Methods for engineering innate-like lymphocytes

Abstract

The present invention relates to a method for introducing nucleic acid into innate-like lymphocytes, including γδ T cells, with a lentiviral vector; a population or composition of genetically engineered innate-like lymphocytes; and a method for treating or preventing disease. Adoptive cell therapy or transfer (ACT) is a form of immunotherapy that involves the isolation of T cells from a patient or donor, genetically modifying and / or expanding the cells, and subsequently reinfusing them back into the patient. For example, T cells can be engineered to express tumor antigen-specific T cell receptors (TCRs) or chimeric antigen receptors (CARs) to redirect the immune response against tumor cells.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to methods for the transfer of nucleic acids into innate-like lymphocytes, including γδ T cells, with lentiviral vectors; populations or compositions of genetically engineered innate-like lymphocytes; and methods for treating or preventing disease. [Background technology]

[0002] background Adoptive cell therapy or transfer (ACT) is a form of immunotherapy that involves the isolation of T cells from a patient or donor, genetically modifying and / or expanding the cells, and subsequently reinfusing them back into the patient. For example, T cells can be engineered to express tumor antigen-specific T cell receptors (TCRs) or chimeric antigen receptors (CARs) to redirect the immune response against tumor cells.

[0003] Most cell engineering approaches to date have been applied to αβ T cells, which are easy to expand and purify from peripheral blood, but such approaches have limitations.

[0004] For example, the use of TCR requires the identification of HLA-matched TCR against the processed antigen presented by tumor cells, and is susceptible to tumor immune evasion strategies (e.g., downregulation of MHC). Furthermore, when using donor (allogeneic) cells, there is a risk of graft-versus-host disease (GvHD). The use of CAR can eliminate the need for HLA matching and antigen presentation against tumor MHC, but the opportunity for immune evasion through antigen loss remains.

[0005] The use of innate or innate-like immune lymphocytes (e.g., γδT and natural killer (NK) cells) may offer several potential advantages. First, tumor recognition and killing is not dependent on the expression of a single antigen, because innate immune cells can recognize a broad range of antigens through their natural receptors. This property reduces the chance of immune escape due to loss of a single antigen and provides an opportunity to target tumors that lack well-defined neoantigens. Second, innate immune cells recognize their target cells in an MHC-independent manner, reducing the risk of alloreactivity and GvHD and allowing the development of "off-the-shelf" allogeneic therapies.

[0006] Combining the properties of innate-like immune cells with engineering strategies to enhance cytotoxicity and / or redirect cells to specific targets is therefore a promising strategy for ACT.

[0007] The clinical and commercial gold standard for lentivirus-based cell therapy involves VSVg-pseudotyped lentiviruses. Clinical and translationally, the use of non-VSVg-pseudotyped lentiviruses is very rare, as virus manufacturers (in the form of contract research organizations (CROs) or contract manufacturing organizations (CDMOs)) offer few such products in their repertoire. This is mainly due to the widespread success of the use of VSVg-pseudotyped lentiviruses for the clinical use of canonical autologous αβ T cells.

[0008] Although they can efficiently deliver transgenes to activated canonical αβ T cells, VSVg-pseudotyped lentiviruses are poor at transducing innate-like lymphocytes (e.g., γδ T cells and NK cells). This is due in large part to the low expression of the VSVg entry receptor (the low-density lipoprotein or "LDL" receptor) on innate lymphocytes. A recent report on optimized production of γδ T cells for immunotherapeutic use recommended a multiplicity of infection (MOI) of 80 for transducing expanding γδ T cells with VSVg-pseudotyped lentivirus; even at this MOI, the group reported an average transduction efficiency of only 20% (Sutton et al., 2016, Cytotherapy, 18(7):881-892). A recent report aimed at optimizing NK cell transduction with VSVg pseudotyped lentivirus reported a transduction efficiency of 11.8% of activated cells at a viral MOI of 10 and subsequently compared various transduction enhancers, including boosters commonly used to increase this efficiency (e.g., vectofusin, dextran, and PGE2). They concluded that of all the boosters screened, statins provided the highest enhancement of transduction efficiency, with rosuvastatin providing a transduction efficiency of 33.3% (Gong et al., 2020, Mol Ther Methods Clin Dev, 17:634-646). In both cases, the use of high viral MOI and / or transduction boosters came with the expected tradeoffs in the quality and number of cell products. Remarkably, statins were found to be inhibitory to NK cell cytotoxicity. Furthermore, the need to obtain large batches of virus (for high MOI transduction) and transduction boosters places high costs and a heavy burden on the cell therapy manufacturing supply chain. This highlights an unmet need for efficient and cost-effective innate-like lymphocyte lentiviral transduction methods that do not harm cell quality or number. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Sutton et al., Cytotherapy (2016) 18(7):881-892 [Non-Patent Document 2] Gong et al., Mol Ther Methods Clin Dev(2020)17:634~646 Summary of the Invention [Means for solving the problem]

[0010] Summary of the Invention The present invention provides a method for efficient transfer of genetic material into innate-like lymphocytes with lentiviral vectors pseudotyped with RD144-HIV chimeric envelope protein (also known as "RD-Pro"). The inventors have found that chimeric RD144-HIV pseudotyped lentiviruses are surprisingly effective in delivering transgenes to stimulated innate-like lymphocytes at low MOIs and with unexpectedly favorable cell viability and proliferation. As previously mentioned, VSVg-based transduction protocols can require MOIs of up to 80. In contrast, chimeric RD144-HIV pseudotyped lentiviral vectors can achieve approximately 70% transduction efficiency at MOIs as low as 1.25, requiring substantially lower viral titers (up to 60-fold lower viral requirements). Furthermore, transduction with chimeric RD144-HIV pseudotyped viruses exhibits less toxicity and results in higher levels of cellular products that are healthier and more effective. Thus, the methods of the present invention allow for a more cost-effective product manufacturing process that still yields better quality cells than standard lentiviral protocols.

[0011] The present invention provides a method for introducing nucleic acid into innate-like lymphocytes with a lentiviral vector, wherein the lentiviral vector comprises an RD114-HIV chimeric envelope protein.

[0012] The use of alternative RD114-derived lentiviral pseudotypes, namely RD114TR pseudotyped lentiviruses, for gamma delta T cell transduction has been described previously (WO2019 / 104269). Interestingly (and in contrast to our findings with RD114-HIV chimeric envelope protein or "RD-Pro"), VSVg pseudotyped lentiviruses resulted in greater transgene expression at day 5 post-transduction compared to RD114TR pseudotyped lentiviruses. This suggests the fact that not all RD114-derived lentiviral envelopes confer the same enhancement to innate lymphocyte transduction as the RD-Pro envelope.

[0013] The innate-like lymphocytes can be γδ T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, mucosal-associated invariant T (MAIT) cells, lymphoid tissue-derived (LTi) cells, intraepithelial lymphocytes (IEL), innate lymphocytes (ILCs) (e.g., group 1 innate lymphocytes, group 2 innate lymphocytes, group 3 innate lymphocytes, B1 cells, marginal zone B cells, and / or cells expressing CD8αα).

[0014] The innate-like lymphocytes may be innate lymphoid cells (ILCs), including γδ T cells, natural killer (NK) cells, natural killer T (NKT) cells, mucosal-associated invariant T (MAIT) cells, lymphoid tissue-derived (LTi) cells, intraepithelial lymphocytes (IELs), group 1 innate lymphocytes, group 2 innate lymphocytes, group 3 innate lymphocytes, B1 cells, marginal zone B cells, and / or cells expressing CD8αα.

[0015] The innate-like lymphocytes may be γδ T cells, natural killer (NK) cells, invariant natural killer T (iNKT) cells, mucosal-associated invariant T (MAIT) cells, lymphoid tissue-derived (LTi) cells, intraepithelial lymphocytes (IEL), innate lymphocytes (ILCs) (e.g., group 1 innate lymphocytes, group 2 innate lymphocytes, group 3 innate lymphocytes, B1 cells, marginal zone B cells, and / or cells expressing CD8αα).

[0016] The innate-like lymphocytes can be γδ T cells, natural killer (NK) cells, mucosal-associated invariant T (MAIT) cells, lymphoid tissue-derived (LTi) cells, intraepithelial lymphocytes (IEL), group 1 innate lymphocytes, group 2 innate lymphocytes, group 3 innate lymphocytes, B1 cells, marginal zone B cells, and / or innate lymphocytes (ILCs), including cells expressing CD8αα. In some embodiments, the innate-like lymphocytes are γδ T cells (e.g., V52+ T cells).

[0017] In some embodiments, the innate-like lymphocytes are γδ T cells (eg, Vδ1+ T cells).

[0018] In some embodiments, the innate-like lymphocytes are γδ T cells (e.g., non-Vδ1+ / Vδ2+ T cells).

[0019] In some embodiments, the innate-like lymphocytes are NK cells.

[0020] In some embodiments, the chimeric envelope protein comprises an RD114 envelope protein, in which the R peptide cleavage sequence is replaced with an HIV-1 matrix / capsid cleavage sequence.

[0021] The RD114-HIV chimeric envelope protein may have the sequence of SEQ ID NO:4.

[0022] The present invention provides methods for introducing nucleic acids into innate-like lymphocytes with lentiviral vectors at a low multiplicity of infection (MOI).

[0023] In some embodiments, the nucleic acid is introduced into innate-like lymphocytes by a lentiviral vector as described herein at an MOI of about 50, about 25, about 10, about 5, about 2.5 or about 1.25.

[0024] In some embodiments, the nucleic acid is introduced into innate-like lymphocytes in the absence of a transduction booster by a lentiviral vector as described herein.

[0025] The present invention provides methods for introducing a gene of interest into innate-like lymphocytes. The gene of interest may comprise any therapeutically relevant nucleotide sequence.

[0026] For example, the gene may encode an immunologically or metabolically active, naturally occurring or synthetic protein or chimeric molecule of human or non-human origin, which may be expressed intracellularly, at the cell membrane, or secreted from the cell.

[0027] In some embodiments, the lentiviral vectors described herein comprise genes encoding chimeric antigen receptors (CARs), chimeric costimulatory receptors, dual CARs, tri-CARs, tethered CARs, non-signaling CARs, truncated CARs, T cell receptors (TCRs), TCRs co-expressed with CARs, scFv-Fc fusion proteins (SFPs), antibodies, DARPINs, nanobodies, tribodies, duabodies, bispecific T cell engagers (BiTEs), transcription factors, intracellular signaling molecules and / or mediators, cytokines, chemokines, integrins, lectins, adhesion molecules, cell surface receptors, cell surface ligands, glucose transporters, ion transporters, membrane proximal and / or membrane distal intracellular enzymes, such as phosphatases, tyrosine kinases, serine threonine kinases, proteases, matrix metalloproteinases, and / or degrons.

[0028] The present invention provides a method for introducing a nucleic acid into an innate-like lymphocyte, the method comprising: (i) obtaining a population of innate-like lymphocytes; and (ii) incubating the innate-like lymphocytes with a lentiviral vector; wherein the lentiviral vector comprises an RD114-HIV chimeric envelope protein.

[0029] The above method can be performed in the absence of transduction enhancers.

[0030] The population of innate-like lymphocytes can be isolated from a subject, for example, from blood or tissue-derived products.

[0031] The methods of the invention may further include stimulating (eg, activating and / or expanding) the innate-like lymphocytes prior to and / or during incubation with the lentiviral vector.

[0032] The innate-like lymphocytes can be activated and / or expanded in the presence of zoledronic acid, interleukin 2 (IL-2), interleukin 15 (IL-15), and / or anti-CD3 antibodies.

[0033] The present invention provides a population of genetically engineered innate-like lymphocytes obtained or obtainable by the methods of the present invention.

[0034] The invention provides compositions (eg, pharmaceutical compositions) comprising the innate-like lymphocyte populations according to the invention.

[0035] Innate-like lymphocytes may be genetically modified to enhance their anti-tumor properties (e.g., cytotoxicity and cytokine production), proliferation, persistence, metabolic fitness (e.g., resistance to hypoxia or glucose starvation), ability to distinguish between healthy and malignant tissue, anti-inflammatory properties, and / or resistance to chemotherapeutic agents.

[0036] For example, the innate-like lymphocytes may be genetically modified to express a chimeric antigen receptor (CAR), a chimeric costimulatory receptor, a dual CAR, a tri-CAR, a tethered CAR, a non-signaling CAR, a truncated CAR, a T cell receptor (TCR), a TCR co-expressed with a CAR, an scFv-Fc fusion protein (SFP), an antibody, a DARPIN, a nanobody, a tribody, a dual body, a bispecific T cell engager (BiTE), a transcription factor, an intracellular signaling molecule and / or a mediator, a cytokine, a chemokine, an integrin, a lectin, an adhesion molecule, a cell surface receptor, a cell surface ligand, a glucose transporter, an ion transporter, a membrane proximal and / or membrane distal intracellular enzyme, e.g., a phosphatase, a tyrosine kinase, a serine threonine kinase, a protease, a matrix metalloproteinase, and / or a degron. The invention provides a method of treating or preventing a disease in a subject, said method comprising administering to the subject an innate-like lymphocyte population or composition according to the invention.

[0037] The invention provides a innate-like lymphocyte population or composition according to the invention for use in treating or preventing disease in a subject.

[0038] The invention provides a innate-like lymphocyte population or composition according to the invention for use in immunotherapy.

[0039] The invention provides the use of a innate-like lymphocyte population or a composition according to the invention for the preparation of a medicament for treating or preventing a disease in a subject.

[0040] The innate-like lymphocytes may be allogeneic.

[0041] The disease may be cancer; an autoimmune disease or immunopathology; an infectious disease, such as a bacterial, fungal or viral infection; associated with organ transplantation (eg, GvHD); or a wound, ulcer, or abscess.

[0042] These and other aspects and embodiments of the invention are described in further detail below. [Brief description of the drawings]

[0043] [Figure 1] Figure 1 - (A) Transduction efficiency of CD3+ / Vδ2+ and CD3+ / Vδ2- cells at 7 days post-transduction comparing transduction with RD114-HIV chimeric envelope (also known as "RD-Pro") pseudotyped lentivirus and VSVg pseudotyped lentivirus (both at MOI 15) using eGFP expression as a reporter of transduction. (B) Viable Vδ2+ cells per ml at 7 days post-transduction in the same conditions as in (A). (C) Viable GFP-expressing Vδ2+ cells per ml at 7 days post-transduction in the same conditions as in (A) and (B). [Diagram 2]Figure 2 - (A) Flow cytometry analysis of CD3 and Vδ2 expression at days 4, 8 and 11 post-transduction with RD114-HIV chimeric envelope ("RD-Pro") pseudotyped lentivirus. (B) GFP expression in CD3+ Vδ2+ cells. (C) % Vδ2+ cells expressing GFP at various MOIs at days 4, 8 and 11 post-transduction. [Diagram 3] Figure 3 - (A) Example of a method for expansion and transduction of mixed populations of γδ T cells. (B) Transduction efficiency of Vδ1+, Vδ2+ and Vδ1- / Vδ2- cells 12 days after transduction with RD114-HIV chimeric envelope ("RD-Pro") pseudotyped lentivirus at an MOI of 4, containing a bicistronic expression cassette including an eGFP reporter. [Figure 4] Figure 4 - (A) Transduction efficiency of V52+ cells with RD114-HIV chimeric envelope ("RD-Pro") pseudotyped lentiviruses at a range of MOIs. One lentivirus (14G2a-SFP) encodes a bicistronic construct containing eGFP and a secreted GD2-binding opsonin, while the other (104) encodes a bicistronic construct containing eGFP and luciferase. (B) V52+ transduction efficiency (% GFP expressing cells) after transduction with 14G2a-SFP lentivirus as in (A). (C) Percentage of surviving cells that are V52+ / GFP+ cells after transduction as in (A). (D) Flow cytometry analysis of CD3 and V52 expression of surviving cells transduced with 14G2a-SFP lentivirus as in (A). (E) Detection of anti-GD2 opsonins in the supernatants of γδ T cells transduced with 14G2a-SFP-encoding lentivirus as in (A), showing the relationship between the viral MOI and the amount of opsonin detected by binding to GD2+ target cells. [Diagram 5]FIG. 5 - (A) Similar to γδ T cells above, expanding NK cells (CD3-CD56+) were transduced with RD114-HIV chimeric envelope protein ("RD-Pro") pseudotyped lentivirus at MOI 4 on day 2 post-stimulation in the absence of transduction boosters. NK cells were transduced with a bicistronic GFP-secreting mitogen construct and GFP expression was measured on day 14 of expansion. Representative data from three different donors are shown (mean transduction efficiency 55%). (B) Efficient NK cell transduction could also be achieved using different activation methods. Representative transduction efficiency data from one donor of either untransduced cells or cells transduced with two different RD-Pro pseudotyped bicistronic GFP-mitogen constructs are shown. As above, NK cells were transduced on day 2 post-culture initiation in the absence of transduction boosters and at MOI of 4. [Figure 6-1] Figure 6 - (A and B) Exemplary expansion protocols for γδ T cells. (C) Transduction efficiency (GFP transgene expression) of Vδ1+, Vδ2+ and Vδ1- / Vδ2- cells of different expansion protocols 12 days after transduction with RD-Pro pseudotyped lentivirus at MOI 4. [Figure 6-2] Figure 6 - (A and B) Exemplary expansion protocols for γδ T cells. (C) Transduction efficiency (GFP transgene expression) of Vδ1+, Vδ2+ and Vδ1- / Vδ2- cells of different expansion protocols 12 days after transduction with RD-Pro pseudotyped lentivirus at MOI 4. [Figure 7] FIG. 7 - (A) NK cells (CD3-CD56+) in zoledronic acid-activated PBMCs were transduced with RD-Pro- and VSVg-pseudotyped lentiviruses at an MOI of 2. GFP expression was measured on day 14 of expansion. Representative data from three different donors are shown. (B) High transduction was maintained with bicistronic and immunologically active tricistronic large vectors at low MOI when NK cells were activated with Miltenyi Biotec's proprietary feeder-free and CD2 / NKp46 GMP compatible NK cell manufacturing kit. [Figure 8] Figure 8 - (A) Transduction efficiency (GFP expression) of NK-T cells at day 14 in zoledronic acid-activated PBMCs transduced with RD-Pro lentivirus at MOI 4. (B) High NK-T cell transduction was maintained with bicistronic and immunologically active tricistronic large vectors at low MOI when NK cells were activated with Miltenyi Biotec's proprietary feeder-free and CD2 / NKp46 GMP compatible NK cell production kit. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Detailed Description of the Invention Innate-like lymphocytes The present invention relates to a method for introducing nucleic acid into innate-like lymphocytes with a lentiviral vector comprising an RD114-HIV chimeric envelope protein.

[0045] The term "innate-like lymphocyte" as used herein refers to lymphocytes that exhibit innate-like immune properties. Antigen recognition and effector functions of innate immune cells are typically mediated through germline-encoded receptors. In contrast, adaptive immune responses rely on gene rearrangement and somatic hypermutation to generate diverse antigen recognition. Innate-like lymphocytes are thought to bridge the gap between innate and adaptive immunity, for example, innate-like lymphocytes can express semi-invariant antigen receptors.

[0046] Examples of innate-like lymphocytes include γδ T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, mucosal-associated invariant T (MAIT) cells, lymphoid tissue-derived (LTi) cells, intraepithelial lymphocytes (IELs), innate lymphocytes (e.g., group 1 innate lymphocytes, group 2 innate lymphocytes, group 3 innate lymphocytes, B1 cells, marginal zone B cells, and / or cells expressing CD8αα).

[0047] In some embodiments, the innate-like lymphocytes do not express the αβ T cell receptor.

[0048] In some embodiments, the innate-like lymphocytes are not canonical αβ T cells.

[0049] In some embodiments, the innate-like lymphocytes express a semi-invariant αβ T cell receptor.

[0050] γδ T cells represent a subset of T cells that express the γδ TCR instead of the αβ TCR. Although human γδ T cells normally comprise only 1–5% of circulating T lymphocytes, they undergo rapid expansion in response to tumors, inflammation, and invading pathogens.

[0051] γδ T cells can be divided into two major subsets - tissue-bound Vδ1-positive cells and peripherally circulating Vδ2-positive cells. The Vδ2 chain typically pairs with the Vγ9 chain, with Vγ9 / Vδ2 T cells accounting for 50-95% of peripheral T cells, in 1-5% of circulating T cells. Vδ1 γδ T cells represent the predominant T cell subset in solid tumors and the second most frequent subset in peripheral blood (1-3% of lymphocytes) after Vγ9 / Vδ2 T cells. Vδ1 T cells reside primarily in mucosal epithelial tissues and constitute approximately 40% of all intraepithelial lymphocytes in the large intestine. Both Vδ1 and Vδ2 subsets have been shown to have antiviral and antitumor activity. Unlike conventional αβ TCR-expressing cells, γδ TCR-expressing cells recognize their targets independent of classical MHC I and II.

[0052] Similar to natural killer (NK) T cells, γδ T cells express NKG2D, which binds to non-classical MHC molecules present on stressed and / or tumor cells, namely MHC class I polypeptide-related sequence A (MICA) and MHC class I polypeptide-related sequence B (MICB). The γδ TCR recognizes various ligands, e.g., stress- and / or tumor-associated phosphoantigens. γδ T cells mediate direct cytolysis of their targets through multiple mechanisms, namely TRAIL, FasL, perforin, and granzyme secretion. Furthermore, γδ T cells expressing CD16 can enhance antibody-dependent cell-mediated cytotoxicity (ADCC).

[0053] In some embodiments, the innate-like lymphocytes according to the invention are γδ T cells.

[0054] In some embodiments, the innate-like lymphocytes according to the invention are V52+ T cells (e.g., Vy9 / V52 T cells).

[0055] In some embodiments, the innate-like lymphocytes are V51+ γδ T cells.

[0056] In some embodiments, the innate-like lymphocytes are non-V51+ / V52+ γ5 T cells, such as V53+, V54+, V55+, V56+, V57+, or V58+ T cells.

[0057] The δ chain can be paired with any appropriate γ chain (e.g., Vγ2, Vγ3, Vγ4, Vγ5, Vγ8 or Vγ9).

[0058] Natural killer cells can be identified by the presence of CD56 and the absence of CD3. NK cells represent approximately 5-20% of all circulating lymphocytes in humans. NK cells are activated by cytokines including IL-12, IL-15, IL-18, IL-2, and CCL5. NK cells have antitumor activity mediated by receptors including NKG2D, NKp44, NKp46, NKp30, and DNAM, and can eliminate virus-infected cells via CD16-mediated ADCC.

[0059] Natural killer T (NKT) cells share characteristics of both T cells and natural killer cells. NKT cells can be identified by the presence of both CD3 and CD56, as well as αβTCR. They constitute approximately 1% of all peripheral blood T cells. Invariant natural killer T cells (also known as "type I" NKT cells) are a subset of NKT cells that express the invariant TCR α chain and a limited number of non-invariant TCR β chains (semi-invariant TCR). In humans, the highly conserved TCR is typically composed of the Va24-Ja18 segment paired with the Vb11 chain. In contrast, type II NKT cells (also referred to as diverse NKT or dNKT) use diverse TCR α and β chains.

[0060] Invariant natural killer T cells (iNKT) or type I NKT cells are a subset of T cells so named because they express cell surface markers associated with NK cells but also possess an invariant or semi-invariant αβ T cell receptor (TCR). They represent approximately 0.1% of human T cells in peripheral blood. In contrast to classical T cells, iNKT cells recognize lipids and glycolipids presented by CD1d, a nonpolymorphic MHC protein expressed on intestinal epithelial cells. iNKT cells mediate their effector functions primarily through rapid cytokine release following activation, including both Th1 (IFN-γ and TNF-α), Th2 (IL-1, IL-4, and IL-13), and Th17 (IL-17, IL-22) cytokines.

[0061] In some embodiments, innate-like lymphocytes according to the present invention are natural killer (NK) cells.

[0062] In some embodiments, innate-like lymphocytes according to the present invention are natural killer T (NKT) cells.

[0063] In some embodiments, innate-like lymphocytes according to the present invention are invariant natural killer T (iNKT) cells.

[0064] Mucosal-associated invariant T (MAIT) cells are important in the defense against bacteria on mucosal surfaces. They express the invariant TCR α chain and a variable but restricted TCR β chain. MAIT cells are found primarily in mucosal tissues (e.g., liver, lung, mesenteric lymph nodes, and intestinal epithelium). In human peripheral blood, they constitute approximately 1-10% of total T lymphocytes. MAIT cells express CD161, interleukin-18 receptor, and chemokine receptors CCR5, CXCR6, and CCR6 on the cell surface; they can be activated via the TCR or by IL-12 and IL-18 in a TCR-independent manner. MAIT cells can release IFN-γ, TNF-α, and IL-17 in response to stimuli and have granzyme b and perforin-mediated cytotoxic activity against infected cells.

[0065] In some embodiments, the innate-like lymphocytes according to the present invention are MAIT cells.

[0066] Innate lymphoid cells (ILCs) are a collective term for cells with lymphoid morphology that do not contain rearranged antigen receptors and lack myeloid-specific phenotypic markers. They do not directly recognize antigens, but instead respond to changes in cytokine expression profiles as a result of infection. ILCs are primarily tissue-resident cells found in both lymphoid and non-lymphoid tissues and rarely in peripheral blood. They are particularly abundant at mucosal surfaces and play an important role in mucosal immunity and homeostasis.

[0067] Based on differences in developmental pathways, phenotypes, and signaling molecules, ILCs are broadly divided into three groups: Group 1 ILCs (ILC1), which include NK cells, are responsive to cytokines (e.g., IL-12 and IL-18) and produce IFN-γ; Group 2 ILCs (ILC2) respond to IL-25, IL-33, and TSLP and produce Th2 cytokines (e.g., IL-4, IL-5, and IL-13); Group 3 ILCs (ILC3) express RORγt and respond to IL-23 and IL-1β through the production of IL-22 and IL-17.

[0068] Lymphoid tissue inducer (LTi) cells share many similar characteristics with ILC3s, but are generally considered a separate lineage due to their unique developmental pathway. Like ILC3s, LTi cells are RORγt-dependent. They participate in the formation of secondary lymph nodes and Peyer's patches by promoting lymphoid tissue development. Activated LTi cells mostly produce IL-17A, IL-17F, and IL-22.

[0069] In some embodiments, innate-like lymphocytes according to the present invention are innate lymphocytes, for example, group 1 innate lymphocytes, group 2 innate lymphocytes, group 3 innate lymphocytes, and / or LTi cells.

[0070] Intraepithelial lymphocytes (IELs) refer to lymphocytes found in the epithelial layer of mucosal linings such as the gastrointestinal and reproductive tracts. IELs can be divided into different subpopulations based on expression of TCR and CD8. Approximately 10-15% of IELs express γδ TCR.

[0071] In some embodiments, innate-like lymphocytes according to the present invention are intraepithelial lymphocytes.

[0072] CD8 is a co-receptor for TCR. CD8 is a dimer, consisting of a pair of CD8 chains. The most common form of CD8 contains CD8α and CD8β chains, but homodimers of the CD8α chain are also expressed in some cells. For example, NK cells and γδ T cells almost exclusively express the CD8αα form. Expression of CD8αα is also an important phenotypic marker of IELs.

[0073] In some embodiments, innate-like lymphocytes according to the invention express CD8αα.

[0074] Innate-like lymphocytes also include B cells expressing semi-invariant B cell receptor (e.g., B1 cells or marginal zone B cells). The B1 cell population includes two subsets based on the expression of CD5: B1a cells (CD5+) and B1b cells (CD5-). B1 cells generally express germline-encoded polyreactive IgM antibodies with restricted V gene segment usage. Marginal zone B cells are found in the spleen, where they surround follicles and are therefore frequently exposed to blood-borne antigens. After exposure to antigen, marginal zone B cells can present antigen, promote T cell activation, or differentiate into plasmablasts. Marginal zone B cells express surface IgM, complement receptors CD35 and CD21, and lipid antigen-presenting molecule CD1d.

[0075] In some embodiments, innate-like lymphocytes according to the invention are B1 cells or marginal zone B cells.

[0076] The present invention further provides a population of genetically engineered innate-like lymphocytes obtained or obtainable by the methods described herein.

[0077] The present invention provides a population of innate-like lymphocytes, wherein said innate-like lymphocytes are genetically engineered with a lentiviral vector comprising an RD114-HIV chimeric envelope protein.

[0078] The innate-like lymphocyte population or composition according to the present invention comprises at least about 10 3 cells, at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 cells, or at least about 10 8 cells, or at least about 10 9 cells, or at least about 10 10 cells, or at least about 10 11 The cell may include cells.

[0079] A natural-like lymphocyte population or composition according to the present invention may comprise at least about 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% natural-like lymphocytes that have been genetically engineered according to the methods of the present invention.

[0080] Lentiviral Vectors Lentiviral vectors are part of the larger group of retroviral vectors. Briefly, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV) (the causative agent of human acquired immune deficiency syndrome (AIDS)) and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype "slow virus" Visna / Maediulus (VMV), as well as the related Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0081] Lentivirus family is different from retrovirus in that lentivirus has the ability to infect both dividing and non-dividing cells.In contrast, other retroviruses (such as MLV) cannot infect non-dividing or slowly dividing cells (such as the cells that constitute muscle, brain, lung and liver tissue).

[0082] A lentiviral vector, as used herein, is a vector that contains at least one component part that can be derived from a lentivirus.Preferably, the component part is involved in the biological mechanism by which the vector infects or transduces a gene of interest into a target cell.

[0083] The term encompasses, for example, envelope-coated lipid nanoparticles and microparticles, vesicles and virus-like particles.

[0084] In one embodiment, the lentiviral vector is derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or visna lentivirus.

[0085] The basic structure of retrovirus and lentivirus genomes share many common features, such as 5'LTR and 3'LTR.Between or within these are located packaging signals that allow genomes to be packaged, primer binding sites, integration sites that allow integration into host cell genomes, and gag, pol and env genes that code packaging components (these are polypeptides required for the assembly of viral particles).Lentiviruses have additional features, such as the rev and RRE sequences in HIV, which allow the RNA transcripts of integrated proviruses to be efficiently exported from the nucleus to the cytoplasm of infected target cells.

[0086] In the provirus, these genes are flanked on both sides by regions called long terminal repeats (LTRs), which are responsible for proviral integration and transcription. LTRs can also act as enhancer-promoter sequences to control the expression of viral genes.

[0087] The LTRs themselves are identical sequences that can be divided into three elements: U3, R and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA. U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.

[0088] In defective retroviral or lentiviral vectors, the genomes gag, pol and env may be absent or non-functional.

[0089] In the representative lentiviral vector as described herein, at least a part of one or more coding regions for proteins essential for viral replication can be removed from the vector.This makes the viral vector replication-defective.A part of the viral genome can also be replaced by a gene of interest to generate a vector that contains the gene of interest and can transduce target cells and / or integrate its genome into target cell genome.

[0090] Optionally, the viral vectors used in the present invention have a minimal viral genome.

[0091] By "minimal viral genome" it is to be understood that viral vectors have been engineered to remove non-essential elements and retain essential elements to provide the functionality required to infect, transduce and deliver a gene of interest to a target host cell. Further details of this strategy can be found in WO1998 / 017815.

[0092] The lentiviral vectors as described herein are pseudotyped. In this respect, pseudotyped can provide one or more advantages. For example, the env gene product of HIV-based vectors restricts these vectors to infect only cells that express a protein called CD4. However, if the env gene in these vectors is replaced with the env sequence from other enveloped viruses, they can have a broad infectious spectrum (Verma and Somia (1997) Nature 389 (6648): 239-242).

[0093] In the present invention, the Env protein is a modified Env protein (e.g., a mutant Env protein or an engineered Env protein). The modification can be made or selected to introduce targeting ability or reduce toxicity or for other purposes (Valsesia-Wittman et al. 1996 J Virol 70:2056-64; Nilson et al. (1996) Gene Ther 3(4):280-286; and Fielding et al. (1998) Blood 91(5):1802-1809 and references cited therein).

[0094] RD114-HIV chimeric envelope protein The lentiviral vector for use in the methods of the present invention comprises an RD114-HIV chimeric envelope protein. In other words, the lentiviral vector of the present invention is pseudotyped with an RD114-HIV chimeric envelope protein.

[0095] The term "envelope protein" or "Env" as used herein refers to the env gene product. Envelope proteins are glycoproteins located on the virion surface that facilitate transduction by binding receptors on the target cell surface and driving subsequent fusion of the target cell and viral membranes.

[0096] The term "chimeric envelope protein" as used herein refers to an envelope protein that includes a portion of a protein derived from another protein. The other protein may or may not be an envelope protein. Chimeric envelope proteins can be made, for example, by joining two or more genes (e.g., two env genes) that originally encoded separate proteins.

[0097] RD114 refers to feline leukemia virus RD114.

[0098] The RD114 receptor, a neutral amino acid transporter (RDR), is widely expressed on hematopoietic stem cells. RD114 is a non-cytotoxic envelope protein, allowing the development of stable lentivirus-producing cell lines.

[0099] An example of a chimeric RD114 envelope protein is an RD114 envelope protein that includes a replacement of the cytoplasmic tail region with a tail region from another viral envelope protein, such as the tail region of murine leukemia virus (also known as "RD114TR"), as described, for example, in (WO2019 / 104269).

[0100] RD114-HIV chimeric envelope protein refers to an RD114 envelope protein that includes a portion of a protein derived from HIV (eg, the HIV Env cytoplasmic tail or the furin cleavage site).

[0101] In some embodiments, the RD114-HIV chimeric envelope protein comprises an RD114 envelope protein, in which the R peptide cleavage sequence in the cytoplasmic tail is replaced with an HIV-1 matrix / capsid cleavage sequence (e.g., as described in Bell et al. Experimental Biology and Medicine 2010;235). This RD114-HIV chimeric envelope protein may also be referred to as RD-Pro.

[0102] Replacement of the protease cleavage sequence of the RD114 envelope protein with the HIV matrix / capsid cleavage sequence is known to enhance the cleavage interaction between the HIV-1 protease and the RD-Pro envelope protein during virion formation. The subsequent increase in cleavage correlates with enhanced titer values ​​for RD-Pro pseudotyped viruses relative to unmodified RD114 pseudotyped viruses.

[0103] The RD114 envelope protein may comprise or consist of the sequence of SEQ ID NO:1.

[0104] The RD114 envelope protein may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO:1. SEQ ID NO:1 (RD114 envelope protein sequence) [ka]

[0105] The R peptide cleavage sequence can consist of or include the sequence VHAMVLAQ (SEQ ID NO:2). The R peptide cleavage sequence can have up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution relative to SEQ ID NO:2.

[0106] The HIV-1 matrix / capsid cleavage sequence may consist of or include the sequence SQNYPIVQ (SEQ ID NO:3). The HIV-1 matrix / capsid cleavage sequence may have up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution relative to SEQ ID NO:3.

[0107] In some embodiments, the RD114-HIV chimeric envelope protein comprises the sequence of SEQ ID NO:4.

[0108] In some embodiments, the RD114-HIV chimeric envelope protein consists of the sequence of SEQ ID NO:4.

[0109] In some embodiments, the RD114 envelope protein has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:4. SEQ ID NO:4 (RD-Pro envelope protein sequence) [ka]

[0110] It will be appreciated by those skilled in the art that some of the amino acid sequences described herein may be varied without significant effect on the structure or function of the protein. When such differences in sequence are contemplated, those skilled in the art will understand how to determine which domains are important regions for retaining the function of the protein.

[0111] It is known that certain amino acid changes are more likely to be tolerated at certain positions in a protein: for example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.

[0112] Typically, conservative substitutions are recognized as follows: substitutions between the aliphatic amino acids Ala (A), Val (V), Leu (L), and Ile (I) with each other; interchanges of the hydroxyl residues Ser (S) and Thr (T), interchanges of the acidic residues Asp (D) and Glu (E), interchanges between the amide residues Asn (N) and Gln (Q), interchanges of the basic residues Lys (K) and Arg (R), and interchanges between the aromatic residues Phe (F) and Tyr (Y).

[0113] identity The term "identity" as used herein refers to the percentage of amino acids or nucleotides of a polypeptide or nucleotide sequence that are identical with a reference sequence, expressed in %.

[0114] The degree of sequence identity between a query sequence and a reference sequence may be determined by 1) aligning the two sequences with any suitable alignment program using a default scoring matrix and default gap penalties, 2) identifying the number of exact matches (where an exact match is when the alignment program identifies an identical amino acid or nucleotide in the two aligned sequences at a given position in the alignment), and 3) dividing the number of exact matches by the length of the reference sequence.

[0115] Suitable computer programs for performing such alignments include, but are not limited to, Vector NTI (Invitrogen Corp.), and the ClustalV, ClustalW and ClustalW2 programs (Higgins DG & Sharp PM (1988), Higgins et al. (1992), Thompson et al. (1994), Larkin et al. (2007). A selection of different alignment tools is available from the ExPASy Proteomics server at www.expasy.org. Another example of software capable of performing sequence alignment is BLAST (Basic Local Alignment Search Tool), which can now be found at http: / / www.ncbi.nlm.nih.gov / and is available from the webpage of the National Center for Biotechnology Information, originally described in Altschul et al. (1990) J. Mol. Biol. 215;403-410.

[0116] Methods for introducing nucleic acids The present invention provides a method for introducing a nucleic acid into an innate-like lymphocyte, the method comprising: (iii) obtaining a population of innate-like lymphocytes; and (iv) incubating said innate-like lymphocytes with a lentiviral vector as described herein. Includes:

[0117] In some embodiments, the method is an in vitro method.

[0118] The lentiviral vector may comprise the nucleic acid, and thus the nucleic acid may be introduced into the innate-like lymphocyte by the lentiviral vector, for example, by transduction or infection, or by endocytosis or membrane fusion of an envelope-coated lipid nanoparticle or microparticle, vesicle or virus-like particle.

[0119] Enveloped lentiviral particles can additionally deliver something other than nucleic acid to a target cell, such as a peptide, carbohydrate or lipid of interest.

[0120] The population of innate-like lymphocytes can be isolated from a subject or from a sample derived from a subject. The sample can be a peripheral blood sample, a cord blood sample, a tumor, a stem cell precursor, a tumor biopsy, a tissue, or a lymph. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a tissue sample (e.g., skin or gut).

[0121] Peripheral blood mononuclear cells are extracted from a subject using, for example, an apheresis machine (Ficoll-Paque TM The innate-like lymphocytes may be collected from the collected sample using, for example, a flow cytometry technique. Cord blood cells may be obtained from the umbilical cord blood during the birth of a subject.

[0122] In some embodiments, the innate-like lymphocytes may be isolated from a blood product, such as a leukapheresate, a leukocyte reduction system chamber (buffy cone), or a buffy coat.

[0123] The cells may be autologous cells.

[0124] The cells can be allogeneic cells (ie, derived from a donor).

[0125] The innate-like lymphocytes can be stimulated, eg, activated and / or expanded, prior to and / or during incubation with the lentiviral vector.

[0126] Accordingly, the present invention provides a method for introducing a nucleic acid into a innate-like lymphocyte, the method comprising: (i) obtaining a population of innate-like lymphocytes (e.g., isolating a population of innate-like lymphocytes from a subject); and (ii) incubating the innate-like lymphocytes with a lentiviral vector as described herein; or (i) obtaining a population of innate-like lymphocytes (e.g., isolating a population of innate-like lymphocytes from a subject); (ii) activating and / or expanding the innate-like lymphocytes; and (iii) incubating the innate-like lymphocytes with a lentiviral vector as described herein; Further provided is a method comprising:

[0127] In some embodiments, the innate-like lymphocytes are activated and / or expanded by culturing the cells in the presence of a bisphosphonate and / or one or more cytokines.

[0128] The bisphosphonate may be zoledronate (zoledronic acid), pamidronate, alendronate, risedronate, ibandronate, incadronate, clodronate, etidronate, or neridronate, salts and / or hydrates thereof, preferably the bisphosphonate is zoledronic acid.

[0129] Suitable cytokines include interleukin (IL-) 2, IL-15, IL-12, IL-7, IL-21, IL-18, IL-19, IL-33, IL-4, IL-9, and / or IL-23.

[0130] In some embodiments, the cells are stimulated with a bisphosphonate and IL-2.

[0131] In some embodiments, the cells are stimulated with zoledronic acid and IL-2.

[0132] In some embodiments, the cells are stimulated with zoledronic acid, IL-2 and IL-15.

[0133] The concentration of the bisphosphonate (e.g., zoledronic acid) during activation and / or expansion of the cells may be from about 0.1 μM to about 500 μM, from about 0.1 μM to about 400 μM, from about 0.1 μM to about 300 μM, from about 0.1 μM to about 200 μM, from about 0.1 μM to about 100 μM, from about 0.5 μM to about 100 μM, from about 1 μM to about 500 μM, from about 1 μM to about 400 μM, from about 1 μM to about 300 μM, from about 1 μM to about 200 μM, from about 1 μM to about 100 μM, from about 1 μM to about 90 μM, from about 1 μM to about 80 μM, M, about 1 μM to about 70 μM, about 1 μM to about 60 μM, about 1 μM to about 50 μM, about 1 μM to about 40 μM, about 1 μM to about 30 μM, about 1 μM to about 20 μM, about 1 μM to about 15 μM, about 1 μM to about 10 μM, about 1 μM to about 9 μM, about 1 μM It can be ~8 μM, about 1 μM to about 7 μM, about 1 μM to about 6 μM, about 1 μM to about 5 μM, about 2 μM to about 5 μM, about 3 μM to about 5 μM, about 2 μM to about 5 μM, about 2 μM to about 10 μM, about 3 μM to about 8 μM, or about 4 μM to about 6 μM.

[0134] In a preferred embodiment, the concentration of the bisphosphonate is 5 μM.

[0135] In a preferred embodiment, the concentration of zoledronic acid is 5 μM.

[0136] The concentration of IL-2 during activation and / or expansion of the cells may be from about 10 IU / ml to about 1000 IU / ml, from about 10 IU / ml to about 500 IU / ml, from about 10 IU / ml to about 400 IU / ml, from about 10 IU / ml to about 300 IU / ml, from about 10 IU / ml to about 200 IU / ml, from about 10 IU / ml to about 150 IU / ml, from about 10 IU / ml to about 100 IU / ml about 20 IU / ml to about 100 IU / ml, about 30 IU / ml to about 100 IU / ml, about 40 IU / ml to about 100 IU / ml, about 50 IU / ml to about 100 IU / ml, about 75 IU / ml to about 125 IU / ml, about 20 IU / ml to about 80 IU / ml, about 25 IU / ml to about 100 IU / ml, or about 50 IU / ml to about 150 IU / ml.

[0137] In a preferred embodiment, the concentration of IL-2 is 100 IU / ml.

[0138] The concentration of IL-15 during activation and / or expansion of the cells can be about 10 ng / ml to about 1 pg / ml, about 10 ng / ml to about 500 ng / ml, about 10 ng / ml to about 400 ng / ml, about 10 ng / ml to about 300 ng / ml, about 10 ng / ml to about 200 ng / ml, about 10 ng / ml to about 150 ng / ml, about 10 ng / ml to about 100 ng / ml, about 20 ng / ml to about 100 ng / ml, about 30 ng / ml to about 100 ng / ml, about 40 ng / ml to about 100 ng / ml, about 50 ng / ml to about 100 ng / ml, about 60 ng / ml to about 100 ng / ml, about 20 ng / ml to about 80 ng / ml, about 30 ng / ml to about 60 ng / ml, or about 50 ng / ml to about 120 ng / ml.

[0139] In a preferred embodiment, the concentration of IL-15 is 70 ng / ml.

[0140] In some embodiments, the cells are stimulated with an anti-CD3 antibody, such as OKT3, 145-2C11, 17A2 or a suitable alternative. Preferably, the anti-CD3 antibody is OKT3.

[0141] The concentration of anti-CD3 antibody during activation and / or expansion of the cells can be from about 0.1 μg / ml to about 50 μg / ml, from about 0.1 μg / ml to about 25 μg / ml, from about 0.1 μg / ml to about 10 μg / ml, from about 0.1 μg / ml to about 5 μg / ml, from about 0.1 μg / ml to about 2.5 μg / ml, from about 0.5 μg / ml to about 2 μg / ml.

[0142] In a preferred embodiment, the concentration of the anti-CD3 antibody (eg, OKT3) is 1 μg / ml.

[0143] In some embodiments, the cells are stimulated with an anti-CD3 antibody (eg, OKT3 or another) and IL-2.

[0144] In some embodiments, the cells are stimulated with an anti-CD3 antibody, a bisphosphonate and IL-2.

[0145] In some embodiments, the cells are stimulated with anti-CD3 antibody, bisphosphonate, IL-2 and IL-15. The seeding density of isolated cells during activation and / or expansion is about 0.01×10 6 cells / cm 2 ~Approx. 1×10 7 cells / cm 2 , about 0.1×10 6 cells / cm 2 ~Approx. 5×10 6 cells / cm 2 , about 0.25×10 6 cells / cm 2 ~Approx. 5×10 6 cells / cm 2 , about 0.5×106 cells / cm 2 ~Approx. 5×10 6 cells / cm 2 , about 0.5×10 6 cells / cm 2 ~Approx. 4×10 6 cells / cm 2 , about 0.5×10 6 cells / cm 2 ~Approx. 3×10 6 cells / cm 2 , about 0.5×10 6 cells / cm 2 ~Approx. 2.5×10 6 cells / cm 2 , about 0.5×10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 , about 0.7×10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2~ Approximately 2×10 6 cells / cm 2 , about 0.9×10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 , about 1×10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 , or about 1.5 × 10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 It could be.

[0146] In some embodiments, the cells are stimulated, activated and / or expanded with an anti-CD2 antibody. In some embodiments, the cells are stimulated, activated and / or expanded with an anti-CD335 (NKp46) antibody. In some embodiments, the cells are stimulated, activated and / or expanded with an anti-CD2 and an anti-CD335 (NKp46) antibody.

[0147] In a preferred embodiment, the seeding density of said isolated cells during activation and / or expansion is 1×10 6 cells / cm 2 It is.

[0148] The duration of activation and / or expansion of the cells can be from 1 to about 20 days, from 1 to about 15 days, from 1 to about 12 days, from 1 to about 10 days, from 2 to about 20 days, from 2 to about 15 days, from 2 to about 12 days, from 2 to about 10 days, from 3 to about 15 days, from about 4 to about 15 days, from about 5 to about 15 days, or from about 10 to about 15 days.

[0149] In a preferred embodiment, the duration of activation and / or expansion of the cells is 12 days.

[0150] In some embodiments, the expansion and / or activation step includes costimulatory agents, which may include ligands that bind to receptors expressed on innate-like lymphocytes, such as NKG2D, CD161, CD70, JAML, DNAX accessory molecule-1 (DNAM-1), ICOS, CD27, CD137, CD30, HVEM, SLAM, CD122, DAP, and CD28.

[0151] Examples of reagents that may be used to promote activation and / or expansion of innate-like lymphocytes may include: anti-CD3, anti-CD2, anti-CD27, anti-CD30, anti-CD70, or anti-OX40 antibodies, IL-2, IL-15, IL-12, IL-9, IL-33, IL-18, or IL-21, CD70 (CD27 ligand), phytohemagglutinin (PHA), concavalin A (ConA), pokeweed (PWM) protein, peanut agglutinin (PNA), soybean agglutinin (SBA), lentil agglutinin (LCA), pea agglutinin (PSA), apple snail agglutinin (HPA), vicia graminea lectin (VGA), or another suitable mitogen capable of stimulating innate-like lymphocyte proliferation.

[0152] In some embodiments, cells expressing αβTCR may be depleted or reduced from the population of innate-like lymphocytes using, for example, magnetic beads coated with anti-αβTCR antibodies.

[0153] The methods of the invention allow for genetic manipulation (eg, by transduction) at a low MOI.

[0154] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of about 1 to about 100, about 1 to about 50, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 2 to about 4. In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of about 0.5 to about 5. In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of about 0.5 to about 2.5. In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of about 1 to about 2.5.

[0155] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than 5.

[0156] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than 3.

[0157] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than 2.5.

[0158] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than two.

[0159] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than 1.5.

[0160] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than 1.25.

[0161] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of about 1.

[0162] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than 1.

[0163] Transduction efficiency can be enhanced by using centrifugal infection (spinoculation) techniques and / or by performing transduction in the presence of transduction enhancers to increase transduction efficiency, e.g., by physically reducing electrostatic repulsion between negatively charged cells and virions, thus increasing cell-virion interactions.

[0164] The term "transduction enhancer" may be used interchangeably with "transduction booster."

[0165] Suitable transduction enhancers are known to those of skill in the art and are described in (登録商標) -1. RetroNectin (登録商標) , dextran, prostaglandin E 2 (P.G.E. 2 ), protamine sulfate, cyclosporine A, rapamycin, and statins (eg, atorvastatin, fluvastatin, simvastatin, pravastatin, and rosuvastatin).

[0166] The use of transduction boosters may also be undesirable as they are often expensive and toxic to lymphocytes, potentially preventing regulatory approval.

[0167] In some embodiments, the method according to the invention does not include the use of additional transduction enhancers, in other words, the innate-like lymphocytes can be incubated with the lentiviral vector in an environment that is free or substantially free of transduction enhancers.

[0168] Thus, in some embodiments, the invention provides a method for introducing a nucleic acid into an innate-like lymphocyte, the method comprising: (i) obtaining a population of innate-like lymphocytes; and (ii) incubating said innate-like lymphocytes with a lentiviral vector as described herein; or (i) obtaining a population of innate-like lymphocytes; (ii) activating and / or expanding the innate-like lymphocytes; and (iii) incubating the innate-like lymphocytes with a lentiviral vector as described herein, wherein the lentiviral vector is incubated with the innate-like lymphocytes at an MOI of less than 5, and wherein the incubation step is free or substantially free of transduction enhancers.

[0169] The seeding density of the isolated cells during incubation with the lentiviral vector was approximately 0.01×10 6 cells / cm 2 ~Approx. 1×10 7 cells / cm 2 , about 0.1×10 6 cells / cm 2 ~Approx. 5×10 6 cells / cm 2 , about 0.25×10 6 cells / cm 2 ~Approx. 5×10 6 cells / cm 2 , about 0.5×10 6 cells / cm 2 ~Approx. 5×10 6 cells / cm 2 , about 0.5×10 6 cells / cm 2 ~Approx. 4×10 6 cells / cm 2 , about 0.5×10 6 cells / cm 2 ~Approx. 3×10 6 cells / cm 2 , about 0.5×10 6 cells / cm 2 ~Approx. 2.5×10 6 cells / cm 2 , about 0.5×10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 , about 0.7×10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm2 , about 0.8×10 6 cells / cm 2 ~about 2×10 6 cells / cm 2 , about 0.9×10 6 cells / cm 2 ~about 2×10 6 cells / cm 2 , about 1×10 6 cells / cm 2 ~about 2×10 6 cells / cm 2 , or about 1.5 × 10 6 cells / cm 2 ~about 2×10 6 cells / cm 2 It could be.

[0170] In a preferred embodiment, the seeding density of the isolated cells during incubation with the lentiviral vector is 1×10 6 cells / cm 2 It is.

[0171] The cells may be incubated with the lentiviral vector, for example, on day 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the expansion / activation step. Preferably, the cells are incubated with the lentiviral vector on day 2 after expansion.

[0172] The duration of the incubation period can be from 1 to about 10 days, from 1 to about 9 days, from 1 to about 8 days, from 1 to about 7 days, from 1 to about 6 days, from 1 to about 5 days, from 1 to about 4 days, from about 2 to about 10 days, from about 2 to about 8 days, from about 2 to about 7 days, from about 3 to about 7 days, or from about 4 to about 6 days.

[0173] Throughout the incubation period, the culture medium may be removed and / or renewed or replenished with a feed medium (eg, a feed medium containing cytokines and / or other appropriate agents).

[0174] Transduction efficiency The transduction efficiency can be determined by the expression of the gene of interest in the target cells (i.e., the innate-like lymphocytes). The expression of the gene of interest can refer to mRNA expression and / or protein expression. For example, the expression of the protein encoded by the gene of interest can be determined by FACS analysis.

[0175] Transduction efficiency can be determined by the expression of a reporter gene.

[0176] Expression of the gene of interest in innate-like lymphocytes transduced according to the method of the present invention can be about 30% to about 100%, about 40% to about 100%, or about 50% to about 100% on day 4 after transduction.

[0177] Expression of the above-mentioned gene of interest in target cells among innate-like lymphocytes transduced according to the method of the present invention can be about 40% to about 100%, about 50% to about 100%, or about 60% to about 100% on day 8 after transduction.

[0178] Expression of the above-mentioned gene of interest in target cells among innate-like lymphocytes transduced according to the method of the present invention can be about 50% to about 100%, about 60% to about 100%, or about 70% to about 100% on day 11 after transduction.

[0179] In some embodiments, the transduction efficiency achieved in innate-like lymphocytes transduced according to the methods of the present invention is approximately 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold higher than the transduction efficiency achieved with VSVg-pseudotyped lentivirus.

[0180] In some embodiments, the transduction efficiency achieved in innate-like lymphocytes transduced according to the methods of the present invention is approximately 2-fold higher than the transduction efficiency achieved with VSVg-pseudotyped lentivirus at day 7 post-transduction.

[0181] In some embodiments, the transduction efficiency achieved in innate-like lymphocytes transduced according to the methods of the present invention is approximately 4-fold higher than the transduction efficiency achieved with VSVg-pseudotyped lentivirus at day 7 post-transduction.

[0182] In some embodiments, the methods of the invention reduce cytotoxicity, resulting in higher cellular product yields and fitness.

[0183] In some embodiments, the number of viable innate-like lymphocytes at day 7 post-transduction is approximately 2-fold higher than that achieved with VSVg-pseudotyped lentivirus.

[0184] Nucleic acid / gene of interest The lentiviral vector used in the methods of the invention may comprise a nucleic acid, which may encode one or more genes of interest.

[0185] Said nucleic acid can comprise DNA or RNA, and can be single-stranded or double-stranded.It is understood by those skilled in the art that many different polynucleotides can code for the same polypeptide as a result of the degeneracy of genetic code.Furthermore, it should be understood that those skilled in the art can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotide of the present invention, to reflect the codon usage of any particular host organism in which the polypeptide of the present invention is to be expressed.

[0186] The polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of the invention.

[0187] The term "gene of interest" as used herein refers to a nucleotide sequence that has any therapeutic or diagnostic application. The term is used interchangeably with the term "nucleotide of interest."

[0188] The gene of interest may encode an immunologically or metabolically active protein. The protein may be naturally occurring or synthetic (i.e., engineered), e.g., a chimeric protein. The protein may be of human or non-human origin.

[0189] The protein may be expressed intracellularly, at the cell membrane, or may be secreted from the cell into which the gene is introduced.

[0190] In the present invention, innate-like lymphocytes can be transduced with genes of interest to genetically modify the cells to express molecules that increase homing to tumors and / or to deliver inflammatory mediators, including but not limited to, cytokines, soluble immunomodulatory receptors and / or ligands, to the tumor microenvironment.

[0191] In some embodiments, the innate-like lymphocytes are genetically modified to enhance their anti-tumor properties (e.g., cytotoxicity and cytokine production). For example, the gene of interest may encode perforin, a protease, a toxin, or a cytokine.

[0192] In some embodiments, the innate-like lymphocytes are genetically modified to enhance their proliferation and / or persistence, for example, to disrupt signaling pathways involved in exhaustion or senescence.

[0193] In some embodiments, the innate-like lymphocytes are genetically modified to enhance their metabolic fitness, for example, the nucleic acid may encode a gene that confers increased resistance to hypoxia or glucose starvation.

[0194] In some embodiments, the innate-like lymphocytes are genetically modified to enhance their ability to distinguish between healthy and malignant tissues. For example, the gene of interest may code for a "NOT gate." Innate-like lymphocytes can be activated by a first receptor that binds to an antigen (e.g., an antigen expressed on malignant tissue). In "NOT gated" cells, the binding of a second receptor to an alternative antigen (e.g., an antigen expressed on healthy tissue) functions to override the activation signal.

[0195] In some embodiments, the innate-like lymphocytes are genetically modified to enhance their anti-inflammatory properties. For example, the gene of interest may encode IL-10 or TGF-β for secretion by the engineered cells.

[0196] Thus, suitable genes of interest include, but are not limited to, enzymes, cofactors, cytokines, chemokines, hormones, antibodies, designed ankyrin repeat proteins (DARPins), nanobodies, tribodies, dualbodies, bispecific T cell engagers (BiTEs), engineered immunoglobulin-like molecules, single chain antibodies, fusion proteins (e.g., scFv-Fc fusion proteins (SFPs)), immune co-stimulatory molecules, immune modulatory molecules, antioxidant molecules, chimeric antigen receptors, chimeric co-stimulatory receptors, dual-CARs, tri-CARs, tether-CARs, non-signaling CARs, truncated CARs, T cell receptors, transdomain negative mutants of a target protein, toxins, conditional toxins, and the like. toxins, antigens, transcription factors, structural proteins, integrins, lectins, adhesion molecules, cell surface receptors, cell surface ligands, reporter proteins, subcellular localization signals, intracellular signaling molecules, tumor suppressor proteins, growth factors, membrane proteins, glucose transporters, ion transporters, receptors, vasoactive proteins and peptides, antiviral proteins and ribozymes, and derivatives thereof (e.g., derivatives having associated reporter groups).

[0197] In some embodiments, the gene of interest may encode a therapeutic protein or a combination of therapeutic proteins.

[0198] In the present invention, innate-like lymphocytes can be transduced with a gene of interest to genetically modify the cells to prevent or reduce expression of a target gene, e.g., to genetically modify the cells to make them resistant to chemotherapeutic agents and / or immune checkpoints, and / or to disrupt signaling pathways.

[0199] In some embodiments, the gene of interest may encode a site-directed nuclease, such as a Cas nuclease, and a guide RNA (gRNA).

[0200] In some embodiments, the gene of interest (or nucleotide of interest) is a small interfering RNA (siRNA) or a mircoRNA (miRNA).

[0201] Chimeric antigen receptor (CAR) CARs, in their conventional form, are proteins that transfer the specificity of monoclonal antibodies (mAbs) to the effector functions of the cells: they are type I transmembrane domain proteins with an antigen-recognizing amino terminus, a spacer, and a transmembrane domain all tethered to a composite endodomain that conveys cell survival and activation signals.

[0202] The most common form of these molecules uses single chain variable fragments (scFv) derived from monoclonal antibodies to recognize target antigens. The scFv is fused to a signaling endodomain via a spacer and a transmembrane domain. Such molecules result in the activation of the cells in response to the recognition of their target by the scFv. When T cells express such CARs, they recognize and kill target cells that express the target antigen. Several CARs have been developed against tumor-associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently in clinical trials for the treatment of various cancers.

[0203] In some embodiments, the innate-like lymphocytes are genetically modified to express a chimeric antigen receptor (CAR), a chimeric costimulatory receptor, a dual CAR, a tri-CAR, a tethered CAR, a non-signaling CAR, and / or a truncated CAR.

[0204] T cell receptor (TCR) The TCR is a molecule found on the surface of certain lymphocytes (e.g., T cells) that is responsible for recognizing antigens bound to MHC molecules. The TCR heterodimer consists of either α and β chains, or γ and δ chains.

[0205] Binding of the TCR to antigen and MHC (peptide-MHC / pMHC) results in activation of the lymphocyte through a series of biochemical events mediated by associated enzymes, co-receptors, and specialized accessory molecules.

[0206] Each chain of the TCR is a member of the immunoglobulin superfamily and has an N-terminal immunoglobulin (Ig) variable (V) domain, an Ig constant (C) domain, a transmembrane domain, and a short cytoplasmic tail at the C-terminus.

[0207] The variable domains of both the TCR α and β chains have three hypervariable or complementarity determining regions (CDRs). CDR3 is the main CDR responsible for the recognition of processed antigens, but CDR1 of the α chain has also been shown to interact with the N-terminal portion of antigenic peptides, whereas CDR1 of the β chain interacts with the C-terminal portion of said peptides. CDR2 is believed to recognize MHC molecules. Framework regions (FRs) are located between the CDRs. These regions provide the structure of the TCR variable region.

[0208] The repertoire of TCR variable regions is generated by combinatorial concatenation of variable (V), joining (J) and diversity (D) genes; and by N region diversification (nucleotides inserted by the enzyme deoxynucleotidyl transferase).

[0209] The α chain is formed from a recombination event between the V and J segments. The β chain is formed from a recombination event involving the V, D and J segments.

[0210] The human TCRα locus (which also includes the TCRδ locus) is located on chromosome 14 (14q11.2). The TCRβ locus is located on chromosome 7 (7q34). The variable region of the TCRα chain is formed by recombination between one of 46 different Vα (variable) segments and one of 58 Jα (joining) segments (Koop et al.; 1994; Genomics; 19:478-493). The variable region of the TCRβ chain is formed from recombination between 54 Vβ, 14 Jβ, and two Dβ (diversity) segments (Rowen et al.; 1996; Science; 272:1755-1762).

[0211] The V and J (and D, where appropriate) gene segments for each TCR chain locus have been identified and the germline sequence of each gene is known and annotated (see, e.g., Scaviner & Lefranc; 2000; Exp Clin Immunogenet; 17:83-96 and Folch & Lefranc; 2000; Exp Clin Immunogenet; 17:42-54).

[0212] Methods for generating TCRs and affinity-enhanced TCRs are known in the art. Affinity-enhanced TCRs are TCRs that have enhanced affinity for peptide-MHC complexes. Methods include, for example, isolating the TCR gene encoding the TCR from a patient sample (e.g., the patient's peripheral blood or tumor-infiltrating lymphocytes) and improving the TCR affinity for peptide-MHC complexes through modification of the TCR sequence (e.g., by in vitro mutagenesis and selection of enhanced affinity (or affinity-matured) TCRs). Methods for identifying optimal affinity TCRs are also known in the art, including immunizing antigen-negative humanized transgenic mice (e.g., TCR / MHC humanized mice such as ABabDII mice) with a diverse human TCR repertoire with an antigen, and isolating antigen-specific TCRs from such immunized transgenic mice (e.g., see Obenaus M et al., Nat Biotechnol. 33(4):402-7, 2015).

[0213] In some embodiments, the gene of interest may encode a T cell receptor (eg, an αβ T cell receptor).

[0214] Immune Checkpoints In some embodiments, the innate-like lymphocyte populations or compositions according to the invention are genetically modified to render them resistant to immune checkpoints.

[0215] For example, the innate-like lymphocytes may be genetically modified to prevent expression of immune checkpoints, including, but not limited to, PD-1, Lag-3, Tim-3, TIGIT, BTLA, CTLA-4, and combinations thereof.

[0216] Chemotherapeutic agents In some embodiments, the innate-like lymphocyte populations or compositions according to the invention are genetically modified to increase their resistance to chemotherapeutic agents.

[0217] Chemotherapeutic entities as used herein refer to entities that are destructive to cells. That is, the entity reduces the viability of the cells. The chemotherapeutic entities may be cytotoxic drugs. Contemplated chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, epothilones, nitrosoureas, ethylenimines / methylmelamines, alkylsulfonates, alkylating agents, antimetabolites, pyrimidine analogs, epipodophyllotoxins, enzymes (e.g., L-asparaginase); biological response modifiers (e.g., IFNα, IL-2, G-CSF, and GM-CSF); platinum coordination complexes (e.g., cisplatin, oxaliplatin, and carboplatin), anthracenediones, substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (including N-methylhydrazine (MIH) and procarbazine), adrenal cortical suppressants, and the like. suppressants) (e.g., mitotane (o,p'-DDD) and aminoglutethimide); antagonists, including hormone and corticosteroid antagonists (e.g., prednisone and equivalents, dexamethasone and aminoglutethimide); progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); estrogens (e.g., diethylstilbestrol and ethinyl estradiol equivalents); antiestrogens (e.g., tamoxifen); androgens (including testosterone propionate and fluoxymesterone / equivalents); antiandrogens (e.g., flutamide, gonadotropin releasing hormone analogs, and leuprolide); and nonsteroidal antiandrogens (e.g., flutamide).

[0218] composition The present invention further provides a composition comprising a population of innate-like lymphocytes according to the present invention.

[0219] The natural-like lymphocyte composition can be a pharmaceutical composition comprising natural-like lymphocytes as defined herein.The pharmaceutical composition can further comprise a pharma- ceutical acceptable carrier, diluent or additive.The pharmaceutical composition can optionally comprise one or more additional pharma- ceutical active polypeptides and / or compounds.Such a formulation can be, for example, in a form suitable for intravenous infusion.

[0220] treatment The term "treatment" refers to the therapeutic use of a innate-like lymphocyte population or composition according to the present invention, where said innate-like lymphocyte population or composition may be administered to a subject with an existing disease or condition to alleviate, reduce or ameliorate at least one symptom associated with the disease and / or to slow down, reduce or block the progression of the disease, the term referring to both the treatment of an existing disease or the prevention of disease, i.e., prophylaxis. Thus, it is recognized that treatment, as referred to herein, may in some embodiments be prophylactic.

[0221] The invention provides a method for treating or preventing a disease in a subject, said method comprising administering to the subject an innate-like lymphocyte population or composition according to the invention.

[0222] The invention provides a innate-like lymphocyte population or composition according to the invention for use in treating or preventing disease in a subject.

[0223] The invention provides a innate-like lymphocyte population or composition according to the invention for use in immunotherapy.

[0224] The invention provides the use of a innate-like lymphocyte population or a composition according to the invention for the preparation of a medicament for treating or preventing a disease in a subject.

[0225] In a preferred embodiment of the invention, the subject described herein is a mammal, preferably a human, cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit or guinea pig, but most preferably, said subject is a human.

[0226] In some embodiments, the innate-like lymphocytes are derived from the subject to be treated (autologous).

[0227] In some embodiments, the innate-like lymphocytes are derived from a donor (allogeneic).

[0228] In one embodiment, the disease is cancer.

[0229] The cancer may be, for example, bladder cancer, gastric cancer, esophageal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, endometrial cancer, kidney cancer (renal cell), lung cancer (small cell, non-small cell, and mesothelioma), brain cancer (e.g., glioma, astrocytoma, glioblastoma), melanoma, lymphoma, small intestine cancer (duodenal and jejunal), leukemia, pancreatic cancer, hepatobiliary tumour, germ cell cancer, prostate cancer, head and neck cancer, thyroid cancer, and sarcoma.

[0230] The cancer may be a childhood cancer, such as craniopharyngioma, ependymoma, Ewing's sarcoma, medulloblastoma, neuroblastoma, soft tissue sarcoma, or Wilms' tumor.

[0231] In some embodiments, the cancer is a childhood cancer.

[0232] Treatment using the compositions and methods of the present invention can also include targeting circulating tumor cells, tumor-derived metastases, and / or liquid tumors.

[0233] Treatment with the innate-like lymphocyte populations or compositions of the invention may help prevent the evolution of therapy-resistant tumor cells that often occurs with standard approaches.

[0234] The method and use for treating cancer according to the present invention may be performed in combination with further cancer therapy. In particular, the innate-like lymphocyte population or composition according to the present invention may be administered in combination with checkpoint blockade therapy, costimulatory antibody, chemotherapy and / or radiation therapy, targeted therapy or monoclonal antibody therapy.

[0235] Checkpoint inhibitors include, but are not limited to, for example, PD-1 inhibitors, PD-L1 inhibitors, Lag-3 inhibitors, Tim-3 inhibitors, TIGIT inhibitors, BTLA inhibitors, and CTLA-4 inhibitors.Co-stimulatory antibodies deliver positive signals through immunoregulatory receptors (including, but not limited to, ICOS, CD137, CD27, OX-40, and GITR).

[0236] "In combination" may refer to the administration of an additional therapy prior to, simultaneously with, or following administration of a innate-like lymphocyte population or composition according to the present invention.

[0237] In one embodiment, the disease is an autoimmune disease or an immunopathology.

[0238] The autoimmune disease can be, for example, celiac disease, type I diabetes, Graves' disease, inflammatory bowel disease, Crohn's disease, multiple sclerosis, psoriasis, rheumatoid arthritis, myasthenia gravis, or systemic lupus erythematosus.

[0239] In one embodiment, the disease is associated with solid organ and / or hematopoietic stem cell transplantation, for example, graft rejection or graft-versus-host disease (GvHD).

[0240] In one embodiment, the disease is an infectious disease, such as a bacterial, fungal or viral infection.

[0241] In one embodiment, the disease is a wound, an ulcer, or an abscess. The innate-like lymphocyte population or composition according to the present invention may be used to aid in wound healing.

[0242] The present invention will now be further illustrated by examples. The examples are meant to serve to aid those skilled in the art in practicing the present invention and are not intended to limit the scope of the invention in any way. EXAMPLES

[0243] Working Example Example 1 - RD-Pro transduces V52+ cells more efficiently than VSVG PBMCs were cultured at 1 × 10 in a 96-well plate. 6 cells / cm 2 They were stimulated with 5 μM zoledronic acid and 100 IU / ml IL-2. On day 2, cells were transduced with RD-Pro pseudotyped virus or VSVg pseudotyped virus containing GFP at an MOI of 15 or mock transduced (no virus). On day 4, 50% of the medium was removed and IL-2 was refreshed.

[0244] Figure 1a shows that at day 5 post-transduction (day 7 of expansion), CD3+V52+ cells were significantly more efficiently transduced by RD-Pro than VSVg. In CD3+V52- cells, there was no difference in transduction efficiency between RD-Pro and VSVg.

[0245] Figures 1b and c show that transduction with RD-Pro yielded more viable cells than transduction with VSVg-pseudotyped virus and, at day 7 of expansion (day 5 post-transduction), more viable GFP-expressing cells per ml.

[0246] Example 2 - Efficient transduction achieved at low MOI PBMCs were cultured at 1 × 10 in a 96-well plate. 6 cells / cm 2and stimulated with 5 μM zoledronic acid and 100 IU / ml IL-2. On day 2, cells were transduced with RD-Pro pseudotyped bicistronic eGFP-luciferase lentiviral vectors at MOIs of 10, 5, 2.5, and 1.25.

[0247] CD3+ V52+ cells were analyzed for GFP expression on days 4, 8 and 11 post-transduction.

[0248] Figure 2b shows that an MOI of 1.25 resulted in approximately 70% transduction efficiency by days 8 and 11 post-transduction. Greater than 90% transduction efficiency was achieved at an MOI of 10 by days 8 and 11 post-transduction.

[0249] Figure 2c shows the effect of a range of MOIs (1.25, 2.5, 5 and 10) on the transduction efficiency of V52+ γδ T cells at days 4, 8 and 11 post-transduction. Stable transduction is achieved at day 8 post-transduction.

[0250] Example 3 - Transduction of V51+, V52+ and V51- / V52- γδ T cells PBMCs were subjected to αβ T cell depletion using magnetic bead-based separation according to the manufacturer's protocol (Miltenyi). The resulting αβ T cell-depleted PBMCs were plated at 1 × 10 6 cells / cm 2 Cells were plated at 1000 ng / ml for 1 h. Figure 3a shows how the cells were stimulated with 1 μg / ml anti-CD3 antibody (OKT3) and 100 IU / ml IL-2. On day 2, cells were transduced with RD-Pro pseudotyped virus containing a bicistronic GFP-luciferase at an MOI of 4. Transduction efficiency of viable γδ T cells (defined as CD3+ / αβ TCR−) from three subsets (Vδ1+, Vδ2+ and Vδ1− / Vδ2−) was determined by flow cytometry on day 14 of expansion (day 12 post-transduction).

[0251] Figure 3b shows GFP expression in V51+, V52+ and V51- / V52- γδ T cell subsets from three representative donors.

[0252] Example 4 - Transduction of V52+ cells with tricistronic constructs PBMCs were cultured at 1 × 10 in a 96-well plate. 6 cells / cm 2 At day 2, cells were transduced with RD-Pro pseudotyped virus containing GFP-luciferase (104) or GFP-14G2a (14G2a), where 14G2a is a secreted opsonin targeting the tumor-associated antigen GD2, at a range of MOIs (1, 2, 4, and 10 for 14G2a, and 5, 10, and 20 for 104). IL-2 was refreshed every 2–3 days. At day 12 post-transduction, transduction efficiency was assessed using flow cytometry, and supernatants from γδ T cells transduced to express the 14G2a opsonin were collected for analysis of secreted protein content.

[0253] Figure 4a shows the transduction efficiency of V52+ cells transduced with lentiviruses containing either eGFP-14G2a or eGFP-luciferase.

[0254] FIG. 4b shows FACS analysis of GFP expression in γδ T cells transduced with lentivirus containing eGFP-14G2a at a range of MOIs.

[0255] Figure 4c shows the percentage of viable cells co-expressing TCR V52 and GFP after transduction with either lentivirus, and Figure 4d shows GFP and V52 expression in a representative FACS blot of cells transduced with eGFP-14G2a lentivirus.

[0256] Figure 4e shows detection of secreted anti-GD2 opsonins in the supernatants of cells transduced with eGFP-14G2a lentivirus. Opsonins were detected by incubating the supernatants with either GD2+ or GD2- target cells and then detecting opsonin binding using a secondary antibody. Opsonin binding was shown to be specific, and the amount of opsonin produced is proportional to the MOI of virus used.

[0257] Example 5 - Transduction of NK cells with bicistronic constructs PBMCs were cultured at 1 × 10 in a 96-well plate. 6 cells / cm 2 The cells were plated at 100 ng / ml for 1 h. They were stimulated with 5 μM zoledronic acid and 100 IU / ml IL-2. On day 2, the cells were transduced with bicistronic RD-Pro pseudotyped virus containing GFP-mitogen at an MOI of 2. IL-2 was refreshed every 2-3 days. On day 14 post-transduction, transduction efficiency was assessed using flow cytometry. NK cells were counted as CD3 negative and CD56 positive cells in the cultures.

[0258] FIG. 5a shows the transduction efficiency of NK cells from three independent donor expansions, achieving a mean transduction efficiency of approximately 55%.

[0259] PBMCs were subjected to αβ T cell depletion using magnetic bead-based separation according to the manufacturer's protocol (Miltenyi). The resulting αβ T cell-depleted PBMCs were plated at 1 × 10 6 cells / cm 2 NK cells were plated at 100 μg / ml. Figure 3a shows how the cells were stimulated with 1 μg / ml anti-CD3 antibody (OKT3) and 100 IU / ml IL-2. On day 2, cells were transduced with RD-Pro pseudotyped virus containing a bicistronic GFP-luciferase at an MOI of 4. Transduction efficiency of viable NK cells (defined as CD3- / CD56+) from the three subsets was determined by flow cytometry on day 14 of expansion (day 12 post-transduction).

[0260] FIG. 5b shows comparable GFP expression of one representative donor NK cells that were either untransduced or transduced with two different bicistronic GFP-mitogen constructs.

[0261] Example 6 - Transduction of γδ T cells across different expansion conditions Figure 6a shows αβ-depleted PBMCs were cultured at 1 × 10 6 cells / cm 2 1 and stimulated with 1 μg / ml anti-CD3 antibody (OKT3) and 100 IU / ml IL-2 as described in Example 3 (OKT-3 expansion).

[0262] Figure 6b shows that αβ-undepleted PBMCs were cultured at 1 × 10 6 cells / cm 2 and stimulated with 5 μM zoledronic acid and 100 IU / ml IL-2 (ZOL-expansion).

[0263] γδ T cells were activated using two translationally related GMP-compatible protocols and transduced with the bicistronic RD-Pro vector at an MOI of 4. GFP transgene expression was measured in Vδ1+, Vδ2+ and Vδ1- / Vδ2- γδ T cell subsets derived from three independent donors (Fig. 6c). As can be seen, RDPro effectively transduces different types of γδ T cells across the different expansion protocols.

[0264] Example 7 - Efficient transduction of NK cells at low MOI Zoledronic acid-activated PBMCs were transduced with RD-Pro- and VSVg-pseudotyped lentiviruses containing the bicistronic construct at an MOI of 2. NK cells were highly GFP positive at culture harvest on day 14 when RD-Pro lentivirus was used (mean transduction efficiency -46%) but not when VSVg-pseudotyped lentivirus was used (mean transduction efficiency -10%) (Figure 7a).

[0265] High transduction was maintained with bicistronic and immunologically active tricistronic large vectors at low MOI when NK cells were activated with Miltenyi Biotec's proprietary feeder-free and CD2 / NKp46 GMP compatible NK cell production kit.

[0266] Example 8 - RD-Pro pseudotyped lentivirus transduces NK-T cells with translationally relevant efficiency at low MOI Zoledronic acid-activated PBMCs were transduced with RD-Pro pseudotyped lentivirus at an MOI of 4. NK T cells (CD3+ CD56+ αβTCR+) were GFP positive at culture harvest on day 14 (Figure 8a).

[0267] High NK-T cell transduction was maintained with bicistronic and immunologically active tricistronic large vectors at low MOI when NK cells were activated with Miltenyi Biotec's proprietary feeder-free and CD2 / NKp46 GMP compatible NK cell production kit (Figure 8b).

Claims

1. A composition for use in a method for introducing nucleic acids into natural-like lymphocytes, wherein the composition comprises a lentiviral vector, and the lentiviral vector comprises an RD114-HIV chimeric envelope protein.

2. The composition according to claim 1, wherein the innate-like lymphocytes are innate lymphocytes (ILCs) comprising γδ T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, mucosa-associated invariant T (MAIT) cells, lymphoid tissue-induced (LTi) cells, intraepithelial lymphocytes (IELs), group 1 innate lymphocytes, group 2 innate lymphocytes, group 3 innate lymphocytes, B1 cells, marginal zone B cells, and / or cells expressing CD8αα.

3. The composition according to claim 1 or claim 2, wherein the native-like lymphocytes are γδT cells.

4. The composition according to claim 3, wherein the native-like lymphocytes are Vδ1+γδT cells.

5. The composition according to claim 3, wherein the native-like lymphocytes are Vδ2+γδT cells.

6. The composition according to claim 3, wherein the native-like lymphocytes are non-Vδ1+ / Vδ2+ γδT cells.

7. The composition according to claim 1 or claim 2, wherein the native-like lymphocytes are NK cells.

8. The composition according to claim 1 or claim 2, wherein the native-like lymphocytes are NKT cells.

9. The composition according to claim 1 or 2, wherein the native-like lymphocytes are not canonical αβT cells.

10. The composition according to claim 1, wherein the chimeric envelope protein comprises an RD114 envelope protein, and the R peptide cleavage sequence is replaced with an HIV-1 matrix / capsid cleavage sequence.

11. The composition according to claim 1, wherein the chimeric envelope protein comprises a sequence having at least 60%, at least 70%, at least 80%, or at least 90% identity with SEQ ID NO:

4.

12. The composition according to claim 1, wherein the nucleic acid is introduced into the native-like lymphocytes by the lentiviral vector at multiple infection degrees (MOI) of about 50, about 25, about 10, about 5, about 2.5, or about 1.

25.

13. The composition according to claim 1, wherein the nucleic acid encodes a target gene.

14. The composition according to claim 13, wherein the gene encodes a naturally occurring or synthetic protein or chimeric molecule of human or non-human origin that is immunologically or metabolically active, either expressed intracellularly, expressed in the cell membrane, or secreted from the cell.

15. The composition according to claim 13 or 14, wherein the gene encodes a chimeric antigen receptor (CAR), a chimeric costimulatory receptor, a dual CAR, a tri CAR, a tethered CAR, a non-signaling CAR, a truncated CAR, a T cell receptor (TCR), a TCR co-expressed with a CAR, an scFv-Fc fusion protein (SFP), an antibody, a DARPIN, a nanobody, a tribody, a duabody, a bispecific T cell engager (BiTE), a transcription factor, an intracellular signaling molecule and / or mediator, a cytokine, a chemokine, an integrin, a lectin, an adhesion molecule, a cell surface receptor, a cell surface ligand, a glucose transporter, an ion transporter, a membrane-proximal and / or membrane-distal intracellular enzyme, such as a phosphatase, a tyrosine kinase, a serine / threonine kinase, a protease, a matrix metalloproteinase, and / or degron.

16. The aforementioned method, (i) the process of obtaining a population of native-like lymphocytes; and (ii) The step of incubating the native-like lymphocytes together with a lentiviral vector. encompassing; The composition according to claim 1, wherein the lentiviral vector comprises the RD114-HIV chimeric envelope protein.

17. The composition according to claim 16, wherein the incubation step is carried out in the absence of a transduction enhancer.

18. The composition according to claim 16, wherein the population of native-like lymphocytes is isolated from human products derived from blood or tissue.

19. The composition according to claim 16, wherein the population of native-like lymphocytes is stimulated, for example, activated and / or expanded, before and / or during incubation with the lentiviral vector.

20. A population of genetically modified natural-like lymphocytes obtained or obtainable by the method of Claim 1.

21. A population of natural-like lymphocytes, wherein these natural-like lymphocytes are genetically modified with a lentiviral vector containing the RD114-HIV chimeric envelope protein.

22. A composition comprising the native-like lymphocyte population described in claim 20.

23. A composition comprising the native-like lymphocyte population described in Claim 21.

24. The aforementioned group or composition is at least about 10 ６ individual cells, at least about 10 ７ individual cells, at least about 10 ８ individual cells, at least about 10 ９ individual cells, at least about 10 １０ A single cell, or at least about 10 １１ A population or composition of native-like lymphocytes according to any one of claims 20 to 23, comprising a single cell.

25. The population or composition of innate lymphocytes according to any one of claims 20 to 23, wherein at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of the innate lymphocytes are genetically modified.

26. The spontaneous lymphocyte population or composition according to any one of claims 20 to 23, wherein the spontaneous lymphocytes consist of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% γδT cells.

27. The spontaneous lymphocyte population or composition according to claim 26, wherein the spontaneous lymphocytes consist of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% Vδ1+ γδT cells.

28. The spontaneous lymphocyte population or composition according to claim 26, wherein the spontaneous lymphocytes consist of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% Vδ2+ γδT cells.

29. The spontaneous lymphocyte population or composition according to claim 26, wherein the spontaneous lymphocytes consist of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% non-Vδ1+ / Vδ2+ γδT cells.

30. The native-like lymphocyte population or composition according to any one of claims 20 to 23, wherein the native-like lymphocytes consist of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% natural killer (NK) cells.

31. The spontaneous lymphocyte population or composition according to any one of claims 20 to 23, wherein the spontaneous lymphocytes consist of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% iNKT cells.

32. The native-like lymphocyte population or composition according to any one of claims 20 to 23, wherein the native-like lymphocytes are genetically modified to enhance their antitumor properties, such as cytotoxicity and cytokine production.

33. The spontaneous lymphocyte population or composition according to any one of claims 20 to 23, wherein the spontaneous lymphocytes are genetically modified to enhance their proliferation and / or persistence.

34. The native-like lymphocyte population or composition according to any one of claims 20 to 23, wherein the native-like lymphocytes are genetically modified to enhance their metabolic fitness, for example, their resistance to hypoxia or glucose starvation.

35. The spontaneous lymphocyte population or composition according to any one of claims 20 to 23, wherein the spontaneous lymphocytes are genetically modified to enhance their ability to distinguish between healthy tissue and malignant tissue, for example, via a "NOT" gate.

36. The native-like lymphocyte population or composition according to any one of claims 20 to 23, wherein the native-like lymphocytes are genetically modified to enhance their anti-inflammatory properties, for example, through the secretion of IL-10 or TGF-β.

37. The spontaneous lymphocyte population or composition according to any one of claims 20 to 23, wherein the spontaneous lymphocytes are genetically modified to increase their resistance to chemotherapeutic drugs.

38. The aforementioned innate-like lymphocytes include chimeric antigen receptors (CARs), chimeric costimulatory receptors, dual CARs, tri-CARs, tethered CARs, non-signaling CARs, truncated CARs, T cell receptors (TCRs), TCRs co-expressed with CARs, scFv-Fc fusion proteins (SFPs), antibodies, DARPINs, nanobodies, tribodies, duabodies, bispecific T cell engagers (BiTEs), transcription factors, intracellular signaling molecules and / or mediators. A population or composition of natural-looking lymphocytes according to any one of claims 20 to 23, which is genetically modified to express cytokines, chemokines, integrins, lectins, adhesion molecules, cell surface receptors, cell surface ligands, glucose transporters, ion transporters, membrane-proximal and / or membrane-distal intracellular enzymes, such as phosphatases, tyrosine kinases, serine / threonine kinases, proteases, matrix metalloproteinases, and / or degrons.

39. A composition for use in treating or preventing a disease in a subject, comprising the native-like lymphocyte population described in claim 20 or 21, or the composition described in claim 22 or 23.

40. A composition comprising a population of native-like lymphocytes according to claim 20 or 21, or the composition according to claim 22 or 23, for use in immunotherapy.

41. The composition for use according to claim 40, wherein the aforementioned native-like lymphocytes are allogeneic.

42. The composition for use according to claim 41, wherein the native-like lymphocytes are allogeneic.

43. Use of a native-like lymphocyte population or composition according to any one of claims 20 to 23 for the preparation of a pharmaceutical for treating or preventing a disease in a subject.

44. The use according to claim 43, wherein the aforementioned native-like lymphocytes are allogeneic.

45. The aforementioned disease is, i) Cancer, ii) Autoimmune disease or immunopathology, iii) Infectious diseases, for example, bacterial infections, fungal infections or viral infections, iv) Diseases associated with organ transplantation, or v) Wounds, ulcers, or abscesses A composition for use according to claim 39, which is more selected.

46. The aforementioned disease is, i) Cancer, ii) Autoimmune diseases or immunopathologies, iii) Infectious diseases, for example, bacterial infections, fungal infections or viral infections, iv) Diseases associated with organ transplantation, or v) Wounds, ulcers, or abscesses A composition for use according to claim 40, which is more selected.

47. The aforementioned disease is, i) Cancer, ii) Autoimmune diseases or immunopathologies, iii) Infectious diseases, for example, bacterial infections, fungal infections or viral infections, iv) Diseases associated with organ transplantation, or v) Wounds, ulcers, or abscesses A more selected use according to claim 43.

48. An in vitro method for introducing nucleic acids into native-like lymphocytes using a lentiviral vector, wherein the lentiviral vector comprises the RD114-HIV chimeric envelope protein.

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