Methods for engineering innate-like lymphocytes

Abstract

The present invention relates to methods for introducing nucleic acids into innate-like lymphocytes, including gamma delta T cells, using lentiviral vectors; a population or composition of genetically engineered innate-like lymphocytes; and a method for treating or preventing a disease.

Description

Technical Field

[0001] The present invention relates to methods for introducing nucleic acids into innate-like lymphocytes (including γδT cells) using lentiviral vectors; populations or compositions of genetically engineered innate-like lymphocytes; and methods for treating or preventing diseases. Background Art

[0002] Adoptive cell therapy or transfer (ACT) is a form of immunotherapy that involves the isolation of T cells from a patient or donor, genetic modification and / or expansion of the cells, and subsequent reinfusion 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 immune responses to tumor cells.

[0003] To date, most cell engineering approaches have been applied to αβ T cells, which are easily expanded and purified from peripheral blood. However, this approach has limitations.

[0004] For example, the use of TCRs requires the identification of HLA-matched TCRs against processed antigens presented by tumor cells and is susceptible to tumor immune escape strategies such as MHC downregulation. In addition, there is a risk of graft-versus-host disease (GvHD) when using donor (allogeneic) cells. Although the use of CARs can eliminate the need for HLA matching and antigen presentation on the tumor MHC, the possibility of immune escape through antigen loss still exists.

[0005] The use of innate or innate-like immune lymphocytes, such as γδ T and natural killer (NK) cells, may offer several potential advantages. First, tumor recognition and killing are not dependent on the expression of a single antigen, as innate immune cells can recognize a broad spectrum of antigens through their innate receptors. This property reduces the chance of immune escape due to loss of a single antigen and provides the possibility of targeting tumors that lack clear neoantigens. Second, innate immune cells recognize their target cells in an MHC-independent manner, thereby reducing the risk of alloreactivity and GvHD and enabling the development of “off-the-shelf” allogeneic therapies.

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

[0007] The clinical and commercial gold standard for lentiviral-based cell therapies includes VSVg-pseudotyped lentiviruses. The use of non-VSVg-pseudotyped lentiviruses in clinical and translational settings is so rare that few virus manufacturers (in the form of contract research organizations (CROs) or contract development and manufacturing organizations (CDMOs)) offer such products in their repertoires. This is largely due to the widespread and successful use of VSVg-pseudotyped lentiviruses in clinical settings with regulated autologous αβ T cells.

[0008] Despite the ability to efficiently deliver transgenes to activated canonical αβ T cells, VSVg-pseudotyped lentiviruses are poor at transducing innate-like lymphocytes, such as γδ T cells and NK cells. This is largely due to the low expression of the VSVg entry receptor (low-density lipoprotein or “LDL” receptor) on innate lymphoid cells. A recent report on the optimized preparation of γδ T cells for immunotherapy use recommended transduction of expanded γδ T cells with VSVg-pseudotyped lentivirus at a multiplicity of infection (MOI) of 80; furthermore, even with this MOI, the average transduction efficiency reported by this group was only 20% (Sutton et al., 2016, Cytotherapy, 18(7): 881-892). A recent report aimed at optimizing NK cell transduction using VSVg-pseudotyped lentivirus reported a transduction efficiency of 11.8% for activated cells at a viral MOI of 10 and went on to compare various transduction enhancers to enhance this efficiency, including commonly used enhancers such as vectofusin, dextran, and PGE2. They concluded that of all the enhancers screened, statins resulted in the greatest enhancement of transduction efficiency, with 33.3% transduction efficiency obtained with rosuvastatin (Gong et al., 2020, Mol Ther Methods Clin Dev, 17: 634-646). In both cases, the use of high viral MOIs and / or transduction enhancers imposes expected costs on the quality and quantity of the cell product. Notably, statins have been found to be inhibitory to NK cell cytotoxicity. In addition, the need for large batches of virus (for high MOI transduction) and access to transduction enhancers leads to high costs and pressures on the cell therapy manufacturing supply chain.

[0009] This highlights the unmet need for efficient and cost-effective lentiviral transduction methods of innate-like lymphocytes that do not compromise cell quality or quantity. Summary of the invention

[0010] The present invention provides methods for efficiently introducing genetic material into innate-like lymphocytes using a lentiviral vector that has been pseudotyped with an RD144-HIV chimeric envelope protein (also referred to as "RD-Pro"). The inventors have found that chimeric RD144-HIV pseudotyped lentivirus is unexpectedly effective in delivering transgenes to stimulated innate-like lymphocytes at low MOIs, and has unexpectedly favorable cell viability and proliferation. As previously described, VSVg-based transduction protocols may require an MOI of up to 80. In contrast, chimeric RD144-HIV pseudotyped lentiviral vectors can achieve approximately 70% transduction efficiency at an MOI as low as 1.25, thus requiring significantly lower viral titers (up to 60 times lower viral requirements). In addition, transduction with chimeric RD144-HIV pseudotyped viruses has been shown to exhibit a small fraction of toxicity, thereby producing higher levels of healthier and more effective cell products. Thus, the methods of the present invention enable a more cost-effective product manufacturing process than standard lentiviral protocols, which further results in cells of better quality.

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

[0012] The use of an alternative RD114-derived lentiviral pseudotype for γδ T cell transduction, namely RD114TR-pseudotyped lentivirus (WO 2019 / 104269), was previously described. Interestingly (and in contrast to our findings with the RD114-HIV chimeric envelope protein, or "RD-Pro"), VSVg-pseudotyped lentivirus resulted in higher transgene expression at day 5 post-transduction compared to RD114TR-pseudotyped lentivirus. This hints at the fact that not all RD114-derived lentiviral envelopes confer similar enhancements to innate lymphoid cell transduction as RD-Pro envelopes.

[0013] Innate-like lymphocytes can be 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 inducer (LTi) cells, intraepithelial lymphocytes (IELs), innate lymphoid cells (ILCs), such as group 1 innate lymphoid cells, group 2 innate lymphoid cells, group 3 innate lymphoid cells, B1 cells, marginal zone B cells and / or cells expressing CD8αα.

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

[0015] Innate-like lymphocytes can be γδ T cells, natural killer (NK) cells, invariant natural killer T (iNKT) cells, mucosa-associated invariant T (MAIT) cells, lymphoid tissue inducer (LTi) cells, intraepithelial lymphocytes (IELs), innate lymphoid cells (ILCs), such as group 1 innate lymphoid cells, group 2 innate lymphoid cells, group 3 innate lymphoid cells, B1 cells, marginal zone B cells and / or cells expressing CD8αα.

[0016] Intrinsic lymphocytes can be γδ T cells, natural killer (NK) cells, mucosa-associated constant T (MAIT) cells, lymphoid tissue induction (LTi) cells, intraepithelial lymphocytes (IEL), intrinsic lymphoid cells (ILC), including group 1 intrinsic lymphoid cells, group 2 intrinsic lymphoid cells, group 3 intrinsic lymphoid cells, B1 cells, marginal zone B cells and / or cells expressing CD8αα. In some embodiments, intrinsic lymphocytes are γδ T cells, such as Vδ2+ T cells.

[0017] In some embodiments, the innate-like lymphocyte is a γδ T cell, such as a Vδ1+ T cell.

[0018] In some embodiments, the innate-like lymphocyte is a γδ T cell, such as a non-Vδ1+ / Vδ2+ T cell.

[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 a method for introducing nucleic acid into innate-like lymphocytes using a lentiviral vector at a low multiplicity of infection (MOI).

[0023] In some embodiments, the nucleic acid is introduced into the innate-like lymphocytes by a lentiviral vector 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 the innate-like lymphocytes by a lentiviral vector described herein in the absence of a transduction booster.

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

[0026] For example, the gene may encode a naturally occurring or synthetic protein or chimeric molecule of immunological or metabolic activity of human or non-human origin. The encoded protein may be expressed within the cell, on the cell membrane, or secreted from the cell.

[0027] In some embodiments, the lentiviral vectors described herein comprise genes encoding a chimeric antigen receptor (CAR), a chimeric co-stimulatory 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, a scFv-Fc fusion protein (SFP), an antibody, a DARPIN, a nanobody, a triabody, a diabody, 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 proximal and / or distal membrane intracellular enzyme, such as a phosphatase, a tyrosine kinase, a serine threonine kinase, a protease, a matrix metalloproteinase, and / or a degron.

[0028] The present invention provides a method for introducing a nucleic acid into an innate-like lymphocyte, comprising the following steps:

[0029] (i) obtaining an innate-like lymphocyte population; and

[0030] (ii) incubation of innate-like lymphocytes with lentiviral vectors;

[0031] The lentiviral vector comprises RD114-HIV chimeric envelope protein.

[0032] The method can be performed in the absence of a transduction enhancing agent.

[0033] The innate-like lymphocyte population can be isolated from a subject, for example from blood or a tissue-derived product.

[0034] The method of the invention may further comprise a step of stimulating, such as activating and / or expanding, the innate-like lymphocytes before and / or during incubation with the lentiviral vector.

[0035] Intrinsic-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.

[0036] The present invention provides a population of genetically engineered innate-like lymphocytes obtained or obtainable by a method of the present invention.

[0037] The present invention provides compositions, such as pharmaceutical compositions, comprising a population of innate-like lymphocytes according to the present invention.

[0038] Intrinsic-like lymphocytes can be genetically modified to enhance their anti-tumor properties (e.g., cytotoxicity and cytokine production), proliferation, persistence, metabolic adaptability (e.g., resistance to hypoxia or glucose starvation), ability to distinguish between healthy and malignant tissues, anti-inflammatory properties, and / or resistance to chemotherapeutic drugs.

[0039] For example, innate-like lymphocytes can be genetically modified to express chimeric antigen receptors (CAR), chimeric costimulatory receptors, dual CAR, three CAR, tethered CAR, non-signaling CAR, truncated CAR, T cell receptor (TCR), TCR co-expressed with CAR, scFv-Fc fusion protein (SFP), antibody, DARPIN, nano antibody, tribody, bibody, bispecific T cell engager (BiTE), transcription factor, intracellular signaling molecule and / or mediator, cytokine, chemokine, integrin, lectin, adhesion molecule, cell surface receptor, cell surface ligand, glucose transporter, ion transporter, near membrane and / or far membrane intracellular enzyme, such as phosphatase, tyrosine kinase, serine threonine kinase, protease, matrix metalloproteinase and / or degradation determinant. The present invention provides a method for treating or preventing a disease in a subject, comprising administering to a subject an innate-like lymphocyte group or composition according to the present invention.

[0040] The present invention provides an innate-like lymphocyte population or a composition according to the present invention for use in treating or preventing a disease in a subject.

[0041] The present invention provides an innate-like lymphocyte population or composition according to the present invention for use in immunotherapy.

[0042] The present invention provides use of the innate-like lymphocyte population or composition according to the present invention in the preparation of a medicament for treating or preventing a disease in a subject.

[0043] The innate-like lymphocytes can be allogeneic.

[0044] The disease may be cancer; an autoimmune disease or immune pathology; an infectious disease, such as a bacterial, fungal or viral infection; associated with organ transplantation, such as GvHD; or a wound, ulcer or abscess.

[0045] These and other aspects and embodiments of the invention are described in further detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 – (A) Transduction efficiency of CD3+ / Vδ2+ and CD3+ / Vδ2- cells at day 7 post-transduction, compared with RD114-HIV chimeric envelope (also called “RD-Pro”) pseudotyped lentivirus and VSVg pseudotyped lentivirus (both at an MOI of 15), using eGFP expression as a reporter of transduction. (B) Live Vδ2+ cells per ml at day 7 post-transduction under the same conditions as in (A). (C) Live Vδ2+ cells expressing GFP per ml at day 7 post-transduction under the same conditions as in (A) and (B).

[0047] Figure 2 – (A) Flow cytometric analysis of CD3 and Vδ2 expression at D4, D8 and D11 after transduction with RD114-HIV chimeric envelope ('RD-Pro') pseudotyped lentivirus. (B) GFP expression in CD3+ Vδ2+ cells. (C) Percentage of Vδ2+ cells expressing GFP at different MOIs at days 4, 8 and 11 after transduction.

[0048] Figure 3 – (A) Example of the method used to expand and transduce a mixed population of γδ T cells. (B) Transduction efficiency of Vδ1+, Vδ2+, and Vδ1- / Vδ2- cells 12 days after transduction at an MOI of 4 with RD114-HIV chimeric envelope (“RD-Pro”) pseudotyped lentivirus containing a bicistronic expression cassette containing an eGFP reporter.

[0049] Figure 4– (A) Transduction efficiency of Vδ2+ cells transduced 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, and the other lentivirus (104) encodes a bicistronic construct containing eGFP and luciferase. (B) Vδ2+ transduction efficiency (% GFP expressing cells) after transduction with 14G2a-SFP lentivirus as in (A). (C) Percentage of viable cells that are Vδ2+ / GFP+ cells after transduction as in (A). (D) Flow cytometric analysis of CD3 and Vδ2 expression in viable cells transduced with 14G2a-SFP lentivirus as in (A). (E) Detection of anti-GD2 opsonins in supernatants of γδ T cells transduced with lentivirus encoding 14G2a-SFP as in (A) showing the relationship between viral MOI and the amount of opsonin detected by binding to GD2+ target cells.

[0050] Figure 5 – (A) Similar to γδ T cells described above, expanding NK cells (CD3-CD56+) were transduced with RD114-HIV chimeric envelope protein (“RD-Pro”) pseudotyped lentivirus at an MOI of 4 and in the absence of a transduction booster on day 2 after stimulation. NK cells were transduced with a bicistronic GFP secreting mitogen construct and GFP expression was measured on day 14 of expansion. Representative data from 3 different donors are shown, with an average transduction efficiency of 55%. (B) Efficient NK cell transduction can also be achieved using different activation methods. Representative transduction efficiency data are shown for untransduced cells from one donor or cells transduced with two different RD-Pro pseudotyped bicistronic GFP mitogen constructs. NK cells were transduced as above at an MOI of 4 in the absence of a transduction booster on day 2 after culture initiation.

[0051] Figure 6 – (A and B) Example expansion schemes for γδ T cells. (C) Transduction efficiency (GFP transgene expression) of Vδ1+, Vδ2+, and Vδ1- / Vδ2- cells from different expansion schemes 12 days after transduction with RD-Pro pseudotyped lentivirus at MOI 4.

[0052] Figure 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 at day 14 of expansion. Representative data from 3 different donors are shown. (B) High transduction was maintained at low MOI using large bicistronic and immunocompetent tricistronic vectors when NK cells were activated using Miltenyi Biotec’s proprietary, feeder-free, CD2 / NKp46 GMP-compliant NK cell manufacturing kit.

[0053] Figure 8 – (A) Transduction efficiency (GFP expression) of NK-T cells in zoledronic acid-activated PBMCs transduced with MOI 4 RD-Pro lentivirus at day 14. (B) High NK-T cell transduction was maintained at low MOI using large bicistronic and immunocompetent tricistronic vectors when NK cells were activated using Miltenyi Biotec’s proprietary, feeder-free, CD2 / NKp46 GMP-compliant NK cell manufacturing kit. DETAILED DESCRIPTION

[0054] Intrinsic-like lymphocytes

[0055] The present invention relates to a method for introducing a nucleic acid into an innate-like lymphocyte using a lentiviral vector comprising a RD114-HIV chimeric envelope protein.

[0056] As used herein, the term "innate-like lymphocytes" refers to lymphocytes that exhibit innate-like immune properties. Antigen recognition and effector functions of innate immune cells are typically mediated by germline-encoded receptors. In contrast, adaptive immune responses rely on gene rearrangements and somatic hypermutations to generate different antigen recognitions. It is believed that innate-like lymphocytes bridge the gap between innate and adaptive immunity, for example, innate-like lymphocytes can express semi-constant antigen receptors.

[0057] Examples of innate-like lymphocytes include 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 inducer (LTi) cells, intraepithelial lymphocytes (IELs), innate lymphoid cells, such as group 1 innate lymphoid cells, group 2 innate lymphoid cells, group 3 innate lymphoid cells, B1 cells, marginal zone B cells and / or cells expressing CD8αα.

[0058] In some embodiments, the innate-like lymphocytes do not express the αβ T cell receptor.

[0059] In some embodiments, the innate-like lymphocyte is not a canonical αβ T cell.

[0060] In some embodiments, the innate-like lymphocytes express a semi-constant αβ T cell receptor.

[0061] γδ T cells represent a subset of T cells that express the γδ TCR instead of the αβ TCR. Human γδ T cells normally make up only 1-5% of circulating T lymphocytes but undergo rapid expansion in response to tumors, inflammation, and invading pathogens.

[0062] γδ T cells can be divided into two major subsets - tissue-bound Vδ1-positive cells and peripheral circulating Vδ2-positive cells. The Vδ2 chain is usually paired with the Vγ9 chain, and Vγ9 / Vδ2 T cells account for 50-95% of peripheral T cells among the 1-5% of T cells in the circulation. Vδ1 γδ T cells represent the major T cell subset in solid tissues and are the second most common subset in peripheral blood after Vγ9 / Vδ2 T cells (accounting for 1-3% of lymphocytes). Vδ1 T cells are mainly found in mucosal epithelial tissues and account for approximately 40% of all intraepithelial lymphocytes in the large intestine. Both Vδ1 and Vδ2 subsets have been shown to have antiviral and antitumor activities. Unlike conventional αβ TCR-expressing cells, γδ TCR-expressing cells do not rely on classical MHC I and II to recognize their targets.

[0063] Similar to natural killer (NK) T cells, γδ T cells express NKG2D, which binds to nonclassical MHC molecules (i.e., MHC class I polypeptide-related sequence A (MICA) and MHC class I polypeptide-related sequence B (MICB)) presented on stressed cells and / or tumor cells. γδ TCRs recognize a variety of ligands, such as stress and / or tumor-associated phosphoantigens. γδ T cells directly mediate cytolysis of targets through a variety of mechanisms (i.e., secretion of TRAIL, FasL, perforin, and granzymes). In addition, CD16-expressing γδ T cells enhance antibody-dependent cell-mediated cytotoxicity (ADCC).

[0064] In some embodiments, the innate-like lymphocytes according to the present invention are γδ T cells.

[0065] In some embodiments, the innate-like lymphocytes according to the present invention are Vδ2+ T cells, such as Vγ9 / Vδ2 T cells.

[0066] In some embodiments, the innate-like lymphocytes are Vδ1+γδ T cells.

[0067] In some embodiments, the innate-like lymphocytes are non-Vδ1+ / Vδ2+ γδ T cells, such as Vδ3+, Vδ4+, Vδ5+, Vδ6+, Vδ7+, or Vδ8+ T cells.

[0068] The δ-chain can be paired with any suitable γ-chain (eg, Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, or Vγ9).

[0069] Natural killer cells can be identified by the presence of CD56 and the absence of CD3. NK cells comprise 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 anti-tumor activity mediated by receptors including NKG2D, NKp44, NKp46, NKp30, and DNAM, and can eliminate virus-infected cells via CD16-mediated ADCC.

[0070] Natural killer T (NKT) cells share the characteristics of both T cells and natural killer cells. NKT cells can be identified by the presence of both CD3 and CD56 and αβ TCR. NKT cells account for about 1% of all peripheral blood T cells. Constant natural killer T cells (also referred to as "type I" NKT cells) are NKT cell subsets expressing constant TCR α chains and a limited number of non-constant TCR β chains (semi-constant TCR). In humans, highly conserved TCRs are usually obtained by pairing the Va24-Ja18 segment with the Vb11 chain. In contrast, type II NKT cells (also referred to as diversified NKT or dNKT) use diversified TCR α and β chains.

[0071] 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 a constant or semi-constant αβ T cell receptor (TCR). They comprise approximately 0.1% of T cells in human peripheral blood. In contrast to classical T cells, iNKT cells recognize lipids and glycolipids presented by CD1d, a non-polymorphic MHC protein expressed on intestinal epithelial cells. iNKT cells mediate their effector functions primarily through the rapid release of cytokines upon activation, including Th1 (IFN-γ and TNF-α), Th2 (IL-1, IL-4, and IL-13), and Th17 (IL-17, IL-22) cytokines.

[0072] In some embodiments, the innate-like lymphocytes according to the present invention are natural killer (NK) cells.

[0073] In some embodiments, the innate-like lymphocytes according to the present invention are natural killer T (NKT) cells.

[0074] In some embodiments, the innate-like lymphocytes according to the present invention are invariant natural killer T (iNKT) cells.

[0075] Mucosa-associated invariant T (MAIT) cells play an important role in the defense of mucosal surfaces against bacteria. They express a constant TCR alpha chain and a variable but restricted TCR beta chain. MAIT cells are mainly found in mucosal tissues, such as in the liver, lungs, mesenteric lymph nodes, and intestinal epithelium. In human peripheral blood, MAIT cells account for approximately 1-10% of the total number of T lymphocytes. MAIT cells express CD161, interleukin 18 receptor, and chemokine receptors CCR5, CXCR6, and CCR6 on the cell surface; and can be activated in a TCR-independent manner by TCR or IL-12 and IL-18. MAIT cells are able to release IFN-γ, TNF-α, and IL-17 in response to stimulation, and have cytotoxic activity against infected cells mediated by granzyme b and perforin.

[0076] In some embodiments, the innate-like lymphocytes according to the present invention are MAIT cells.

[0077] Intrinsic lymphoid cells (ILC) are a general term for cells with lymphoid morphology that do not contain rearranged antigen receptors and lack bone marrow-specific phenotypic markers. They do not directly recognize antigens, but respond to changes in cytokine expression profiles as a result of infection. ILCs are mainly tissue-resident cells, present in both lymphoid and non-lymphoid tissues, and rarely present in peripheral blood. They are particularly abundant on mucosal surfaces and play a key role in mucosal immunity and homeostasis.

[0078] According to the differences in developmental pathways, phenotypes and signaling molecules, ILCs are generally divided into three groups. Group 1 ILCs (ILC1) contain NK cells, are responsive to cytokines such as 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 such as IL-4, IL-5 and IL-13. Group 3 ILCs (ILC3) express RORγt and respond to IL-23 and IL-1β by producing IL-22 and IL-17.

[0079] Lymphoid tissue induced (LTi) cells have many similar features to ILC3s, however, they are generally considered a separate lineage due to their unique developmental pathways. Like ILC3s, LTi cells are also dependent on RORγt. They participate in the formation of secondary lymph nodes and Peyer's patches by promoting lymphoid tissue development. Activated LTi cells primarily produce IL-17A, IL-17F, and IL-22.

[0080] In some embodiments, the innate-like lymphocytes according to the present invention are innate lymphoid cells, such as Group 1 innate lymphoid cells, Group 2 innate lymphoid cells, Group 3 innate lymphoid cells and / or LTi cells.

[0081] Intraepithelial lymphocytes (IELs) refer to lymphocytes present in mucosa, such as in the epithelial layer of the gastrointestinal tract and reproductive tract. IELs can be divided into different subgroups according to the expression of TCR and CD8. About 10-15% of IELs express γδ TCR.

[0082] In some embodiments, the innate-like lymphocytes according to the present invention are intraepithelial lymphocytes.

[0083] 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, however homodimers of CD8α chains are also expressed on 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.

[0084] In some embodiments, the innate-like lymphocytes according to the present invention express CD8αα.

[0085] Intrinsic-like lymphocytes also include B cells expressing semi-constant B cell receptors, such as B1 cells or marginal zone B cells. According to the expression of CD5, the B1 cell population includes two subgroups: B1a cells (CD5+) and B1b cells (CD5-). B1 cells usually express polyreactive IgM antibodies encoded by germline, and the use of V gene segments is restricted. Marginal zone B cells are present in the spleen, they surround follicles, and are therefore often exposed to blood-borne antigens. After exposure to antigens, marginal zone B cells can present antigens and promote T cell activation, or can differentiate into plasmablasts. Marginal zone B cells express surface IgM, complement receptors CD35 and CD21, and lipid antigen presenting molecules CD1d.

[0086] In some embodiments, the innate-like lymphocyte according to the present invention is a B1 cell or a marginal zone B cell.

[0087] The present invention also provides a population of genetically engineered innate-like lymphocytes obtained or obtainable by the methods described herein.

[0088] The present invention provides a population of innate-like lymphocytes, wherein the innate-like lymphocytes have been genetically engineered using a lentiviral vector comprising a RD114-HIV chimeric envelope protein.

[0089] The innate-like lymphocyte population or composition according to the present invention may comprise 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 108 cells, or at least about 10 9 cells, or at least about 10 10 cells or at least about 10 11 cells.

[0090] A population or composition of innate-like lymphocytes according to the 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% of innate-like lymphocytes that have been genetically engineered according to the methods of the invention.

[0091] Lentiviral vectors

[0092] Lentiviral vectors are part of a larger group of retroviral vectors. In short, lentiviruses can be divided into primate groups and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype "lentivirus" visna / maedi virus (VMV), as well as the related caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and the recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0093] The lentivirus family differs from retroviruses in that lentiviruses are able to infect both dividing and non-dividing cells. In contrast, other retroviruses (e.g., MLV) are unable to infect non-dividing cells or slowly dividing cells, such as those that make up, for example, muscle, brain, lung, and liver tissue.

[0094] As used herein, a lentiviral vector is a vector comprising at least one component that can be derived from a lentivirus. Preferably, the component is involved in the biological mechanism by which the vector carrying the gene of interest infects or transduces a target cell.

[0095] The term encompasses, for example, lipid-encapsulated nano- and microparticles, vesicles, and virus-like particles.

[0096] In one embodiment, the lentiviral vector is derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or Visna lentivirus.

[0097] The basic structure of retroviral and lentiviral genomes share many common features, such as the 5' LTR and 3' LTR. Between or within these genes are packaging signals that allow the genome to be packaged, primer binding sites, integration sites that allow the genome to integrate into the host cell genome, and gag, pol, and env genes that encode packaging components - these are polypeptides required for the assembly of viral particles. Lentiviruses also have additional features, such as the rev and RRE sequences in HIV, which enable efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of infected target cells.

[0098] In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). LTRs are responsible for the integration and transcription of the provirus. LTRs can also serve as enhancer-promoter sequences and can control the expression of viral genes.

[0099] The LTR itself is an identical sequence that can be divided into three elements: U3, R, and U5. U3 is derived from a unique sequence at the 3' end of the RNA. R is derived from a repeated sequence at both ends of the RNA. U5 is derived from a unique sequence at the 5' end of the RNA. The sizes of these three elements can vary greatly in different retroviruses.

[0100] In defective retroviral or lentiviral vector genomes, gag, pol, and env may be absent or nonfunctional.

[0101] In the typical lentiviral vector described herein, at least a portion of one or more coding regions of proteins essential for viral replication can be removed from the vector. This makes the viral vector replication defective. Portions of the viral genome can also be replaced by a gene of interest to generate a vector comprising a gene of interest that is capable of transducing a target cell and / or integrating its genome into the target cell genome.

[0102] Optionally, the viral vector used in the present invention has a minimal viral genome.

[0103] "Minimal viral genome" is understood to mean that the viral vector has been manipulated to remove non-essential elements and retain essential elements to provide the functions required for infection, transduction and delivery of genes of interest to target host cells. Further details of this strategy can be found in WO1998 / 017815.

[0104] The lentiviral vectors described herein have been pseudotyped. In this regard, pseudotyping can bring one or more advantages. For example, the env gene product of HIV-based vectors will limit these vectors to infect only cells expressing a protein called CD4. However, if the env gene in these vectors has been replaced by env sequences from other enveloped viruses, they may have a wider spectrum of infection (Verma and Somia (1997) Nature 389 (6648): 239-242).

[0105] In the present invention, the Env protein is a modified Env protein, such as a mutant or engineered Env protein. Modifications may be made or selected to introduce targeting capabilities 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).

[0106] RD114-HIV chimeric envelope protein

[0107] The lentiviral vector used in the method of the present invention comprises the RD114-HIV chimeric envelope protein. In other words, the lentiviral vector of the present invention has been pseudotyped with the RD114-HIV chimeric envelope protein.

[0108] As used herein, the term "envelope protein" or "Env" refers to the env gene product. Envelope protein is a glycoprotein located on the surface of viral particles that facilitates transduction by binding to receptors on the surface of target cells and driving subsequent fusion of the target cell and viral membranes.

[0109] As used herein, the term "chimeric envelope protein" refers to an envelope protein that includes a portion of a protein from another protein. The other protein may or may not be an envelope protein. For example, a chimeric envelope protein can be produced by connecting two or more genes that originally encoded separate proteins, such as two env genes.

[0110] RD114 refers to feline leukemia virus RD114.

[0111] The RD114 receptor, the neutral amino acid transporter (RDR), is widely expressed on hematopoietic stem cells. RD114 is a non-cytotoxic envelope protein, thus allowing the development of stable lentiviral production cell lines.

[0112] An example of a chimeric RD114 envelope protein is an RD114 envelope protein comprising a cytoplasmic tail region replaced with a tail region from another viral envelope protein, such as a tail region of a murine leukemia virus (also referred to as "RD114TR"), for example as described in (WO 2019 / 104269).

[0113] RD114-HIV chimeric envelope protein refers to RD114 envelope protein that contains a portion of a protein derived from HIV, such as HIV Env cytoplasmic tail or furin cleavage site.

[0114] 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 by an HIV-1 matrix / capsid cleavage sequence, such 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.

[0115] It is known that replacement of the protease cleavage sequence of the RD114 envelope protein with the HIV matrix / capsid cleavage sequence can 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 an increase in the titer values ​​of the RD-Pro pseudotyped virus relative to the unmodified RD114 pseudotyped virus.

[0116] The RD114 envelope protein may comprise the sequence of SEQ ID NO: 1 or consist of the sequence of SEQ ID NO: 1.

[0117] The RD114 envelope protein may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:1.

[0118] SEQ ID NO: 1 (RD114 envelope protein sequence)

[0119] MKLPTGMVILCSLIIVRAGFDDPRKAIALVQKQHGKPCECSGGQVSEAPPNSIQQVTCPGKTAYLMTNQKWKCRVTPKISPSGGELQNCPCNTFQDSMHSSCYTEYRQCRRINKTYYTATLLKIRSGSLNEVQIL QNPNQLLQSPCRGSINQPVCWSATAPIHISDGGGPLDTKRVWTVQKRLEQIHKAMTPELQYHPLALPKVRDDLSLDARTFDILNTTFRLLQMSNFSLAQDCWLCLKLGTPTPLAIPTPSLTYSLADSLANASCQII PPLLVQPMQFSNSSCLSSPFINDTEQIDLGAVTFTNCTSVANVSSPLCALNGSVFLCGNNMAYTYLPQNWTRLCVQASLLPDIDINPGDEPVPIPAIDHYIHRPKRAVQFIPLLAGLGITAAFTTGATGLGVSVTQ YTKLSHQLISDVQVLSGTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYANKSGIVRNKIRTLQEELQKRRESLATNPLWTGLQGFLPYLLPLLGPLLTLLLILTIGPCVFSRLMAFINDRLNV VHAMVLAQ QYQALKAEEEAQD

[0120] The R peptide cleavage sequence may consist of or comprise the sequence VHAMVLAQ (SEQ ID NO: 2). The R peptide cleavage sequence may have up to 5, up to 4, up to 3, up to 2, up to 1 amino acid substitutions relative to SEQ ID NO: 2.

[0121] The HIV-1 matrix / capsid cleavage sequence may consist of or comprise 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, up to 1 amino acid substitutions relative to SEQ ID NO: 3.

[0122] In some embodiments, the RD114-HIV chimeric envelope protein comprises the sequence of SEQ ID NO:4.

[0123] In some embodiments, the RD114-HIV chimeric envelope protein consists of the sequence of SEQ ID NO:4.

[0124] In some embodiments, the RD114 envelope protein is 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% identical to SEQ ID NO:4.

[0125] SEQ ID NO:4 (RD-Pro envelope protein sequence)

[0126] MKLPTGMVILCSLIIVRAGFDDPRKAIALVQKQHGKPCECSGGQVSEAPPNSIQQVTCPGKTAYLMTNQKWKCRVTPKNLTPSGGELQNCPCNTFQDSMHSSCYTEYRQCRANNKTYYTATLLKIRSGSLNEVQIL QNPNQLLQSPCRGSINQPVCWSATAPIHISDGGGPLDTKRVWTVQKRLEQIHKAMHPELQYHPLALPKVRDDLSLDARTFDILNTTFRLLQMSNFSLAQDCWLCLKLGTPTPLAIPTPSLTYSLADSLANASCQII PPLLVQPMQFSNSSCLSSPFINDTEQIDLGAVTFTNCTSVANVSSPLCALNGSVFLCGNNMAYTYLPQNWTGLCVQASLLPDIDIIPGDEPVPIPAIDHYIHRPKRAVQFIPLLAGLGITAAFTTGATGLGVSVTQ YTKLSHQLISDVQVLSGTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYANKSGIVRNKIRTLQEELQKRRESLASNPLWTGLQGFLPYLLPLLGPLLTLLLILTIGPCVFSRLMAFINDRLNV SQNYPIVQ QYQALKAEEEAQD*

[0127] Those of ordinary skill in the art will recognize that some of the amino acid sequences described herein can be varied without significantly affecting the structure or function of the protein. If such sequence differences are considered, the skilled person will understand how to determine which domains are key regions that retain protein function.

[0128] It is well known that certain positions in proteins may tolerate certain amino acid variations. For example, most buried amino acid residues require nonpolar side chains, yet few side chain surface features are usually conserved.

[0129] Typical conservative substitutions are substitutions between the aliphatic amino acids Ala (A), Val (V), Leu (L) and Ile (I); interchange of the hydroxyl residues Ser (S) and Thr (T), exchange of the acidic residues Asp (D) and Glu (E), substitutions between the amide residues Asn (N) and Gln (Q), exchange of the basic residues Lys (K) and Arg (R), and substitutions between the aromatic residues Phe (F) and Tyr (Y).

[0130] Identity

[0131] As used herein, the term "identity" refers to the proportion (expressed as a percentage) of amino acids or nucleotides that are identical to a reference sequence in a polypeptide or nucleotide sequence.

[0132] The degree of sequence identity between a query sequence and a reference sequence can be determined by 1) aligning the two sequences using 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 one in which 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.

[0133] Computer programs suitable 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). The ExPASy proteomics server at www.expasy.org provides a range of different alignment tools. Another example of software that can perform sequence alignments is BLAST (Basic Local Alignment Search Tool), which is available from the National Center for Biotechnology Information webpage, which can currently be found at http: / / www.ncbi.nlm.nih.gov / and which was first described in Altschul et al. (1990) J. Mol. Biol. 215; 403-410.

[0134] Nucleic acid introduction method

[0135] The present invention provides a method for introducing nucleic acid into innate-like lymphocytes. The method comprises the following steps:

[0136] (iii) obtaining an innate-like lymphocyte population; and

[0137] (iv) incubating the innate-like lymphocytes with the lentiviral vector described herein.

[0138] In some embodiments, the method is an in vitro method.

[0139] The lentiviral vector may comprise a nucleic acid. Thus, the nucleic acid may be introduced into the innate-like lymphocytes by the lentiviral vector, for example by transduction or infection, or by endocytosis or membrane fusion of an envelope-coated lipid nano- or micro-particle, vesicle or virus-like particle.

[0140] Enveloped lentiviral particles can also deliver substances other than nucleic acids to target cells, such as peptides of interest, carbohydrates, or lipids.

[0141] The innate-like lymphocyte population can be isolated from a subject or from a sample of a subject. The sample can be a peripheral blood sample, an umbilical cord blood sample, a tumor, a stem cell precursor, a tumor biopsy, a tissue, or lymph fluid. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a tissue sample, such as skin or intestine.

[0142] Peripheral blood mononuclear cells can be collected from a subject, for example, using an apheresis machine comprising a Ficoll-Paque™ PLUS (GE Healthcare) system or another suitable device / system. Intrinsic-like lymphocytes can be purified from the collected sample, for example, using flow cytometry techniques. Umbilical cord blood cells can be obtained from umbilical cord blood during birth of the subject.

[0143] In some embodiments, innate-like lymphocytes can be isolated from a blood product (eg, a leukoreduction fluid, a leukoreduction system chamber (buffy cone), or a buffy coat).

[0144] The cells may be autologous cells.

[0145] The cells can be allogeneic (ie, derived from a donor).

[0146] Prior to and / or during incubation with the lentiviral vector, the innate-like lymphocytes may be stimulated, e.g., activated and / or expanded.

[0147] Therefore, the present invention also provides a method for introducing a nucleic acid into an innate-like lymphocyte, comprising the following steps:

[0148] (i) obtaining a population of innate-like lymphocytes, for example isolating a population of innate-like lymphocytes from a subject; and

[0149] (ii) incubating the innate-like lymphocytes with the lentiviral vector described herein;

[0150] or

[0151] (i) obtaining a population of innate-like lymphocytes, for example, isolating a population of innate-like lymphocytes from a subject;

[0152] (ii) activating and / or expanding the innate-like lymphocytes; and

[0153] (iii) incubating the innate-like lymphocytes with the lentiviral vector described herein.

[0154] In some embodiments, innate-like lymphocytes are activated and / or expanded by culturing the cells in the presence of a bisphosphonate and / or one or more cytokines.

[0155] The bisphosphonate may be zoledronate (zoledronic acid), pamidronate, alendronate, risedronate, ibandronate, incadronate, clodronate, etidronate or neridronate, a salt thereof and / or a hydrate thereof, preferably the bisphosphonate is zoledronic acid.

[0156] 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.

[0157] In some embodiments, the cells are stimulated with a bisphosphonate and IL-2.

[0158] In some embodiments, cells are stimulated with zoledronic acid and IL-2.

[0159] In some embodiments, cells are stimulated with zoledronic acid, IL-2, and IL-15.

[0160] During cell activation and / or expansion, the concentration of a bisphosphonate, such as zoledronic acid, can 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, from about 1 µM to about 70 µM, from about 1 µM to about 60 µM, from about 1 µM to about 50 µM, from about 1 µM to about 40 µM, from about 1 µM to about 30 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, from about 1 µM to about 70 µM, from about 1 µM to about 60 µM, from about 1 µM to about 50 µM, from about 1 µM to about 40 µM, from about 1 µM to about 30 µM, from about 1 From about 1 µM to about 20 µM, from about 1 µM to about 15 µM, from about 1 µM to about 10 µM, from about 1 µM to about 9 µM, from about 1 µM to about 8 µM, from about 1 µM to about 7 µM, from about 1 µM to about 6 µM, from about 1 µM to about 5 µM, from about 2 µM to about 5 µM, from about 3 µM to about 5 µM, from about 2 µM to about 5 µM, from about 2 µM to about 10 µM, from about 3 µM to about 8 µM, or from about 4 µM to about 6 µM.

[0161] In a preferred embodiment, the concentration of the bisphosphonate is 5 μM.

[0162] In a preferred embodiment, the concentration of zoledronic acid is 5 μM.

[0163] The concentration of IL-2 during cell activation and / or expansion can be from about 10 lU / ml to about 1000 lU / ml, from about 10 lU / ml to about 500 lU / ml, from about 10 lU / ml to about 400 lU / ml, from about 10 lU / ml to about 300 lU / ml, from about 10 lU / ml to about 200 lU / ml, from about 10 lU / ml to about 150 lU / ml, from about 10 lU / ml to about 100 lU / ml, from about 20 lU / ml to about 100 lU / ml, from about 30 lU / ml to about 100 lU / ml, from about 40 lU / ml to about 100 lU / ml, from about 50 lU / ml to about 100 lU / ml, from about 75 lU / ml to about 125 lU / ml, from about 20 lU / ml to about 80 lU / ml, from about 25 1U / ml to about 100 1U / ml or from about 50 1U / ml to about 150 1U / ml.

[0164] In a preferred embodiment, the concentration of IL-2 is 100 IU / ml.

[0165] The concentration of IL-15 during cell activation and / or expansion can be from about 10 ng / ml to about 1 pg / ml, from about 10 ng / ml to about 500 ng / ml, from about 10 ng / ml to about 400 ng / ml, from about 10 ng / ml to about 300 ng / ml, from about 10 ng / ml to about 200 ng / ml, from about 10 ng / ml to about 150 ng / ml, from about 10 ng / ml to about 100 ng / ml, from about 20 ng / ml to about 100 ng / ml, from about 30 ng / ml to about 100 ng / ml, from about 40 ng / ml to about 100 ng / ml, from about 50 ng / ml to about 100 ng / ml, from about 60 ng / ml to about 100 ng / ml, from about 20 ng / ml to about 80 ng / ml, from about 30 ng / ml to about 60 ng / ml or from about 50 ng / ml to about 120 ng / ml.

[0166] In a preferred embodiment, the concentration of IL-15 is 70 ng / ml.

[0167] In some embodiments, 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.

[0168] The concentration of anti-CD3 antibodies during cell activation and / or expansion 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 2.5 µg / ml, from about 0.5 µg / ml to about 2 µg / ml.

[0169] In a preferred embodiment, the concentration of the anti-CD3 antibody (eg, OKT3) is 1 μg / ml.

[0170] In some embodiments, cells are stimulated with an anti-CD3 antibody (eg, OKT3 or another) and IL-2.

[0171] In some embodiments, cells are stimulated with an anti-CD3 antibody, a bisphosphonate, and IL-2.

[0172] In some embodiments, cells are stimulated with anti-CD3 antibodies, bisphosphonates, IL-2, and IL-15. During activation and / or expansion, the seeding density of isolated cells can be from about 0.01 x 10 6 cells / cm 2 About 1x10 7 cells / cm 2 , from about 0.1x10 6 cells / cm 2 About 5x10 6 cells / cm 2 , from about 0.25x10 6 cells / cm 2 About 5x10 6 cells / cm 2 , from about 0.5x10 6 cells / cm 2 About 5x10 6 cells / cm 2 , from about 0.5x10 6 cells / cm 2 About 4x10 6 cells / cm 2 , from about 0.5x10 6 cells / cm 2 About 3x10 6 cells / cm 2 , from about 0.5x10 6 cells / cm 2 About 2.5x10 6 cells / cm 2 , from about 0.5x10 6 cells / cm 2 About 2x10 6 cells / cm 2 , from about 0.6x10 6 cells / cm 2 About 2x10 6 cells / cm 2 , from about 0.7x10 6 cells / cm 2 About 2x10 6 cells / cm 2 , from about 0.8x10 6 cells / cm 2 About 2x10 6 cells / cm 2 , from about 0.9x10 6 cells / cm 2 About 2x106 cells / cm 2 , from about 1x10 6 cells / cm 2 About 2x10 6 cells / cm 2 , or from about 1.5x10 6 cells / cm 2 About 2x10 6 cells / cm 2 .

[0173] In some embodiments, cells are stimulated, activated and / or expanded with anti-CD2 antibodies. In some embodiments, cells are stimulated, activated and / or expanded with anti-CD335 (NKp46) antibodies. In some embodiments, cells are stimulated, activated and / or expanded with anti-CD2 and anti-CD335 (NKp46) antibodies.

[0174] In a preferred embodiment, during activation and / or expansion, the seeding density of the isolated cells is 1×10 6 cells / cm 2 .

[0175] The duration of cell activation and / or expansion can be from 1 day to about 20 days, 1 day to about 15 days, 1 day to about 12 days, from 1 day to about 10 days, from 2 days to about 20 days, from 2 days to about 15 days, from 2 days to about 12 days, from 2 days to about 10 days, from 3 days to about 15 days, from about 4 days to about 15 days, from about 5 days to about 15 days, or from about 10 days to about 15 days.

[0176] In a preferred embodiment, the duration of cell activation and / or expansion is 12 days.

[0177] In some embodiments, the expansion and / or activation step includes a co-stimulatory agent. These co-stimulatory agents can 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.

[0178] Examples of agents that can be used to promote activation and / or expansion of innate-like lymphocytes can 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), concanavalin A (ConA), pokeweed (PWM), peanut protein agglutinin (PNA), soybean agglutinin (SBA), lentil (lens culinaris) agglutinin (LCA), pea (pisumsativum) agglutinin (PSA), helix pomatia (helix pomatia) agglutinin (HPA), vicia graminea (VGA) or other suitable mitogens capable of stimulating the proliferation of innate-like lymphocytes.

[0179] In some embodiments, cells expressing αβ TCR are depleted or reduced from the innate-like lymphocyte population, for example using magnetic beads coated with anti-αβ TCR antibodies.

[0180] The methods of the invention enable genetic engineering (eg, by transduction) at low MOI.

[0181] 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.

[0182] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than 5.

[0183] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than 3.

[0184] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than 2.5.

[0185] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than 2.

[0186] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than 1.5.

[0187] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than 1.25.

[0188] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of about 1.

[0189] In some embodiments, the innate-like lymphocytes are transduced with a lentiviral vector at an MOI of less than 1.

[0190] Transduction efficiency can be enhanced by using centrifugal infection (spinning) techniques and / or by performing transduction in the presence of transduction enhancers, for example, by physically reducing electrostatic repulsion between negatively charged cells and viral particles, thereby increasing cell-virion interactions.

[0191] The term "transduction enhancing agent" may be used interchangeably with "transduction boosting agent."

[0192] Suitable transduction enhancers are known to those skilled in the art and may include VectoFusin ® -1. RetroNectin ® , dextran, prostaglandin E2 (PGE2), protamine sulfate, cyclosporine A, rapamycin, and statins (such as atorvastatin, fluvastatin, simvastatin, pravastatin, and rosuvastatin).

[0193] The use of transduction boosters may be undesirable because they are often expensive, toxic to lymphocytes, and may hamper regulatory approval.

[0194] In some embodiments, the methods according to the present invention do not involve the use of additional transduction enhancing agents. In other words, the innate-like lymphocytes can be incubated with the lentiviral vector in an environment free of or substantially free of transduction enhancing agents.

[0195] Thus, in some embodiments, the present invention provides a method for introducing a nucleic acid into an innate-like lymphocyte comprising the steps of:

[0196] (i) obtaining an innate-like lymphocyte population; and

[0197] (ii) incubating the innate-like lymphocytes with the lentiviral vector described herein;

[0198] or

[0199] (i) obtaining an innate-like lymphocyte population;

[0200] (ii) activating and / or expanding the innate-like lymphocytes; and

[0201] (iii) incubating the innate-like lymphocytes with the lentiviral vector described herein;

[0202] Wherein the lentiviral vector is incubated with the innate-like lymphocytes at an MOI of less than 5, and wherein the incubating step is free of or substantially free of a transduction enhancer.

[0203] During incubation with the lentiviral vector, the seeding density of the isolated cells can be from about 0.01 x 10 6 cells / cm 2 About 1x10 7 cells / cm 2 , from about 0.1x10 6 cells / cm 2 About 5x10 6 cells / cm 2 , from about 0.25x10 6 cells / cm 2 About 5x10 6 cells / cm 2 , from about 0.5x10 6 cells / cm 2 About 5x10 6 cells / cm 2 , from about 0.5x10 6 cells / cm 2 About 4x10 6 cells / cm 2 , from about 0.5x10 6 cells / cm 2 About 3x10 6 cells / cm 2 , from about 0.5x10 6 cells / cm 2 About 2.5x10 6 cells / cm 2 , from about 0.5x10 6 cells / cm 2 About 2x10 6 cells / cm 2 , from about 0.6x10 6 cells / cm 2 About 2x10 6 cells / cm 2 , from about 0.7x10 6 cells / cm 2 About 2x10 6 cells / cm 2 , from about 0.8x10 6 cells / cm 2 About 2x106 cells / cm 2 , from about 0.9x10 6 cells / cm 2 About 2x10 6 cells / cm 2 , from about 1x10 6 cells / cm 2 About 2x10 6 cells / cm 2 or from about 1.5×10 6 cells / cm 2 About 2×10 6 cells / cm 2 .

[0204] In a preferred embodiment, the seeding density of the isolated cells during incubation with the lentiviral vector is 1×10 6 cells / cm 2 。

[0205] The cells can be incubated with the lentiviral vector at, for example, 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 at day 2 after expansion.

[0206] The duration of the incubation phase can be from 1 day to about 10 days, from 1 day to about 9 days, from 1 day to about 8 days, from 1 day to about 7 days, from 1 day to about 6 days, from 1 day to about 6 days, from 1 day to about 5 days, from 1 day to about 4 days, from about 2 days to about 10 days, from about 2 days to about 8 days, from about 2 days to about 7 days, about 3 days to about 7 days, or about 4 to about 6 days.

[0207] Throughout the incubation period, the culture medium may be removed and / or renewed, or supplemented with feed medium, for example, containing cytokines and / or other suitable agents.

[0208] Transduction efficiency

[0209] Transduction efficiency can be determined by the expression of a gene of interest in target cells (i.e., innate-like lymphocytes). The expression of a gene of interest can refer to mRNA expression and / or protein expression. For example, the expression of a protein encoded by a gene of interest can be determined by FACS analysis.

[0210] Transduction efficiency can be determined by reporter gene expression.

[0211] At day 4 after transduction, expression of the gene of interest in the innate-like lymphocytes transduced according to the methods of the present invention can be about 30% to about 100%, about 40% to about 100%, or about 50% to about 100%.

[0212] At day 8 after transduction, the expression of the gene of interest in the target cells of innate-like lymphocytes transduced according to the methods of the present invention can be about 40% to about 100%, about 50% to about 100%, or about 60% to about 100%.

[0213] At day 11 after transduction, the expression of the gene of interest in the target cells of innate-like lymphocytes transduced according to the methods of the present invention can be about 50% to about 100%, about 60% to about 100%, or about 70% to about 100%.

[0214] In some embodiments, the transduction efficiency achieved in innate-like lymphocytes transduced according to the methods of the invention is about 1, 2, 3, 4, or 5 times higher than the transduction efficiency achieved using a VSVg-pseudotyped lentivirus.

[0215] In some embodiments, at day 7 post-transduction, the transduction efficiency achieved in innate-like lymphocytes transduced according to the methods of the invention is about 2-fold higher than the transduction efficiency achieved with VSVg-pseudotyped lentivirus.

[0216] In some embodiments, at day 7 post-transduction, the transduction efficiency achieved in innate-like lymphocytes transduced according to the methods of the invention is about 4-fold higher than the transduction efficiency achieved with VSVg-pseudotyped lentivirus.

[0217] In some embodiments, the methods of the invention have reduced cytotoxicity, resulting in higher cell product yield and adaptability.

[0218] In some embodiments, the number of viable innate-like lymphocytes at day 7 post-transduction is about 2-fold higher than that achieved with VSVg-pseudotyped lentivirus.

[0219] Nucleic acid / gene of interest

[0220] The lentiviral vector used in the method of the present invention may contain a nucleic acid. The nucleic acid may encode one or more genes of interest.

[0221] Nucleic acids may comprise DNA or RNA and may be single-stranded or double-stranded. It will be appreciated by the skilled artisan that, due to the degeneracy of the genetic code, many different polynucleotides may encode the same polypeptide. In addition, it will be appreciated that the skilled artisan may use conventional techniques to make nucleotide substitutions (which will not affect the polypeptide sequence encoded by the polynucleotides of the invention) to reflect the codon usage of any particular host organism in which the polypeptides of the invention will be expressed.

[0222] The polynucleotides may be modified by any method available in the art. Such modifications may be performed to enhance the in vivo activity or lifespan of the polynucleotides of the invention.

[0223] As used herein, the term "gene of interest" refers to a nucleotide sequence having any therapeutic or diagnostic application. The term is used interchangeably with the term "nucleotide of interest".

[0224] The gene of interest may encode an immunologically or metabolically active protein. The protein may be naturally occurring or synthetic (i.e. engineered), such as a chimeric protein. The protein may be of human or non-human origin.

[0225] The protein can be expressed intracellularly, on the cell membrane, or secreted from the cell into which the gene has been introduced.

[0226] 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 delivery of inflammatory mediators to the tumor microenvironment, including but not limited to cytokines, soluble immunomodulatory receptors and / or ligands.

[0227] In some embodiments, the innate-like lymphocytes have been genetically modified to enhance their anti-tumor properties, such as cytotoxicity and cytokine production. For example, the gene of interest can encode perforin, a protease, a toxin, or a cytokine.

[0228] In some embodiments, the innate-like lymphocytes have been genetically modified to enhance their proliferation and / or persistence, for example, to disrupt signaling pathways involved in exhaustion or senescence.

[0229] In some embodiments, the innate-like lymphocytes have been genetically modified to enhance their metabolic adaptability. For example, the nucleic acid can encode a gene that confers increased resistance to hypoxia or glucose starvation.

[0230] In some embodiments, the intrinsic lymphocytes have been genetically modified to enhance their ability to distinguish between healthy and malignant tissues. For example, the gene of interest can encode a "non-gated". The intrinsic lymphocytes can be activated by a first receptor that binds to an antigen (e.g., an antigen expressed on malignant tissue). In "non-gated" cells, the binding of a second receptor to an alternative antigen (e.g., an antigen expressed on healthy tissue) is used to override the activation signal.

[0231] In some embodiments, the innate-like lymphocytes have been 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.

[0232] Thus, suitable genes of interest include, but are not limited to, sequences encoding enzymes, cofactors, cytokines, chemokines, hormones, antibodies, designed ankyrin repeat proteins (DARPins), nanobodies, triabodies, diabodies, bispecific T cell engagers (BiTEs), engineered immunoglobulin-like molecules, single chain antibodies, fusion proteins, such as scFv-Fc fusion proteins (SFPs), immune co-stimulatory molecules, immune modulatory molecules, antioxidant molecules, chimeric antigen receptors, chimeric co-stimulatory receptors, dual CARs, tria CARs, tethered CARs, non-signaling CARs, truncated CARs, T cell receptors, domain-spanning negative mutants of target proteins, toxins, conditional 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 with associated reporter groups).

[0233] In some embodiments, the gene of interest may encode a therapeutic protein or a combination of therapeutic proteins.

[0234] In the present invention, innate-like lymphocytes can be transduced with a gene of interest to genetically modify the cells to prevent or reduce the expression of the target gene. For example, in order to genetically modify the cells to make them resistant to chemotherapeutic agents and / or immune checkpoints and / or disrupt signaling pathways.

[0235] In some embodiments, the gene of interest can encode a site-directed nuclease, such as a Cas nuclease and a guide RNA (gRNA).

[0236] In some embodiments, the gene of interest (or nucleotide of interest) is a small interfering RNA (siRNA) or a micro RNA (miRNA).

[0237] Chimeric Antigen Receptor (CAR)

[0238] CARs are proteins that in their general form graft the specificity of a monoclonal antibody (mAb) onto the effector function of a cell. Their general form is a type I transmembrane domain protein with an antigen recognition amino terminus, a spacer, a transmembrane domain, all connected to a complex intracellular domain that transmits cell survival and activation signals.

[0239] The most common form of these molecules uses a single-chain variable fragment (scFv) derived from a monoclonal antibody to recognize the target antigen. ScFv is fused to the signal transduction intracellular domain through a spacer and a transmembrane domain. These molecules cause cells to activate in response to scFv recognizing its target. When T cells express such CARs, they recognize and kill target cells expressing the target antigen. Several CARs for tumor-associated antigens have been developed, and adoptive transfer methods using such T cells expressing CARs are currently in clinical trials for the treatment of various cancers.

[0240] In some embodiments, the innate-like lymphocytes have been genetically modified to express a chimeric antigen receptor (CAR), a chimeric co-stimulatory receptor, a dual CAR, a triple CAR, a tethered CAR, a non-signaling CAR, and / or a truncated CAR.

[0241] T cell receptor (TCR)

[0242] TCR is a molecule present on the surface of certain lymphocytes (such as T cells) that is responsible for recognizing antigens bound to MHC molecules. TCR heterodimers are composed of α and β chains or γ and δ chains.

[0243] Binding of TCR to antigen and MHC (peptide-MHC / pMHC) leads to the activation of lymphocytes through a series of biochemical events mediated by related enzymes, co-receptors and specialized accessory molecules.

[0244] Each chain of the TCR is a member of the immunoglobulin superfamily and possesses 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.

[0245] The variable domains of both TCR α chain and β chain have three hypervariable or complementary determining regions (CDRs). CDR3 is the main CDR responsible for recognizing and processing antigens, although it has also been shown that the CDR1 of the α chain interacts with the N-terminal portion of the antigenic peptide, while the CDR1 of the β chain interacts with the C-terminal portion of the peptide. It is believed that CDR2 recognizes MHC molecules. The framework region (FR) is located between the CDRs. These regions provide the structure of the TCR variable region.

[0246] Repertoires of TCR variable regions are generated by: combinatorial joining of variable (V), joining (J), and diversity (D) genes; and N region diversification (nucleotide insertion by deoxynucleotidyl transferase).

[0247] The α chain is formed by a recombination event between the V and J segments. The β chain is formed by a recombination event involving the V, D, and J segments.

[0248] 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 by recombination between 54 Vβ, 14 Jβ, and 2 Dβ (diversity) segments (Rowen et al.; 1996; Science; 272: 1755-1762).

[0249] The V and J (and D, as appropriate) gene segments of 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).

[0250] Methods for generating TCR and affinity-enhanced TCR are known in the art. Affinity-enhanced TCR is a TCR with enhanced affinity for peptide-MHC complexes. Methods include, for example, separating TCR genes encoding TCR from patient samples (such as patient peripheral blood or tumor infiltrating lymphocytes), and improving TCR affinity for peptide-MHC complexes by modifying TCR sequences (such as by in vitro mutagenesis and selecting affinity-enhanced (or affinity maturation) TCR). The method for identifying the best affinity TCR is related to antigen-negative humanized transgenic mice (such as TCR / MHC humanized mice, such as ABabDII mice) with multiple human TCR libraries immunized with antigens, and separating antigen-specific TCR from such immune transgenic mice is also known in the art (see, for example, Obenaus M et al., Nat Biotechnol.33 (4): 402-7, 2015).

[0251] In some embodiments, the gene of interest may encode a T cell receptor, such as an αβ T cell receptor.

[0252] Immune checkpoints

[0253] In some embodiments, the innate-like lymphocyte population or composition according to the invention has been genetically modified to render them resistant to immune checkpoints.

[0254] For example, innate-like lymphocytes can be genetically modified to prevent the expression of immune checkpoints including but not limited to PD-1, Lag-3, Tim-3, TIGIT, BTLA, CTLA-4, and combinations of these.

[0255] Chemotherapeutic agents

[0256] In some embodiments, an innate-like lymphocyte population or composition according to the invention has been genetically modified to increase its resistance to chemotherapeutic drugs.

[0257] As used herein, a chemotherapeutic entity refers to an entity that is destructive to cells, i.e., an entity that reduces cell viability. A chemotherapeutic entity may be a cytotoxic drug. Chemotherapeutic agents contemplated include, but are not limited to, alkylating agents, anthracyclines, epothilones, nitrosoureas, ethyleneimines / methylmelamine, alkyl sulfonates, alkylating agents, antimetabolites, pyrimidine analogs, epipodophyllotoxins, enzymes such as L-asparaginase; biological response modifiers such as IFNα, IL-2, G-CSF, and GM-CSF; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin, anthracenediones, substituted ureas such as hydroxyurea, methylcarbazine containing N-methylhydrazine (MIH) and procarbazine; hydrazine derivatives, adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; hormones and antagonists, including adrenocortical steroid antagonists, such as prednisone and its equivalents, dexamethasone and aminoglutethimide; progestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, etc.; estrogens, such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens, such as tamoxifen; androgens, including testosterone propionate and fluoxymesterone / equivalents; antiandrogens, such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide.

[0258] Composition

[0259] The present invention also provides a composition comprising the innate-like lymphocyte population according to the present invention.

[0260] The innate-like lymphocyte composition can be a pharmaceutical composition comprising innate-like lymphocytes as defined herein. The pharmaceutical composition can further comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition can optionally comprise one or more additional pharmaceutically active polypeptides and / or compounds. Such a formulation can be, for example, in a form suitable for intravenous infusion.

[0261] treat

[0262] The term "treatment" relates to the therapeutic use of the innate-like lymphocyte population or composition according to the present invention. Herein, the innate-like lymphocyte population or composition can be administered to a subject with an existing disease or condition to alleviate, reduce or improve at least one symptom associated with the disease and / or to slow down, reduce or block the progression of the disease. The term refers to the treatment of an existing disease or the prevention of a disease, i.e., prevention and treatment. Therefore, it should be recognized that the treatment referred to herein may be preventive and therapeutic in some embodiments.

[0263] The present invention provides a method for treating or preventing a disease in a subject, comprising administering to the subject an innate-like lymphocyte population or a composition according to the present invention.

[0264] The present invention provides an innate-like lymphocyte population or a composition according to the present invention for use in treating or preventing a disease in a subject.

[0265] The present invention provides an innate-like lymphocyte population or composition according to the present invention for use in immunotherapy.

[0266] The present invention provides use of the innate-like lymphocyte population or composition according to the present invention in the preparation of a medicament for treating or preventing a disease in a subject.

[0267] In a preferred embodiment of the present 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, the subject is a human.

[0268] In some embodiments, the innate-like lymphocytes are derived from the subject to be treated (autologous).

[0269] In some embodiments, the innate lymphocytes are derived from a donor (allogeneic).

[0270] In one embodiment, the disease is cancer.

[0271] The cancer can 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 carcinoma, non-small cell carcinoma and mesothelioma), brain cancer (e.g., glioma, astrocytoma, glioblastoma), melanoma, lymphoma, small intestine cancer (duodenal cancer and jejunal cancer), leukemia, pancreatic cancer, hepatobiliary tumors, germ cell cancer, prostate cancer, head and neck cancer, thyroid cancer and sarcoma.

[0272] The cancer can be a pediatric cancer, such as craniopharyngioma, ependymoma, Ewing's sarcoma, medulloblastoma, neuroblastoma, soft tissue sarcoma, or Wilms' tumor.

[0273] In some embodiments, the cancer is a pediatric cancer.

[0274] Treatment using the compositions and methods of the invention may also encompass targeting circulating tumor cells, tumor-derived metastases, and / or liquid tumors.

[0275] Treatment with the innate-like lymphocyte populations or compositions of the invention can help prevent the evolution of treatment-resistant tumor cells that often occurs with standard approaches.

[0276] The method and use for treating cancer according to the present invention can be combined with other cancer therapies. Specifically, the innate-like lymphocyte population or composition according to the present invention can be administered in combination with checkpoint blockade therapy, co-stimulatory antibodies, chemotherapy and / or radiotherapy, targeted therapy or monoclonal antibody therapy.

[0277] 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 immunomodulatory receptors (including but not limited to ICOS, CD137, CD27, OX-40, and GITR).

[0278] "In combination" may mean that the additional therapy is administered before, simultaneously with, or after the administration of the innate-like lymphocyte population or composition according to the invention.

[0279] In one embodiment, the disease is an autoimmune disease or immunopathology.

[0280] The autoimmune disease may be, for example, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, Crohn's disease, multiple sclerosis, psoriasis, rheumatoid arthritis, myasthenia gravis or systemic lupus erythematosus.

[0281] In one embodiment, the disease is associated with solid organ and / or hematopoietic stem cell transplantation, such as transplant rejection or graft-versus-host disease (GvHD).

[0282] In one embodiment, the disease is an infectious disease, such as a bacterial, fungal or viral infection.

[0283] In one embodiment, the disease is a wound, an ulcer or an abscess.The innate-like lymphocyte population or composition according to the invention can be used to aid wound healing.

[0284] The present invention will now be further described by way of examples, which are intended to help those of ordinary skill in the art to practice the present invention and are not intended to limit the scope of the present invention in any way.

[0285] Example

[0286] Example 1 - RD-Pro transduces Vδ2+ cells more efficiently than VSVG

[0287] PBMCs were cultured at 1x10 6 cells / cm 2 Plate in 96-well plates. Stimulate cells with 5 µM zoledronic acid and 100 IU / ml IL-2. On day 2, transduce cells with RD-Pro pseudotyped virus containing GFP or VSVg pseudotyped virus or mock transduction (no virus) at an MOI of 15. On day 4, remove 50% of the medium and refresh IL-2.

[0288] Figure 1 a shows that RD-Pro transduced CD3+Vδ2+ cells significantly more efficiently than VSVg on day 5 after transduction (day 7 of expansion). There was no difference in the transduction efficiency of RD-Pro and VSVg in CD3+Vδ2- cells.

[0289] Figure 1 b and c show that transduction with RD-Pro resulted in more viable cells and more viable cells expressing GFP per ml at day 7 of expansion (day 5 post-transduction) compared with transduction with VSVg-pseudotyped virus.

[0290] Example 2 - Highly efficient transduction at low MOI

[0291] PBMCs were cultured at 1x10 6 cells / cm 2 Cells were plated in 96-well plates and 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 vector at MOIs of 10, 5, 2.5, and 1.25.

[0292] CD3+ Vδ2+ cells were analyzed for GFP expression on days 4, 8, and 11 after transduction.

[0293] Figure 2 b shows that an MOI of 1.25 generated approximately 70% transduction efficiency at days 8 and 11 after transduction. An MOI of 10 achieved over 90% transduction efficiency at days 8 and 11 after transduction.

[0294] Figure 2 c shows the effect of a range of MOIs (1.25, 2.5, 5, and 10) on the transduction efficiency of Vδ2+ γδ T cells at days 4, 8, and 11 after transduction. Stable transduction was achieved at day 8 after transduction.

[0295] Example 3 - Transduction of Vδ1+, Vδ2+ and Vδ1- / Vδ2- γδ T cells

[0296] PBMCs were depleted of αβ T cells using magnetic bead-based separation according to the manufacturer's protocol (Miltenyi). The resulting αβ T cell-depleted PBMCs were isolated at 1×10 6 cells / cm 2 Plated in 96-well plates. Figure 3 a shows the method by which 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 bicistronic GFP-luciferase at MOI 4. The transduction efficiency of live 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 after transduction).

[0297] Figure 3 b shows the expression of GFP in Vδ1+, Vδ2+, and Vδ1- / Vδ2- γδ T cell subsets from three representative donors.

[0298] Example 4 - Transduction of Vδ2+ cells with tricistronic constructs

[0299] PBMCs were cultured at 1x10 6 Cells / cm2 were plated in 96-well plates. Cells were stimulated with 5 µM zoledronic acid and 100 IU / ml IL-2. On day 2, cells were transduced with RD-Pro pseudotyped viruses containing GFP-luciferase (104) or GFP-14G2a (14G2a) at a range of MOIs (1, 2, 4, and 10 for 14G2a, and 5, 10, and 20 for 104), where 14G2a is a secreted opsonin targeting the tumor-associated antigen GD2. IL-2 was refreshed every 2-3 days. On day 12 after transduction, transduction efficiency was assessed using flow cytometry, and supernatants of γδ T cells transduced to express 14G2a opsonin were collected for analysis of secreted protein content.

[0300] Figure 4 a shows the transduction efficiency of Vδ2+ cells transduced with lentivirus containing eGFP-14G2a or eGFP-luciferase.

[0301] Figure 4 b shows FACS analysis of GFP expression in γδ T cells transduced with lentivirus containing eGFP-14G2a at a range of MOIs.

[0302] Figure 4 c shows the percentage of live cells co-expressing TCR Vδ2 and GFP after transduction with either lentivirus. Figure 4d shows representative FACS plots of GFP and Vδ2 expression in cells transduced with eGFP-14G2a lentivirus.

[0303] Figure 4 e shows the detection of secreted anti-GD2 opsonin in the supernatant of cells transduced with eGFP-14G2a lentivirus. The opsonin was detected by incubating the supernatant with GD2+ or GD2- target cells and subsequently detecting opsonin binding using a secondary antibody. It was shown that opsonin binding was specific and the amount of opsonin produced was coordinated with the MOI of the virus used.

[0304] Example 5 - Transduction of NK cells with bicistronic constructs

[0305] PBMCs were cultured at 1x10 6 Cells were plated at 100 cells / cm2 in 96-well plates. Cells were stimulated with 5 µM zoledronic acid and 100 IU / ml IL-2. On day 2, cells were transduced with bicistronic RD-Pro pseudotyped virus containing a GFP mitogen at an MOI of 2. IL-2 was refreshed every 2-3 days. On day 14 after transduction, transduction efficiency was assessed using flow cytometry. CD3-negative and CD56-positive cells in the culture were counted as NK cells.

[0306] Figure 5 a shows the transduction efficiency of NK cells expanded from 3 independent donors, achieving an average transduction efficiency of approximately 55%.

[0307] PBMCs were depleted of αβ T cells using magnetic bead-based separation according to the manufacturer's protocol (Miltenyi). The resulting αβ T cell-depleted PBMCs were isolated at 1×10 6 cells / cm 2 Plated in 96-well plates. Figure 3 a shows the method by which 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 bicistronic GFP-luciferase at MOI 4. The transduction efficiency of live NK cells (defined as CD3- / CD56+) from three subsets was determined by flow cytometry on day 14 of expansion (day 12 after transduction).

[0308] Figure 5 b shows comparative GFP expression of one representative donor NK cells that were untransduced or transduced with two different bicistronic GFP-mitogen constructs.

[0309] Example 6 - Transduction of γδ T cells under different expansion conditions

[0310] Figure 6a shows the method described in Example 3, in which αβ-depleted PBMCs were cultured at 1×10 6 cells / cm 2 Plated in 96-well plates and stimulated with 1 μg / ml anti-CD3 antibody (OKT3) and 100 IU / ml IL-2 (OKT-3 amplified).

[0311] Figure 6 b shows a method in which non-αβ-depleted PBMCs were cultured at 1×10 6 cells / cm 2 Plated in 96-well plates and stimulated with 5 µM zoledronic acid and 100 IU / ml IL-2 (ZOL-amplified).

[0312] 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. Expression of the GFP transgene was measured in Vδ1+, Vδ2+, and Vδ1- / Vδ2- γδ T cell subsets from three independent donors ( Figure 6 c). It can be seen that RDPro effectively transduces different types of γδ T cells in different expansion schemes.

[0313] Example 7 - Efficient transduction of NK cells at low MOI

[0314] 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 when the culture was harvested on day 14 when the RD-Pro lentivirus was used (mean transduction efficiency of approximately 46%), but not when the VSVg-pseudotyped lentivirus was used (mean transduction efficiency of approximately 10%). Figure 7 a).

[0315] Maintain high transduction at low MOI using large bicistronic and immunologically active tricistronic vectors when activating NK cells using Miltenyi Biotec’s proprietary, feeder-free, CD2 / NKp46 GMP-compliant NK Cell Manufacturing Kit.

[0316] Example 8 - RD-Pro pseudotyped lentivirus transduces NK-T cells at low MOI to achieve translation-related efficiency

[0317] Zoledronic acid-activated PBMCs were transduced with RD-Pro pseudotyped lentivirus at an MOI of 4. When cultures were harvested on day 14, NK T cells (CD3+ CD56+ αβTCR+) were GFP positive ( Figure 8 a).

[0318] When activating NK cells using Miltenyi Biotec's proprietary, feeder-free, CD2 / NKp46 GMP-compliant NK cell manufacturing kit, high NK-T cell transduction is maintained at low MOI using large bicistronic and immunocompetent tricistronic vectors ( Figure 8 b).

Claims

1. A method for introducing a nucleic acid into an innate-like lymphocyte using a lentiviral vector, wherein the lentiviral vector comprises a RD114-HIV chimeric envelope protein.

2. The method of claim 1, wherein the innate-like lymphocytes are 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), innate lymphoid cells (ILCs), including group 1 innate lymphoid cells, group 2 innate lymphoid cells, group 3 innate lymphoid cells, B1 cells, marginal zone B cells and / or cells expressing CD8αα.

3. The method of claim 1 or claim 2, wherein the innate-like lymphocytes are γδ T cells. The method according to claim 3 , wherein the innate-like lymphocytes are Vδ1+γδ T cells. The method according to claim 3 , wherein the innate-like lymphocytes are Vδ2+γδ T cells. The method of claim 3 , wherein the innate-like lymphocytes are non-Vδ1+ / Vδ2+γδ T cells.

7. The method of claim 1 or claim 2, wherein the innate-like lymphocytes are NK cells.

8. The method of claim 1 or claim 2, wherein the innate-like lymphocytes are NKT cells.

9. The method according to any one of claims 1 to 8, wherein the innate-like lymphocytes are not canonical αβ T cells.

10. The method of any one of claims 1 to 9, wherein the chimeric envelope protein comprises an RD114 envelope protein, wherein the R peptide cleavage sequence is replaced with an HIV-1 matrix / capsid cleavage sequence.

11. The method of any one of claims 1 to 10, wherein the chimeric envelope protein comprises a sequence that is at least 60%, at least 70%, at least 80%, at least 90% identical to SEQ ID NO:

4.

12. The method according to any one of claims 1 to 11, wherein the nucleic acid is introduced into the innate-like lymphocytes by the lentiviral vector at a multiplicity of infection (MOI) of about 50, about 25, about 10, about 5, about 2.5 or about 1.

25.

13. The method according to any one of claims 1 to 12, wherein the nucleic acid encodes a gene of interest.

14. The method of claim 13, wherein the gene encodes an immunologically or metabolically active naturally occurring or synthetic protein or chimeric molecule of human or non-human origin that is expressed within a cell, on a cell membrane, or secreted from a cell.

15. A method according to claim 13 or claim 14, wherein the gene encodes a chimeric antigen receptor (CAR), a chimeric co-stimulatory 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, a scFv-Fc fusion protein (SFP), an antibody, a DARPIN, a nanobody, a triabody, a diabody, 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 proximal and / or distal membrane intracellular enzyme, such as a phosphatase, a tyrosine kinase, a serine threonine kinase, a protease, a matrix metalloproteinase and / or a degradation determinant.

16. A method for introducing a nucleic acid into an innate-like lymphocyte according to any one of claims 1 to 15, wherein the method comprises the following steps: (i) obtaining an innate-like lymphocyte population; and (ii) incubating the innate-like lymphocytes with a lentiviral vector; The lentiviral vector comprises RD114-HIV chimeric envelope protein.

17. The method of claim 16, wherein the incubating step is performed in the absence of a transduction enhancing agent.

18. The method of claim 16 or claim 17, wherein the innate-like lymphocyte population is isolated from blood or a tissue-derived human product.

19. The method according to any one of claims 16 to 18, wherein the innate-like lymphocyte population is stimulated, such as activated and / or expanded, before and / or during incubation with the lentiviral vector.

20. A population of genetically engineered innate-like lymphocytes obtained or obtainable by a method according to any one of the preceding claims.

21. A population of innate-like lymphocytes, wherein the innate-like lymphocytes have been genetically engineered with a lentiviral vector comprising a RD114-HIV chimeric envelope protein.

22. A composition comprising the innate-like lymphocyte population according to claim 20 or claim 21.

23. The innate-like lymphocyte population or composition according to any one of claims 20 to 22, wherein the population or composition comprises at least about 10 6 cells, at least about 10 7 cells, at least about 10 8 cells, at least about 10 9 cells, at least about 10 10 cells or at least about 10 11 cells.

24. The innate-like lymphocyte population or composition 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%, at least 90% of the innate-like lymphocytes have been genetically engineered.

25. The innate-like lymphocyte population or composition of any one of claims 20 to 24, wherein the innate-like lymphocytes are at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% γδ T cells.

26. The innate-like lymphocyte population or composition of claim 25, wherein the innate-like lymphocytes are at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% Vδ1+γδ T cells.

27. The innate-like lymphocyte population or composition of claim 25, wherein the innate-like lymphocytes are at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% Vδ2+γδ T cells.

28. The innate-like lymphocyte population or composition of claim 25, wherein the innate-like lymphocytes are at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% non-Vδ1+ / Vδ2+γδ T cells.

29. The innate-like lymphocyte population or composition of any one of claims 20 to 24, wherein the innate-like lymphocytes are at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% Natural Killer (NK) cells.

30. The innate-like lymphocyte population or composition of any one of claims 20 to 24, wherein the innate-like lymphocytes are at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% iNKT cells.

31. The innate-like lymphocyte population or composition according to any one of claims 20 to 30, wherein the innate-like lymphocytes have been genetically modified to enhance their anti-tumor properties, such as cytotoxicity and cytokine production.

32. The innate-like lymphocyte population or composition according to any one of claims 20 to 31, wherein the innate-like lymphocytes have been genetically modified to enhance their proliferation and / or persistence.

33. The innate-like lymphocyte population or composition according to any one of claims 20 to 32, wherein the innate-like lymphocytes have been genetically modified to enhance their metabolic adaptability, such as resistance to hypoxia or glucose starvation.

34. The innate-like lymphocyte population or composition according to any one of claims 20 to 33, wherein the innate-like lymphocytes have been genetically modified to enhance their ability to distinguish between healthy tissue and malignant tissue, for example via "NOT" gating.

35. The innate-like lymphocyte population or composition according to any one of claims 20 to 34, wherein the innate-like lymphocytes have been genetically modified to enhance their anti-inflammatory properties, for example via secretion of IL-10 or TGF-β.

36. The innate-like lymphocyte population or composition according to any one of claims 20 to 35, wherein the innate-like lymphocytes have been genetically modified to enhance their resistance to chemotherapeutic drugs.

37. A population or composition of innate-like lymphocytes according to any one of claims 20 to 36, wherein the innate-like lymphocytes have been genetically modified to express a chimeric antigen receptor (CAR), a chimeric co-stimulatory 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, a scFv-Fc fusion protein (SFP), an antibody, a DARPIN, a nanobody, a triabody, a diabody, 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 proximal and / or distal membrane intracellular enzyme, such as a phosphatase, a tyrosine kinase, a serine threonine kinase, a protease, a matrix metalloproteinase and / or a degradation determinant.

38. A method for treating or preventing a disease in a subject, comprising administering to the subject the innate-like lymphocyte population or composition according to any one of claims 20 to 37.

39. The method of claim 38, wherein the innate-like lymphocytes are allogeneic.

40. The innate-like lymphocyte population or composition according to any one of claims 20 to 37, for use in treating or preventing a disease in a subject.

41. The innate-like lymphocyte population or composition according to any one of claims 20 to 37, for use in immunotherapy.

42. A population or composition of innate-like lymphocytes for use according to claim 40 or claim 41, wherein the innate-like lymphocytes are allogeneic.

43. Use of the innate-like lymphocyte population or composition according to any one of claims 20 to 37 in the preparation of a medicament for treating or preventing a disease in a subject.

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

45. The method of claim 38 or claim 39, the innate-like lymphocyte population or composition for use according to any one of claims 40 to 42, or the use according to claim 43 or claim 44, wherein the disease is cancer.

46. ​​The method of claim 38 or claim 39, the innate-like lymphocyte population or composition for use according to any one of claims 40 to 42, or the use according to claim 43 or claim 44, wherein the disease is an autoimmune disease or an immune pathology.

47. The method of claim 38 or claim 39, the innate-like lymphocyte population or composition for use according to any one of claims 40 to 42, or the use according to claim 43 or claim 44, wherein the disease is an infectious disease, such as a bacterial, fungal or viral infection.

48. The method of claim 38 or claim 39, the innate-like lymphocyte population or composition for use according to any one of claims 40 to 42, or the use according to claim 43 or claim 44, wherein the disease is associated with organ transplantation.

49. The method of claim 38 or claim 39, the innate-like lymphocyte population or composition for use according to any one of claims 40 to 42, or the use according to claim 43 or claim 44, wherein the disease is a wound, an ulcer or an abscess.

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