaged into lentivirus and further transfected into K562 cells to obtain the 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 confirm that each lentivirus was effectively transfected into K562 cells.

[0054] Table 1 Constructed engineered K562 cells and required viruses

[0055] Example 2 Activation and Expansion of NK92 Cells

[0056] 2.1 Treatment of engineered K562 cells with mitomycin

[0057] On the day of cell activation (Day 0), the Mock K562 (Mock), NKp46 CAR K562 (46), and NKp46 CAR-SHT K562 (46SHT) cells prepared in Example 1 were transferred to centrifuge tubes, centrifuged at 300g for 5 minutes at room temperature, and the supernatant was discarded. 25 μg / ml mitomycin (diluted with PBS) was added and treated at 37°C for 45 minutes. After treatment, the cells were resuspended in PBS and centrifuged at 300g for 5 minutes at room temperature, and the supernatant was discarded. This step was repeated 4 times to obtain engineered K562 cells treated with mitomycin.

[0058] 2.2 Activation of NK92 cells

[0059] Count the NK92 cells and engineered K562 cells after treatment with mitomycin. With a ratio of NK92 cells: K562 cells = 1:0.65, remove the NK92 cells and K562 cells, transfer them to centrifuge tubes, centrifuge at 300g for 5 minutes at room temperature, and discard the supernatant. Add NK92 medium (XVIVO-15 + 12% FBS + 900IU / ml IL2) to the NK92 cells and K562 cells, resuspend them, mix the two together and add them to the cell culture plate until the NK92 cell density reaches 2.5×10 5 Cells were cultured in a cell culture incubator at 37°C. Every 2-3 days, half of the medium was replaced with NK92 medium. Cell counts were performed on days 4, 8, and 12 of culture. The results are shown in Figure 1.

[0060] 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 independent of the intracellular domain.

[0061] Example 3 Activation and expansion of PBNK cells (after sorting)

[0062] One day before cell activation (Day-1), thaw PBMC cells and add 10 ml of PBMC culture medium (XVIVO-15 + 5% FBS). Centrifuge at 300 g for 5 minutes at room temperature and discard the supernatant. Add PBMC culture medium to adjust the cell density to 1×10 6 Cells were cultured at 37°C overnight in a cell culture incubator. On Day 0, the day of activation, the suspended cells from the overnight cultured PBMCs were transferred to a centrifuge tube and centrifuged at 400 g for 5 minutes at room temperature. The supernatant was discarded and magnetic bead separation buffer was added. PBNK cells were isolated using the NK cell isolation kit (Miltenyi Biotec, 130-092-657).

[0063] Count the PBNK cells and the engineered K562 cells treated with mitomycin in Example 2.1. Remove the corresponding cells at a ratio of 1:4 between PBNK cells and K562 cells, transfer them to centrifuge tubes, centrifuge at 300g for 5 minutes at room temperature, and discard the supernatant. Add PBNK culture medium (XVIVO-15 + 10% FBS + 900 IU / ml IL2) and adjust the PBNK density to 2.5×10 5Cells were transferred to a cell culture plate at 400 nmol / ml. Cultured in a cell incubator at 37°C. Every 2-3 days, the medium was half-changed with PBNK medium. Cell counts were performed on day 8 of culture. The results are shown in Figure 2.

[0064] It can be seen that compared with Mock K562, the cell proliferation times of 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 promoting effect on the expansion of sorted PBNK cells, and this effect is not dependent on the intracellular domain.

[0065] Example 4 Activation and Expansion of PBNK Cells (Unsorted)

[0066] One day before cell activation (Day-1), thaw PBMC cells and add 10 ml of PBMC culture medium (XVIVO-15 + 5% FBS). Centrifuge at 300 g for 5 minutes at room temperature and discard the supernatant. Add PBMC culture medium to adjust the cell density to 1×10 6 Cells / ml. Place in a cell culture incubator and culture overnight at 37°C. On the day of cell activation (Day 0), transfer the suspended cells from the overnight cultured PBMCs to a centrifuge tube and centrifuge at 400g for 5 minutes at room temperature. Discard the supernatant. A portion of the suspension was analyzed by flow cytometry to determine the absolute count of PBNK cells. This count was used as the starting value for PBNK expansion.

[0067] PBMC cells and Mock K562 (Mock), 4-1BBL+NKp46 CAR K562 (bbl46), NKp46 CAR+mbIL21 K562 (4621), and 4-1BBL+NKp46 CAR+mbIL21 K562 (bbl4621) treated with mitomycin in Example 2.1 were counted. The corresponding cells were removed 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 medium (XVIVO-15 + 10% FBS + 900IU / ml IL2) was added and the PBMC density was adjusted to 6×10 5 Cells were transferred to a cell culture plate at 400 nmol / ml. Cultured in a cell incubator at 37°C. Every 2-3 days, the medium was half-changed with PBNK medium. Cell counts were performed on the 6th day of culture. The results are shown in Figure 3.

[0068] 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 all significantly enhanced the activation effect of PBNK cells. Notably, 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 fold of PBNK cells.

[0069] Example 5 Killing of K562 cells by NK92 cells activated by different engineered K562 cells

[0070] 5.1 Activation of NK92 cells using engineered K562 cells

[0071] Four days before the experiment (Day-4), engineered K562 cells were treated with mitomycin according to the method described in Example 2.1. The cells were divided into two groups:

[0072] Group 1: non-transfected K562 cells (NT), NKp46 CAR K562 (46), and NKp46 CAR-SHT K562 (46SHT);

[0073] The second group: 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).

[0074] Following the activation protocol described in Example 2.2, these engineered K562 cells treated with mitomycin were used to activate NK92 cells. NK92 culture medium (XVIVO-15 + 12% FBS + 900 IU / ml IL-2) was added and the cells were cultured in a cell culture incubator at 37°C. Half of the culture medium was replaced with NK92 culture medium every 2-3 days.

[0075] 5.2 Using activated NK92 cells to kill K562 cells

[0076] On the day of the experiment (Day 0), 50 ml of R10 medium (45 ml RPMI 1640 + 5 ml FBS) was prepared and the number of NK92 cells and K562 cells stably expressing luciferase was counted.

[0077] 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 1×10 5 NK92 cells from each group were transferred to centrifuge tubes, centrifuged at 300g for 5 minutes at room temperature, and the supernatant was discarded. R10 medium was added to adjust the cell density to 5×10 5 Cells were collected at 100 μl / well of each NK92 cell suspension and K562 cells stably expressing luciferase. The suspensions were transferred to corresponding wells of a 96-well plate. Incubated at 37°C for 4 hours. After 4 hours, 100 μl was transferred from each well to a white 96-well plate. Luciferase substrate was added. Fluorescence was read using a microplate reader. The results are shown in Figure 4.

[0078] 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 significant difference between them, indicating that activating NK cells with 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, additional expression of mbIL21, or 4-1BBL and mbIL21 can further enhance the killing effect of NK92 cells on K562, indicating that additional expression of some exogenous cell activation molecules, such as mbIL21, or 4-1BBL and mbIL21, on the basis of anti-NKp46 CAR can further enhance the activation effect of NK cells and enhance the tumor cell killing activity of NK cells.

[0079] Example 6 Detection of CD107a Positive Rate After Co-incubation of NK92 Cells Activated by Differently Engineered K562 Cells with K562 Cells

[0080] 6.1 Activation of NK92 cells using engineered K562 cells

[0081] Four days before the experiment (Day-4), engineered K562 cells were treated with mitomycin according to the method described in Example 2.1. The cells were divided into two groups:

[0082] Group 1: non-transfected K562 cells (NT), NKp46 CAR K562 (46), and NKp46 CAR-SHT K562 (46SHT);

[0083] The second group: 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).

[0084] Following the activation protocol described in Example 2.2, these engineered K562 cells treated with mitomycin were used to activate NK92 cells. NK92 culture medium (XVIVO-15 + 12% FBS + 900 IU / ml IL-2) was added and the cells were cultured in a cell culture incubator at 37°C. Half of the culture medium was replaced with NK92 culture medium every 2-3 days.

[0085] 6.2 Detection of CD107a positivity after co-incubation of activated NK92 cells with K562 cells

[0086] One day in advance (Day-1), replace the culture medium of all NK92 cells with low-concentration IL-2 medium (90 IU / ml IL-2). On the day of the experiment (Day 0), prepare 50 ml of X10 medium (45 ml X-VIVO 15 + 5 ml FBS). Prepare Golgi Stop diluent: add 2 μl of Golgi Stop to 3 ml of X10 medium. Count the number of NK92 cells and wild-type K562 cells.

[0087] Add 1×10 5 NK92 cells and 1×10 5 Wild-type K562 cells were plated with a CD107a fluorescent antibody in a total volume of 170 μl per well and incubated at 37°C for 1 hour. 20 μl of Golgi Stop solution was added to each well and incubated at 37°C for 2.5 hours. Finally, 5 μl of CD56 fluorescent antibody was added and incubated at 37°C for 30 minutes. The samples were washed with FACS buffer. The CD56+ cell population in the samples was analyzed by flow cytometry, and the percentage of CD107+ cells was calculated. The results are shown in Figure 5.

[0088] 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 improved after co-incubation with wild-type K562, and the difference between the NK cells activated by each engineered K562 cell was not much, 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 by the present invention was significantly enhanced.

[0089] 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.

[0090] It should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may be modified and varied in various ways. It is understood by those skilled in the art that any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. An engineered trophoblast cell expressing a cell surface molecule that specifically recognizes NKp46, wherein the cell surface molecule at least comprises an antibody targeting NKp46 and a transmembrane domain.

2. The engineered trophoblast cell of claim 1, wherein the antibody is selected from the group consisting of whole antibody, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibody, diabody, sdAb and nanobody.

3. The engineered trophoblast cell according to claim 2, wherein 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.

4. The engineered trophoblast cell according to claim 3, wherein the light chain variable region has at least 90% identity with the amino acid sequence shown in SEQ ID NO:7, and wherein the heavy chain variable region has at least 90% identity with the amino acid sequence shown in SEQ ID NO:8; preferably, the antibody targeting NKp46 has at least 90% identity with the amino acid sequence shown in SEQ ID NO:

9.

5. The engineered trophoblast cell according to any one of claims 1 to 4, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

6. The engineered trophoblast cell according to any one of claims 1-5, wherein the cell surface molecule further comprises a hinge region between the antibody and the transmembrane domain.

7. The engineered trophoblast cell according to claim 6, wherein the hinge region is selected from the hinge regions of the following proteins: CD8α, CD28, FcγRIIIα receptor, IgG4 and IgG1.

8. The engineered trophoblast cell according to any one of claims 1-7, wherein the cell surface molecule further comprises an intracellular domain.

9. The engineered trophoblast cell of claim 8, wherein the intracellular domain comprises a co-stimulatory domain and / or a primary signaling domain.

10. The engineered trophoblast cell of claim 9, wherein the primary signaling domain is selected from the signaling domains of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, and any combination thereof.

11. The engineered trophoblast cell of claim 9 or 10, wherein the co-stimulatory domain is selected from the co-stimulatory signaling domains of 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.

12. The engineered trophoblast cell according to any one of claims 1-11, further expressing at least one cell activation molecule, wherein the cell activation molecule is selected from secreted or membrane-bound cytokines, co-stimulatory ligands, activating receptor agonists and other molecules with cell activation function.

13. The engineered trophoblast cell according to claim 12, wherein 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.

14. The engineered trophoblast cell according to claim 12, wherein 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 is present in a secretory or membrane-bound form.

15. The engineered trophoblast cell according to any one of claims 1 to 14, wherein the trophoblast cell is selected from PBMC, RPMI8866, HFWT, K562, EBV-LCL, 721.221 and NK92 cells.

16. A cell culture medium comprising the engineered trophoblast cells according to any one of claims 1 to 15.

17. The cell culture medium according to claim 16, wherein the cells are selected from natural NK cells or engineered NK cells.

18. The cell culture medium of claim 17, wherein the engineered NK cells express a chimeric antigen receptor, a T cell receptor, a T cell receptor fusion protein, a T cell antigen coupler, an immune mobilizing monoclonal T cell receptor, or a combination thereof. The cell culture medium according to claim 17 or 18, wherein the NK cells are selected from primary NK cells, NK cell lines and NKT cells.

20. A method for expanding NK cells, comprising co-culturing NK cells with the engineered trophoblast cells described in any one of claims 1-15.

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