阅读说明：本技术 用于nk细胞转导的方法 (Methods for NK cell transduction ) 是由 R·巴里 M·格兰青 W·伦格 N·默克 V·胡珀特 于 2018-12-19 设计创作，主要内容包括：本发明公开了使用假型化逆转录病毒载体颗粒或其病毒样颗粒将生物材料转移至激活的NK细胞中的体外方法,其包括a)NK细胞的激活,和b)将所述假型化逆转录病毒颗粒或其病毒样颗粒添加至所述激活的NK细胞的步骤,其中所述假型化逆转录病毒载体颗粒或其病毒样颗粒包含能够与造血细胞膜结合并融合的修饰的狒狒内源性逆转录病毒(BaEV)包膜糖蛋白,由此将生物材料转移至所述激活的NK细胞中。优选地,通过将IL-1家族细胞因子添加至NK细胞来进行NK细胞的激活。(The present invention discloses an in vitro method for transferring biological material into activated NK cells using a pseudotyped retroviral vector particle or a virus like particle thereof, comprising a) activation of NK cells, and b) a step of adding said pseudotyped retroviral vector particle or a virus like particle thereof to said activated NK cells, wherein said pseudotyped retroviral vector particle or a virus like particle thereof comprises a modified baboon endogenous retrovirus (BaEV) envelope glycoprotein capable of binding to and fusing with a hematopoietic cell membrane, thereby transferring biological material into said activated NK cells. Preferably, the activation of NK cells is performed by adding IL-1 family cytokines to NK cells.)

1. An in vitro method for transferring biological material into activated NK cells with pseudotyped retroviral vector particles or virus like particles thereof, comprising the steps of:

a) activation of NK cells, and

b) adding said pseudotyped retroviral particles or virus-like particles thereof to said activated NK cells,

wherein the pseudotyped retroviral vector particle, or virus-like particle thereof, comprises a modified baboon endogenous retrovirus (BaEV) envelope glycoprotein capable of binding to and fusing with a hematopoietic cell membrane, thereby transferring biological material into the activated NK cells.

2. The method of claim 1, wherein said NK cells are enriched for CX3CR1 negative NK cells from a sample comprising CX3CR1 positive and CX3CR1 negative NK cells prior to activation of said NK cells.

3. The method of claim 1 or 2, wherein the pseudotyped retroviral vector particle, or virus-like particle thereof, comprises at least:

a chimeric envelope glycoprotein comprising or consisting of a fusion of the transmembrane and extracellular domains of a baboon endogenous retrovirus (BaEV) envelope glycoprotein with the cytoplasmic tail domain of a Murine Leukemia Virus (MLV) envelope glycoprotein; or

A modified BaEV envelope glycoprotein, wherein the cytoplasmic tail domain lacks a fusion inhibitory R peptide.

4. The method according to any one of claims 1 to 3, wherein said activation of said NK cells is achieved by adding at least one cytokine or feeder cells or membrane granules of feeder cells to said NK cells or using NK cell activating agents.

5.The method of claim 4, wherein the at least one cytokine is IL-2 and/or IL-15.

6. The method according to any one of claims 1 to 5, wherein the activation of NK cells is achieved by adding a cytokine combination comprising at least one NK cell activating cytokine and an IL-1 family cytokine, thereby resulting in short term activation of NK cells.

7. The method of claim 6, wherein the combination of cytokines is IL2 and/or IL-15 and an IL-1 family cytokine.

8. The method of claim 7, wherein the IL-1 family cytokine is IL-18, IL-33, or IL-1 β.

9.The method according to any one of claims 1 to 8, wherein the transduction efficiency of said NK cells by said pseudotyped retroviral vector particles is at least 50%.

10. The method of any one of claims 1 to 9, wherein the biological material is one or more nucleic acids if the NK cells are transduced with the retroviral particles.

11. The method of any one of claims 1 to 10, wherein the retroviral particle, or virus-like particle thereof, is a lentiviral vector particle, or a virus-like particle thereof.

12. The method of any one of claims 1 to 11, wherein the method is performed in an automated process in a closed system.

13. A pseudotyped retroviral particle, or a virus-like particle thereof, for use in treating a human having a disorder by transferring a biological material in vivo into an activated NK cell, wherein the pseudotyped retroviral vector particle, or virus-like particle thereof, comprises a modified baboon endogenous retrovirus (BaEV) envelope glycoprotein capable of binding to and fusing with a hematopoietic cell membrane, thereby transferring the biological material into the activated NK cell.

14.A combination of compositions for treating a human having a disorder by transferring a biological material into activated NK cells in vivo, comprising:

a) a first composition comprising at least one cytokine that activates NK cells, and

b) a composition comprising a pseudotyped retroviral vector particle, or a virus-like particle thereof, wherein the pseudotyped retroviral vector particle, or virus-like particle thereof, comprises a modified endogenous retrovirus baboon (BaEV) envelope glycoprotein capable of binding to and fusing with a hematopoietic cell membrane.

15.The combination of compositions according to claim 14, wherein said first composition comprises a combination of cytokines comprising at least one NK cell activating cytokine and an IL-1 family cytokine.

Technical Field

The present invention relates generally to the field of transferring biological material into cells, and in particular to transferring biological material into activated Natural Killer (NK) cells.

Background

Natural killer cells (hereinafter also abbreviated as "NK cells") are a unique lymphocyte population capable of detecting and destroying both virally infected cells and malignant degenerating cells (also referred to as tumor cells). In addition, NK cells produce and secrete cytokines when contacted with tumor cells. These functional characteristics make NK cells an attractive drug for the treatment of cancer. Clinical trials have emphasized the potential for clinical use of NK cells (Miller et al, 2005; Rubnitz et al, 2010; Childs & Berg 2013).

The use of donor NK cells for patients ("allogeneic use") requires cell isolation procedures to separate the desired NK cell effects from the undesired, counter-directed effects of non-NK cells (e.g., T cells). These methods are well established (Leung 2014) and can be automated in closed sterile systems (Apel et al, 2013).

Genetic manipulation is a promising strategy to further improve NK cell characteristics (Childs & Carlsten, 2015) that are involved in multiple clinical implications, e.g. in cancer treatment. For example, the use of immune cells expressing Chimeric Antigen Receptors (CARs) is a promising therapeutic option for cancer, as demonstrated in clinical trials using CAR T cells. CAR NK cells may be a good alternative to CAR T cells, however, genetic manipulation of primary NK cells represents a significant technical challenge (Klingemann 2014).

Transfection methods, such as electroporation, result in efficient transgene delivery, but transgene expression is transient. Therefore, transfection of NK cells cannot be used for clinical applications when a durable effect is required. Furthermore, transfection results in a high cell death rate, which represents another major disadvantage.

On the other hand, viral vector-based NK cell transduction is one option for persistent genetic modification. Retroviral transduction of NK cells is possible but requires high viral titers while the efficiency is still low (Suerth et al, 2015). Even more importantly, due to insertional mutagenesis of retroviral vectors, clinical use of the retroviral pathway is a key safety issue related to genotoxic effects revealed in clinical studies (Aiuti & Roncarolo 2009; Aiuti et al, 2009).

However, within the group of retroviral vectors, lentiviral vectors are less genotoxic and represent a safer choice for clinical use (Montini et al, 2009; Papayannakos and Daniel, 2013). Unfortunately, even after stimulation with different cytokine combinations and using high lentiviral vector titers, only about 10% of NK cells can be transduced, which can be explained by the antiviral defense mechanisms of NK cells (Sutlu et al, 2012). This transduction efficiency can be increased to about 40% using high lentiviral vector titers and protamine sulfate and BX795 (inhibitor of TBK1/IKKe complex) (Sutlu et al, 2012). However, TBK 1is essential for mitosis, and it is generally known that BX795 causes cell cycle arrest, leading to non-proliferating cells (Pilai et al, 2015; Bai et al, 2015). Thus, key and undesirable side effects make BX795 unsuitable for clinical NK cell transduction, where stable transduction and appropriate NK cell functionality, including robust cell proliferation, are urgently needed.

In WO2013/045639a1, methods are disclosed for transducing quiescent HSCs and quiescent T and B cells using a viral vector pseudotyped with a modified baboon endogenous retrovirus (BaEV) envelope glycoprotein.

In summary, efficient and durable methods of genetic modification of NK cells and/or viral vector particle systems that can be clinically applied for therapy represent an urgent need that cannot be met by current technology.

Disclosure of Invention

Surprisingly, it was found that primary human NK cells can be efficiently transduced after activation by using retroviral vector particles, such as the lentiviral vector particles pseudotyped with modified baboon endogenous retrovirus (BaEV) envelope glycoprotein as disclosed herein, even without the need for inhibitors of NK cell anti-viral defense mechanisms, whereas resting NK cells cannot be efficiently transduced by these vector particles. Transduction is detectable even at low retroviral vector particle titers (e.g., lentiviral vector particle titers) and can reach over 90%, which is not seen with other viral vector particles reported to date. The use of low vector particle titres leads to better practicality compared to prior art methods. For example, lentiviral vector particles with a modified baboon envelope protein disclosed herein have a much higher transduction efficiency (7-10 fold higher) than vector particles with a VSV-G envelope protein. The transduction methods disclosed herein do not trigger cell death or exhibit other toxic effects on the transduced NK cells. Furthermore, this approach does not affect NK cell mitosis, so transduced NK cells are highly proliferative over a long period, at the same level as their untransduced counterparts. Taken together, the absence of undesirable side effects and their clinically compliant nature of the pseudotyped retroviral vector particles (e.g., lentiviral vector particles) used makes the methods disclosed herein unique for stable genetic modification of NK cells for therapeutic applications.

Unexpectedly, activation of human NK cells and the use of retroviral vector particles pseudotyped with the modified baboon endogenous retrovirus (BaEV) envelope glycoprotein disclosed herein resulted in a high frequency of transduced NK cells. Preferably, the NK cells are activated by soluble forms or surface-bound stimulants or by feeder cells (including parts of feeder cells such as membrane granules). More preferably, NK cells are activated by at least one growth factor, such as a cytokine, or a combination of growth factors, such as cytokines, for example, common-gamma chain cytokines, including but not limited to IL-2, IL-7, IL-15, IL-21, or IL-1 family cytokines (including but not limited to IL-1 α, IL1 β, IL-18, IL-33, IL-36, IL37, and IL 38). Even more surprisingly, the transduction efficiency can be further increased by combining different cytokines with IL-1 family cytokines, for example, IL-2 and/or IL-15 with IL-1 family cytokines (e.g., IL-18, IL-33, or IL-1 β). The addition of IL-1 family cytokines (such as IL-18, IL-33 or IL-1. beta.) to other well-known NK cell activating cytokines results in short-term activation of NK cells, allowing effective transduction of NK cells earlier than using prior art NK activation methods, i.e., in the absence of IL-1 family cytokines. This is a surprising finding: under the conditions disclosed herein, the percentage of activated NK cells transduced with the viral vector particles pseudotyped with the modified baboon endogenous retrovirus (BaEV) envelope glycoprotein disclosed herein is not only very high, but also remains stable for several weeks without reducing transgene expression. The duration of transgene expression can be maintained, for example, for 2, 4, 6, 8 or more weeks. Biological material can also be transferred to activated NK cells by virus-like particles pseudotyped with the modified baboon endogenous retrovirus (BaEV) envelope glycoprotein disclosed herein, rather than viral vector particles.

Even more surprisingly, enrichment of CX3CR 1-negative NK cells from samples comprising CX3CR 1-negative and CX3CR 1-positive NK cells prior to activation and transduction of these NK cells resulted in excellent proliferation and transduction using modified baboon endogenous retrovirus (BaEV) envelope glycoprotein pseudotyped viral vector particles as disclosed herein. Enrichment of CX3CR 1-negative NK cells can be achieved by isolating CX3CR 1-positive and CX3CR 1-negative NK cells from a sample or NK cell population comprising CX3CR 1-positive and CX3CR 1-positive cells.

The transduction methods for activating NK cells disclosed herein can also be carried out entirely as an automated process, preferably in a closed system under GMP conditions.

Drawings

FIG. 1: BaEV pseudotyped vectors allow high NK cell transduction, as demonstrated by GFP expression, but NK cells need to be activated.

FIG. 2A: CD3 used in the experiment⁺T cells and CD3^-/CD56⁺NK cells were isolated from PBMCs, resulting in highly purified cell populations.

FIG. 2B: t cell transduction with VSV-G pseudotyped lentiviral vectors was effective, but it was very ineffective against NK cells even at high titers.

FIG. 2C: transduction of NK-92 cell lines with VSV-G pseudotyped lentiviral vectors was similarly ineffective as primary NK cells, but surprisingly NK-92 could be efficiently transduced with BaEV pseudotyped vectors.

FIG. 2D: pseudotyping of lentiviral vectors with baboon envelope can very efficiently transduce both T cells and activated primary NK cells.

FIG. 3A: surprisingly, short-term activation of NK cells with IL-33 allows for a higher NK cell transduction rate than using BaEV pseudotyped vectors in the case of IL-33.

FIG. 3B: similar to the BaEV pseudotyped vectors, short-term activation of NK cells with IL-33 increased the transduction rate of NK cells even when VSV-G pseudotyped lentiviral vectors were used.

FIG. 4: in a similar manner to IL-33, activation of NK cells with IL-18 increased the transduction rate of NK cells.

FIG. 5: activated NK cells transduced with the BaEV pseudotyped vector showed the same rate of amplification as non-transduced NK cells, demonstrating that transduction itself does not induce cell death and does not affect cell proliferation.

FIG. 6: transgene expression following transduction of NK cells with BaEV pseudotyped vectors is long-term stable.

FIG. 7A: activation of NK cells and use of BaEV pseudotyping allow for very high transduction rates, which makes it possible to generate NK cells with high CAR expression.

FIG. 7B: different target cells showed different expression profiles for the surface marker CD19, making it a suitable target for functional testing of NK cells expressing CD 19-CAR.

FIG. 7C: the transduction method allows the generation of NK cells expressing CD19-CAR with unaltered high cytotoxic potential demonstrated by the killing of K562 cells and by CD19 positive RS 4; 11 and Raji cell killing demonstrated significantly improved CD 19-specific cytotoxic activity.

FIG. 8: native as well as activated NK cells were subjected to western blotting to examine the expression of baboon envelope receptor ASCT 2. Activated, but not native, NK cells expressed the receptor for baboon envelope ASCT 2.

FIG. 9A: cell proliferation and transduction assays showed that only proliferating, but not quiescent NK cells were transductable by the baboon pseudotyped lentiviral vector.

FIG. 9B: a subset of NK cells that are proliferative and transducible is CD56^{Bright Light (LIGHT)}. In contrast, the non-proliferative and non-transducible NK cell is CD56^Darkness。

FIG. 10: comparison of several NK cell receptors between the proliferating and transduced, proliferating but non-transduced and quiescent NK cell subsets showed a significantly different phenotype of quiescent NK cells compared to proliferating NK cells. Proliferating cells are more bright (bright) CD56 with a tendency to lower CD16 expression, higher expression of activation receptors NKp30, NKp44 and NKG2D and higher expression of TRAIL and NKG 2A.

FIGS. 11A-B: proliferation of NK cells and marker screening of cell surface proteins of the quiescent subset. NK cells from 5 donors were used to analyze 371 surface markers by flow cytometry. In direct comparison to resting NK cells, proliferative and GFP-positive NK cells expressed 32 markers at higher levels and 5 markers at lower levels (fig. 11A). The most obvious markers for proliferative and transducible NK cell subsets were the absence of CX3CR1 expression and high frequency of strongly TRAIL expressing cells (fig. 11A and 11B).

FIG. 12A: prior to transduction and culture, different subsets of NK cells were isolated based on CX3CR1 expression using MACS sorting. In the absence of sorting, the expression of CX3CR 1in donor-derived blood-derived NK cells was 86% and CX3CR1^negNK cells were 14%. After sorting, CX3CR1^negThe mean frequency of NK cells was reduced to 2% by enrichment for CX3CR1, while on the other hand, depletion of the marker resulted in 91% CX3CR1^negEnrichment of NK cell population.

FIG. 12B: we observed significant proliferation differences shortly after culture of differentially sorted NK cell fractions. In unsorted, CX3CR 1-enriched (CX3CR 1-positive) and CX3CR 1-depleted (CX3CR 1-negative) NK cell subsets, the percentage of NK cell proliferation was 28, 11 and 81, respectively.

FIG. 12C: NK cell CX3CR1^negThe fractions are highly transducible. Unsorted, CX3CR 12 days after transduction of sorted NK cell fractions^{Enrichment of}And CX3CR1^{Exhaustion of}The transduction rates of the NK cell subsets were 21%, 7% and 69%, respectively.

FIG. 13: ten days post lentiviral transduction of NK cells using BaEV-pseudotyped lentiviral vectors and IL-1 β expression of Chimeric Antigen Receptor (CAR).

Detailed Description

In one aspect, the present invention provides an in vitro method for transferring biological material into activated NK cells using a pseudotyped retroviral vector or a virus like particle thereof, said method comprising the steps of:

a) activation of NK cells, and

b) adding said pseudotyped retroviral vector particles or virus-like particles thereof to said activated NK cells,

wherein the pseudotyped retroviral vector particle, or virus-like particle thereof, comprises a modified baboon endogenous retrovirus (BaEV) envelope glycoprotein capable of binding to and fusing with a hematopoietic cell membrane, thereby transferring biological material into the activated NK cells.

The method, wherein the NK cell is a human NK cell.

The method, wherein the NK cells to be activated are in a sample comprising NK cells, e.g., a whole blood sample or a Peripheral Blood Mononuclear Cell (PBMC) sample (e.g., which may be obtained from the buffy coat).

The method, wherein prior to activation of said NK cells, the NK cells may be enriched for CX3CR1 negative NK cells from a population or sample comprising CX3CR1 positive and CX3CR1 negative NK cells.

Can be isolated by isolating from a sample containing CX3CR1 positive and CX3CR1 negativeThe population or sample of sex NK cells is depleted of CX3CR1 negative cells to perform said enrichment of CX3CR1 negative NK cells or of a subset/subpopulation of CX3CR1 negative NK cells, wherein the depleted fraction is the target fraction comprising CX3CR1 negative cells, e.g. isolated by isolation methods, such as cell isolation methods, e.g.,(Miltenyi Biotec GmbH), or fluorescence-activated cell sorting, such as flow cytometry using anti-CX 3CR1 antibodies or fragments thereof.

The CX3CR1 negative NK cell subset (or CX3CR1 negative NK cells) is preferentially transduced with the method as disclosed herein, since it achieves a very high transduction rate (see example 9).

The method, wherein the NK cells to be activated in step a) are CX3CR1 negative NK cells. Preferably, the CX3CR1 negative NK cells comprise less than 20%, 15%, 10%, 5% or 1% CX3CR1 positive cells in a composition comprising NK cells for activation within the methods disclosed herein.

Pseudotyped retroviral vector particles comprising a modified baboon endogenous retrovirus (BaEV) envelope glycoprotein capable of binding to and fusing with a hematopoietic cell membrane or virus-like particles thereof are well known in the art and are for example disclosed in WO2013/045639a 1.

The pseudotyped retroviral particle, or virus-like particle thereof, wherein the modified baboon endogenous retrovirus (BaEV) envelope glycoprotein capable of binding to and fusing with a hematopoietic cell membrane may be:

a chimeric envelope glycoprotein comprising or consisting of a fusion of the transmembrane and extracellular domains of a baboon endogenous retrovirus (BaEV) envelope glycoprotein with the cytoplasmic tail domain of a Murine Leukemia Virus (MLV) envelope glycoprotein; or a modified BaEV envelope glycoprotein, wherein the cytoplasmic tail domain lacks a fusion inhibitory R peptide.

The pseudotyped retroviral vector particle, or virus-like particle thereof, may comprise at least: a chimeric envelope glycoprotein comprising or consisting of a fusion of the transmembrane and extracellular domains of a baboon endogenous retrovirus (BaEV) envelope glycoprotein with the cytoplasmic tail domain of a Murine Leukemia Virus (MLV) envelope glycoprotein; or a modified BaEV envelope glycoprotein, wherein the cytoplasmic tail domain lacks a fusion inhibitory R peptide.

The method wherein said activation of said NK cells can be achieved by adding at least one cytokine or feeder cells or membrane granules of feeder cells to said NK cells or using NK cell activating agents. Generally, after 2-3 days, the effect of NK cell activation leads to NK cell proliferation.

The method, wherein the at least one cytokine is selected from the group consisting of common-gamma chain cytokines including but not limited to IL-2, IL-7, IL-15, IL-21, or IL-1 family cytokines including but not limited to IL-1a (IL-1 α), IL-1b (IL-1 β), IL-18, IL-33, IL-36, IL37, and IL 38.

The at least one cytokine may be IL-2.

The at least one cytokine may be IL-15.

The cytokine may be IL-2 and IL 15.

The method, wherein NK cells are activated by a cytokine or combination of cytokines (such as IL-2 and/or IL-15) in combination with an IL-1 family cytokine, thereby resulting in earlier activation ("short term activation") of the NK cells compared to activation in the absence of the IL-1 family cytokine.

The method, wherein activation of the NK cells is achieved by adding a combination of cytokines comprising at least one cytokine that activates NK cells and an IL-1 family cytokine, thereby resulting in short term activation of NK cells.

The method wherein the combination of cytokines is an IL-2 and/or IL-15 and IL-1 family cytokine. The use of one or more NK cell activating cytokines (e.g., IL-2 and/or IL-15) together with IL-1 family cytokines (e.g., IL-18 or IL-33) results in short term activation, which shortens the period of time between the start of a culture process, e.g., for NK cell activation, and the point of initiation that allows for efficient transduction of activated NK cells using the retroviral vector particles disclosed herein. Surprisingly, the combination of an IL-1 family cytokine with one or more NK cell activating cytokines (e.g., IL-2 and/or IL-15) and transduction with retroviral particles disclosed herein results in a higher transduction rate than retroviral particles transduced into NK cells activated in a longer process (i.e., without benefiting from a short-term activation process induced by the presence of an IL-1 family cytokine (e.g., IL-8, IL-33, or IL-1 β)).

The IL-1 family cytokine may be selected from the group consisting of IL-1 α, IL-1 β, IL-1Ra, IL-18, IL-36Ra, IL-36 α, IL37, IL-36 β, IL-36 γ, IL38 and IL-33.

Preferably, the IL-1 family cytokine may be IL-18, IL-33 or IL-1 β.

The method, wherein the IL-1 family cytokine is IL-18, thereby resulting in short-term activation within 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours (1 day), 28 hours, 36 hours, 42 hours, or 48 hours (2 days).

The method, wherein the IL-1 family cytokine is IL-33, thereby resulting in short-term activation within 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours (1 day), 28 hours, 36 hours, 42 hours, or 48 hours (2 days).

For example, addition of an IL-1 family cytokine (e.g., IL-18, IL-33, or IL-1 β) to a culture medium containing NK cells at the beginning of a cell culture system, along with other cytokines known to activate NK cells, results in activation of the NK cells as early as 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours (1 day), 28 hours, 36 hours, 42 hours, or 48 hours (2 days) after addition of the IL-1 family cytokine. Thus, addition of an IL-1 family cytokine to NK cells, preferably in cell culture medium, results in short-term activation of NK cells within 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours (1 day), 28 hours, 36 hours, 42 hours or 48 hours (2 days).

The above cytokines may be administered to the cell culture medium containing the NK cells at concentrations well known and commonly used by those skilled in the art.

The method wherein said activation of said NK cells can be achieved by adding an IL-1 family cytokine to said NK cells without other cytokines or feeder cells or membrane granules of feeder cells or with NK cell activating agents.

The method, wherein said activation of said NK cells can be achieved by adding to said NK cells an IL-1 cytokine and feeder cells or membrane granules of feeder cells or using NK cell activating agents.

The method, wherein the transduction efficiency of NK cells by said pseudotyped retroviral particle is at least 50%, at least 60%, at least 70%, at least 80% or at least 90%.

The method, wherein the biological material is one or more nucleic acids if the NK cell is transduced with the retroviral vector.

The method, wherein the retroviral vector particle, or virus-like particle thereof, is a lentiviral vector particle, or a virus-like particle thereof.

The method, wherein the method is performed in an automated process in a closed system.

In another aspect, the invention provides a pseudotyped retroviral particle, or virus-like particle thereof, for use in treating a human having a disorder by transferring a biological material in vivo into an activated NK cell, wherein the pseudotyped retroviral vector particle, or virus-like particle thereof, comprises a modified endogenous retrovirus baboon (BaEV) envelope glycoprotein disclosed herein capable of binding to and fusing with a hematopoietic cell membrane, thereby transferring the biological material into the activated NK cell.

The disorder can be a disease treatable or treatable with the NK cells.

In a further aspect, the invention provides a combination of compositions for treating a human suffering from a disorder by in vivo transfer of a biological material into activated NK cells, said combination comprising

a) A first composition comprising at least one cytokine that activates NK cells, and

b) a composition comprising a pseudotyped retroviral vector particle, or a virus-like particle thereof, wherein the pseudotyped retroviral vector particle, or virus-like particle thereof, comprises a modified endogenous retrovirus (BaEV) envelope glycoprotein of baboon as disclosed herein that is capable of binding to and fusing with a hematopoietic cell membrane.

The disorder can be a disorder that can be treated or treatable with the NK cell.

The combination of compositions, wherein the first composition comprises a combination of cytokines comprising at least one cytokine that activates NK cells and an IL-1 family cytokine.

A combination of said compositions, wherein said combination of cytokines comprises IL-2 and/or IL-5 and an IL-1 family cytokine.

The combination of compositions, wherein the IL-1 family cytokine is IL-18, IL-33, or IL-1 β.

A combination of said compositions for use in the preparation of engineered NK cells.

The combination of compositions, wherein the biological material is a nucleic acid encoding a chimeric antigen receptor.

In another aspect, the invention provides the use of a pseudotyped retroviral vector particle, or a virus-like particle thereof, for transferring biological material to activated NK cells, wherein the pseudotyped retroviral vector particle, or virus-like particle thereof, comprises a modified endogenous retrovirus baboon (BaEV) envelope glycoprotein capable of binding to and fusing with a hematopoietic cell membrane as disclosed herein.

Use of said pseudotyped retroviral vector particles or virus-like particles thereof for the preparation of engineered NK cells.

Use of the pseudotyped retroviral vector particle or a virus-like particle thereof, wherein the biological material is a nucleic acid encoding a chimeric antigen receptor.

In a further aspect, the present invention provides transduced and/or engineered NK cells obtainable by the methods disclosed herein.

The engineered NK cell, wherein the NK cell is engineered to express a chimeric antigen receptor.

In one aspect, the invention provides a pharmaceutical composition of engineered NK cells obtained by the methods disclosed herein.

The pharmaceutical composition, wherein the engineered NK cell expresses a chimeric antigen receptor.

In another aspect, the invention provides a combination of cytokines for short-term activation of NK cells comprising at least one NK cell activating cytokine and an IL-1 family cytokine.

The combination of cytokines may be IL2 and/or IL-15 and IL-1 family cytokines.

The IL-1 family cytokine may be IL-18, IL-33 or IL-1 β.

All definitions, features and embodiments defined herein in relation to one aspect of the invention (e.g. the first aspect of the invention) apply mutatis mutandis to other aspects of the invention disclosed herein.

Definition of

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The retroviral family is a viral family having a single-stranded, diploid, positive-sense RNA genome that is reverse transcribed into DNA intermediates that are subsequently integrated into the host cell genome. Viruses of the retroviral family are particles having an envelope with a diameter of 80-120 nm.

(retro-/lenti-/gammaretro-) viral vectors are replication-defective viral particles derived from the corresponding viral family. They contain Gag and Pol proteins, single-stranded RNA genomes, and are typically pseudotyped with heterologous envelope proteins derived from other viruses, e.g., with baboon endogenous retrovirus (BaEV) envelope glycoproteins disclosed herein. The RNA genome of the viral vector does not contain any viral genes to produce viral progeny, but contains the psi elements and LTRs required for efficient packaging and reverse transcription in DNA. The DNA intermediate may contain a gene of interest under the control of a suitable promoter (e.g., CMV promoter), and the gene of interest is expressed when the DNA is integrated into the genome of the host cell. The process of entry into a host cell, delivery of the RNA genome, integration and expression of a target gene is called transduction. The minimal requirement for gamma retrovirus or lentivirus based viral vectors has been well described in the art.

In addition, integrase-deficient retroviral vectors (ID-RV) have been developed that are incapable of integrating the retroviral vector genome in the host cell genome. ID-RV is derived from a conventional retroviral vector, but does not contain retroviral integrase or contains a mutant form of retroviral integrase. Upon entry into a host cell, the retroviral vector genome is reverse transcribed in the cytoplasm and delivered to the nucleus, but is not stably integrated into the host cell genome. ID-RV is a useful tool for transient expression of a gene of interest. The definition of retroviral vectors and transduction also extends to integration defective retroviral vectors and uses thereof.

Lentiviruses are a genus of the family retroviridae that cause chronic and fatal diseases (characterized by long latency periods) in human and other mammalian species. The most famous lentivirus is the human immunodeficiency virus, HIV, which can efficiently infect non-dividing cells, and thus lentivirus-derived retroviral vectors are one of the most efficient methods of gene delivery.

The gammaretroviridae family is a genus of the family retroviridae. Representative species are murine leukemia virus and feline leukemia virus.

Virus-like particles (VLPs) are similar to virus particles, but do not infect or transduce, as they do not contain viral genetic material encoding the proteins of the virus-like particles. In particular, VLPs in the case of retroviral vectors do not comprise psi positive nucleic acid molecules. Some virus-like particles may contain nucleic acids that are different from their genomes. Expression of viral structural proteins (such as the envelope or capsid) can lead to assembly of virus-like particles (VLPs). As with retroviral vectors, VLPs can also be pseudotyped using the same envelope constructs as used for retroviral vectors. VLPs can be used to deliver proteins, and nucleic acids can be delivered to the cytoplasm of target cells. In particular, VLPs are useful as vaccines.

The term "VLP uptake" as used herein refers to binding of a VLP to a target cell membrane, thereby releasing a nucleic acid molecule, protein or peptide into a target cell.

The term "activation" as used herein refers to the induction of a physiological change by a cell that enhances the function, proliferation and/or differentiation of the target cell.

The term "pseudotyped" or "pseudotyped" as used herein refers to a vector particle bearing envelope glycoproteins derived from other viruses with an envelope. The host range of the lentiviral vectors or vector particles of the invention can thus be extended or altered depending on the type of cell surface receptor used for the glycoprotein.

The gag, pol and env proteins required to generate retroviral vector assembly vector particles are supplied in trans by a packaging cell line, such as HEK-193T. This is typically accomplished by transfecting the packaging cell line with one or more plasmids containing the gag, pol, and env genes. For the generation of pseudotyped vectors, the env genes (which were originally derived from the same retrovirus as the gag and pol genes and as the RNA molecule or expression vector) were exchanged for envelope proteins of a different enveloped virus.

The baboon endogenous retrovirus or BaEV is a type C retrovirus that is present in multiple proviral copies in baboon DNA. In WO2013045639a1, the wild-type BaEV envelope glycoprotein (unmodified BaEV envelope glycoprotein) and the BaEV envelope glycoprotein with defined mutations (modifications) are described in detail, which integrate on the lentiviral surface at a higher level than the wild-type BaEV glycoprotein.

The term "BaEV envelope glycoprotein" as used herein refers to a wild-type form of a BaEV envelope glycoprotein or a mutant of said wild-type BaEV envelope glycoprotein which is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to said wild-type BaEV envelope glycoprotein, provided that said mutant glycoprotein retains the ability of the wild-type glycoprotein to bind to and fuse with a hematopoietic cell membrane.

Preferably, the wild-type BaEV envelope glycoprotein consists of the sequence SEQ ID NO: 1. As known to those skilled in the art, BaEV envelope glycoproteins are composed of a cytoplasmic tail domain, a transmembrane domain, and an extracellular domain. The regions of the envelope glycoprotein sequence corresponding to the cytoplasmic tail domain, transmembrane domain and extracellular domain can be readily determined by the skilled artisan. Typically, the cytoplasmic tail domain is located between amino acids 530 to 564 of the wild-type BaEV envelope glycoprotein. Typically, the transmembrane domain is located between amino acids 507 to 529 of the wild-type BaEV envelope glycoprotein. Typically, the extracellular domain is located between amino acids 1 to 506 of the wild-type BaEV envelope glycoprotein.

In a particular embodiment of the invention, the cytoplasmic tail domain of the BaEV envelope glycoprotein lacks a fusion inhibitory R peptide.

In the context of the present invention, the expression "fusion inhibiting R peptide" refers to the C-terminal part of the cytoplasmic tail domain of the envelope glycoprotein, which carries the tyrosine endocytosis signal-YXXL- (SEQ ID NO:2) and which is cleaved by viral proteases during virion maturation, thus enhancing the membrane fusion of the envelope glycoprotein. The fusion inhibitory R peptide of the BaEV envelope glycoprotein is typically located between amino acids 547 to 564 of the wild-type BaEV envelope glycoprotein.

Thus, in a particularly preferred embodiment, the modified BaEV envelope glycoprotein in which the cytoplasmic tail domain lacks the fusion inhibitory R peptide comprises or consists of the amino acid sequence SEQ ID No. 3.

In another specific embodiment, the cytoplasmic tail domain of the BaEV envelope glycoprotein is replaced by the cytoplasmic tail domain of the Murine Leukemia Virus (MLV) envelope glycoprotein.

The murine leukemia virus envelope glycoprotein is preferably the envelope glycoprotein of strain 4070A.

In the context of the present invention, the term "MLV envelope glycoprotein" refers to a wild-type form of the MLV envelope glycoprotein or a mutant of said wild-type MLV envelope glycoprotein which is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to said wild-type MLV envelope glycoprotein, with the proviso that said mutant glycoprotein retains the ability of the wild-type envelope glycoprotein to interact with a viral core protein, in particular with a lentiviral core protein.

The region of the envelope glycoprotein sequence corresponding to the cytoplasmic tail domain can be readily determined by the skilled artisan. Typically, the cytoplasmic tail domain of the MLV envelope glycoprotein is located between amino acids 622 and 654 of the wild-type MLV envelope glycoprotein.

Thus, in a particularly preferred embodiment, a chimeric envelope glycoprotein comprising or consisting of a fusion of the transmembrane and extracellular domains of a BaEV envelope glycoprotein with the cytoplasmic tail domain of an MLV envelope glycoprotein comprises or consists of the amino acid sequence of SEQ ID No. 4.

As contemplated herein, the expression "biological material" refers to one or more compounds that are susceptible to altering the structure and/or function of a cell. In the context of the present invention, it is preferred that the biological material is one or more nucleic acids, which in the case of lentiviral vector particles may be comprised within the genome of the vector particle.

As contemplated herein, "transfer" refers to the ability of the carrier particle or carrier-like particle to initially deliver the biological material to the membrane or cytoplasm of a target cell upon binding to the target cell. In this context, the target cells are typically NK cells.

The term "natural killer cell (NK cell)" as used herein is typically a human NK cell and is defined as a Large Granular Lymphocyte (LGL) and a third class of cells that constitute differentiation from a common lymphoid progenitor-producing B and T lymphocytes. NK cells are known to differentiate and mature in bone marrow, lymph nodes, spleen, tonsils and thymus, where they subsequently enter the circulation. NK cells differ phenotypically, by origin and by corresponding effector function from natural killer T cells (NKTs); in general, NKT cell activity promotes NK cell activity by secreting IFN γ. In contrast to NKT cells, NK cells do not express the T cell antigen receptor (TCR) or pan T marker CD3 or the surface immunoglobulin (Ig) B cell receptor, but they typically express the surface markers CD16(Fc γ RIII) and CD56 in humans and NK1.1 or NK1.2 in C57BL/6 mice. Up to 80% of human NK cells also express CD 8. Continuously growing NK cell lines can be established from cancer patients and common NK cell lines are e.g. NK-92, NKL and YTS.

The term "titer" or "transduction efficiency" is used as a means to characterize and compare vector particles with respect to their ability to transduce their target cells. Thus, a vector particle with "increased titer" or "increased transduction efficiency" is able to transduce a higher number of cells at a given amount of vector particle compared to other vector particles with the same amount.

The term "activated NK cell" as used herein refers to a change in a cell as compared to a native, freshly isolated, non-activated NK cell by stimulation conditions, thereby presenting itself with an altered gene expression profile or altered cell signaling, resulting in NK cells with different cellular characteristics as compared to non-activated NK cells. An "activated NK cell" may, for example, increase the expression of certain surface receptors, such as NKp44, NKG2D, DNAM-1 or TRAIL, or alter the expression of cytokine receptors, for example, up-regulate CD 25. Activated NK cells may change their cell cycle phase from resting cells in G0 phase to G1, S or G2 phase, or may start to proliferate or proliferate faster.

The term "CX 3CR1 negative NK cells" or "CX 3CR1^negNK cells "or" CX3CR1^-NK cells "or" CX3CR1 depleted NK cells "or" CX3CR1^{Exhaustion of}NK cells "refers to a population of NK cells that do not express the marker CX3CR1 on their cell surface.

CX3C chemokine receptor 1(CX3CR1), also known as fractalkine receptor or G-protein coupled receptor 13(GRP13), is a protein encoded by the CX3CR1 gene in humans. As the name suggests, this receptor binds the chemokine CX3CL1 (also known as neuronectin or fractalkine).

The term "short-term activation" as used herein refers to the possibility of activating NK cells more rapidly by adding an Il-1 family cytokine, e.g. to a cell culture medium comprising NK cells to be activated, compared to standard NK cell activation using e.g. a different cytokine known to activate NK cells but without Il-1 family cytokines. In the presence of the IL-1 family, NK cell activation has occurred after 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours (1 day), 28 hours, 36 hours, 42 hours, or 48 hours (2 days).

The term "NK cell activating agent" refers to a molecule such as a particle, bead or nanomatrix to which has been coupled one or more stimulating agents that provide an activation signal to NK cells (e.g., stimulating antibodies to CD2 and CD 335). Examples of such NK cell activating reagents are the NK cell activation/amplification kit from Miltenyi Biotec (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, order No.130-094-483) or the nanomatrix described in EP2824112B 1.

The interleukin-1 family (IL-1 family) is a group of 11 cytokines that play an important role in regulating immune and inflammatory responses to infection or sterile invasion.

The IL-1 family cytokines are IL-1 α, IL-1 β, IL-1Ra, IL-18, IL-36Ra, IL-36 α, IL-37, IL-36 β, IL-36 γ, IL-38, and IL-33. As disclosed herein, the addition of IL-1 family cytokines to standard (prior art) NK cell activation processes using, for example, other cytokines accelerates NK cell activation, resulting in short-term activation of NK cells.

The IL-1 family of cytokines may also be variants thereof having some amino acids deleted, added or substituted while still retaining the function of the cytokine. Thus, included in this definition are variants of the wild-type cytokine amino acid sequence having at least 70%, or at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity at the amino acid sequence level. In the context of the present invention, "sequence identity" can be determined using pairwise alignments using alignment programs for amino acid sequences well known in the art.

The IL-1 family cytokine may also be a functional fragment of a full-length cytokine that has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity at the amino acid sequence level to the corresponding portion of the full-length cytokine ("IL-1 family cytokine or functional fragment thereof").

Generally, all amino acid variations (i.e., substitutions, additions or deletions of amino acids of an Il-1 family cytokine) are included under this definition that do not result in a loss of properties of the Il-1 family cytokine disclosed herein.

The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence in a cell driven by its promoter.

The term "feeder cells" refers to cells that are added to a culture of target cells (i.e., natural killer cells herein) to support their survival and/or growth. Feeder cells provide the complete and functional extracellular matrix and matrix-related factors, and secrete known and unknown cytokines into the conditioned medium. Feeder cells are usually growth arrested to prevent their proliferation in culture, but maintain their survival. Growth arrest may be achieved by irradiation with an effective dose or treatment with an effective dose of a chemical substance, such as mitomycin C. There are several feeder cells including irradiated peripheral blood mononuclear cells (hereinafter also abbreviated as "PBMC"), NK cell-depleted PBMC, cancer cell lines, genetically engineered cancer cell lines, and lymphocytes immortalized by natural infection with Epstein-Barr virus (hereinafter also abbreviated as "EBV").

As used herein, the term "culturing" includes providing the chemical and physical conditions (e.g., temperature, gas), and growth factors required for NK cell maintenance. Typically, culturing NK cells includes providing conditions for expansion (proliferation) of NK cells. Examples of chemical conditions that can support NK cell expansion include, but are not limited to, buffers, serum, nutrients, vitamins, antibiotics, cytokines, and other growth factors, which are routinely provided in (or can be manually administered to) cell culture media suitable for NK cell expansion. In one embodiment, the NK cell culture medium comprises NK MACS Research medium (Miltenyi Biotec GmbH) supplemented with 5% human serum type AB (Life Technologies), 500U/mL IL-2(Miltenyi) and 10ng/mL IL-15 (Miltenyi). Other media suitable for expanding NK cells are known in the art.

As used herein, the term "cell culture medium" includes liquids that provide the chemical conditions required for NK cell maintenance. Examples of chemical conditions that can support NK cell expansion include, but are not limited to, solutions, buffers, serum components, nutrients, vitamins, cytokines, and other growth factors that are routinely provided in (or can be manually administered to) cell culture media. Suitable media for culturing NK cells as known in the art include NK MACS (Miltenyi), TexMACS (Miltenyi), CellGro SCGM (CellGenix), X-Vivo 10, X-Vivo 15, BINKIT NK Cell InitialMedium (Cosmo Bio USA), AIM-V (Invitrogen), DMEM/F12, NK Cell Culture Medium (uplicate technologies). The terms "IL-2" and "IL-15" as used herein refer to the cytokines interleukin-2 and interleukin-15 and derivatives thereof, e.g., IL-2 superfine, IL-2 diphtheria toxin fusion protein, or IL-15 Ra sushi.

The terms IL-2 and/or IL-15 generally refer to members of the cytokine 4 α -helix bundle family that bind to the heterotrimeric receptors for IL2 and IL15, sharing a common γ chain and IL2/IL15R β (also known as IL2R β, CD 122).

As used herein, the term "adding (repeatedly) an effective concentration of IL-2 and/or IL-15" refers to an IL-2 concentration in the cell culture medium of from 1U/mL to 5000U/mL, preferably from 10U/mL to 1000U/mL, more preferably from 50 to 500U/mL, and/or to an IL-5 concentration in the cell culture medium of from 0.1 to 1000ng/mL, preferably from 1 to 200ng/mL, more preferably from 10 to 100 ng/mL. Typically, the concentration of IL-2 and/or IL-15 decreases over time during the culture process, so IL-2 and/or IL-15 can be repeatedly added to the cell culture medium to maintain the level of cytokines at an effective concentration. Conventionally, IL-2 and/or IL-15 can be added again to the cell culture by medium exchange. In this case, "repeatedly" means at least one repetition throughout the cultivation. It is not necessary to add IL-2 and/or IL-15 at the beginning of the culture process, but at least at the first medium change, i.e.after 7 days. However, it is nevertheless advantageous to add IL2 and/or IL-15 at the beginning of the cultivation process.

As used herein, the term "expansion" or "proliferation" refers to the growth of cells and the multiplication of cell numbers. As used herein, expansion or proliferation involves an increase in the number of NK cells that occurs during culture.

As used herein, the term "culturing process" or "culturing" refers to the culture and expansion of NK cells, wherein the starting day (starting point) of the culturing process (i.e., NK cell expansion) is defined as day 0. The culturing process may be continued as long as possible according to the needs of the operator and may be performed as long as the cell culture medium has conditions that allow the cells to survive and/or grow and/or proliferate.

As used herein, the term "initiation of a culture process" refers to the initiation of an expansion process by the addition of growth factors (e.g., IL-2 and/or IL-15) to the cell culture medium, i.e., the initiation of cells to initiate their proliferation process. Addition of IL-1 family cytokines (e.g., IL-18 or IL-33) accelerates the process of NK cell activation and provides for efficient transduction, therefore IL-1 family cytokines should be administered to the medium along with other well-known NK cell activating cytokines. Preferably, an effective concentration of an IL-1 family cytokine is added to the cell culture medium only once. Alternatively, an effective concentration of an IL-1 family cytokine is repeatedly added (e.g., twice or more frequently) to the cell culture medium.

As used herein, the term "(repeatedly) adding an effective concentration of an IL-1 family" means that the concentration of the IL-1 family cytokine in the cell culture medium is from 1U/mL to 5000U/mL, preferably from 10U/mL to 1000U/mL, more preferably from 50 to 500U/mL. The term "membrane granules of feeder cells" as used herein refers to a membrane preparation of feeder cells containing NK cell stimulating surface molecules of the feeder cells. Membrane particles of feeder cells can be used to avoid the possible disadvantages of live feeder cells. Feeder cell membrane particles can be generated by lysing and destroying cells using nitrogen cavitation, followed by density gradient centrifugation to separate and purify cell membrane fractions as exemplarily described by Oyer et al (Oyer 2015). Generally, in the methods disclosed herein, feeder cells can be replaced with membrane particles of the feeder cells. Preferably, the membrane particles are used in amounts equivalent to those obtainable by the use of live feeder cells. Preferably, the concentration of membrane particles used may be between 100 and 400. mu.g/mL, more preferably between 150 and 250. mu.g/mL. The addition of membrane particles can be done at the same frequency as the addition of live feeder cells.

As used herein, the terms "engineered cell" and "genetically modified cell" (herein specifically NK cells) may be used interchangeably. The term denotes an inclusion and/or a tableExpressing an exogenous gene or nucleic acid sequence that, in turn, alters the genotype or phenotype of the cell or its progeny. The term especially refers to the fact that cells can be manipulated by recombinant methods well known in the art to stably or transiently express peptides or proteins that are not expressed in these cells in their native state. The term "closed system" as used herein refers to any closed system that reduces the risk of contamination of the cell culture while performing the culturing process (e.g., introducing new materials and performing cell culturing steps, such as proliferation, differentiation, activation and/or isolation of cells). Such systems allow for manipulation under GMP or GMP-like conditions ("sterility") to obtain clinically useful cell compositions. Exemplary use of CliniMACS herein(Miltenyi Biotec GmbH, Germany) as closed system. This system is disclosed in WO 2009/072003. It is not intended to limit the use of the method of the present invention toProdigy. The term "automated method" or "automated process" as used herein refers to any process that is automated through the use of equipment and/or computers and computer software that would otherwise or may be performed manually by an operator. The already automated method (process) requires less manual intervention and takes less human time. In some cases, the method of the present invention is automated if at least one step of the method is performed without any manual support or intervention. Preferably, the methods of the present invention are automated if all of the steps of the methods disclosed herein are performed without human support or intervention. Preferably, the automated process is asProdigy is performed on a closed system. The CAR comprises a single chain variable fragment (scFv) of an antibody specific for a particular target antigen, coupled to the cytoplasmic domain of a cell signaling molecule through a hinge and a transmembrane region. The most common form of stranguriaThe basophil activating moiety includes a cellular co-stimulatory (e.g., CD28, CD137, OX40, ICOS, and CD27) domain in tandem with a cellular triggering (e.g., CD3 ξ) moiety CAR-mediated adoptive immunotherapy allows CAR-transplanted cells to directly recognize a desired antigen on target cells in a non-HLA restricted manner.