Patent Application
20220025327
The present invention in general relates to a method of differentiating hematopoietic stem cells (HSC) into mature natural killer (NK) cells; wherein said method is in particular characterized in that mature NK cells are obtainable very early during the differentiation method and, in addition, have enhanced antibody-dependent cellular cytotoxic (ADCC) activity (
Innate lymphoid cells (ILC) are a novel lymphoid cell subfamily belonging to the innate immune system. The different ILC are developmentally related and characterized by a lymphoid morphology, the lack of gene-dependent rearrangement of antigen receptors and the absence of myeloid and dendritic phenotypical markers. Like helper T-cell subsets, ILC can be divided into three different groups according to distinct phenotypes, cytokine-secretion profiles and essential transcription factors [1,2].
Natural killer (NK) cells, which are considered as the prototypical ILC, are important cytotoxic cells [1]. They provide wide anti-tumor and anti-microbial protection upon activation by the release of cytolytic granules containing perforin and granzyme B. Besides cytotoxic effects, NK cells also contribute to immunomodulation by producing cytokines, including IFN-γ [3,4]. NK cells, like other lymphocytes, originate from CD34+ hematopoietic stem cells (HSC) in the bone marrow that differentiate through a common lymphoid progenitor stage. In secondary lymphoid tissues, human NK cell development is pursued whereby the cells sequentially develop into stage 1 (CD34+CD45RA+CD117−CD94−) pro-NK cells, followed by stage 2 or pre-NK cells (CD34+CD45RA+CD117+CD94−). Stage 1 and stage 2 cells are multipotent as they have T-cell, dendritic cell and NK cell developmental potential. Stage 3 cells (CD34−CD117+CD94−CD16−) are committed NK cell precursors since they can no longer develop into T-cells and dendritic cells. Stage 4 (CD34−CD56brightCD94+CD16−) and stage 5 (CD34−CD56dimCD94+CD16+) are mature NK cells [5,6]. Differentiation and maturation of NK cells and ILC is a complex molecular process tightly regulated by transcription factors. Many essential factors have been identified in the transcriptional control of murine ILC differentiation, thanks to the generation and availability of transcription factor-deficient mice [7]. In contrast to mice, the current knowledge on the role of transcription factors in human NK and ILC differentiation is extremely limited.
T-bet and Eomesodermin (Eomes) are two T-box transcription factors. T-bet is a protein encoded by the Tbx21 gene that is only expressed in hematopoietic cells. Eomes also plays an important role in vertebrate embryogenesis and shares homology with T-bet. T-bet is known as a master regulator essential for T-cell effector functions, including IFN-γ production and cytotoxicity. Moreover, T-bet and Eomes play a critical role in differentiation, maintenance and function of murine NK cells and ILC [8]. T-bet-deficient mice and Eomesflox/flox Vav-Cre+ mice show decreased numbers of NK cells that mainly have an immature phenotype [9,11]. Mice lacking both T-bet and Eomes completely fail to develop NK cells [11]. These knockout mouse models show that both T-bet and Eomes are indispensable for NK cell development and terminal NK cell maturation. Furthermore, T-bet and Eomes are needed to maintain a mature NK cell phenotype, highlighted by the loss of maturity markers after induced deletion of T-bet/Eomes in mature NK cells [10]. Next to NK cells, particular subsets of ILC depend on T-bet and/or Eomes for their development. CD127+ ILC1 and natural cytotoxicity receptor (NCR)+ ILC3 express T-bet but lack Eomes. Eomesflox/flox Vav-Cre+ mice have decreased numbers of NK cells but maintain ILC1. In contrast, T-bet-deficient mice have fewer NK cells, but completely lack ILC1. Mice lacking both T-bet and Eomes show a complete lack of ILC1. Also, no NCR+ ILC3 develop in the intestine of T-bet-deficient mice [9-12].
Because of their anti-tumor role, NK cells are abundantly researched as promising agents for cancer immunotherapy. Of the different NK cell-based therapeutic strategies one is the adoptive transfer of HSC into cancer patients. The other is to first differentiate HSC in vitro into mature NK cells that are then expanded to obtain sufficient NK cell numbers for transplantation. Whereas different approaches using NK cells in cancer therapy have already been used in the clinic, there still are some major limitations leading to relapse. Analysis of adoptively transferred mature NK cells in different murine tumor models revealed an exhaustion of the transferred NK cells, resulting in decreased cytotoxicity and IFN-γ production [13]. Importantly, this exhausted NK cell phenotype could be attributed to downregulation of the transcription factors T-BET and EOMES [13]. More recently, research proved that reduced T-BET and EOMES expression is also responsible for the NK cell functional impairment after HSC transplantation in leukemia patients. Reduction of T-BET and EOMES expression is already observed early after HSC transplantation. Downregulation of these transcription factors in NK cells is associated with increased nonrelapse mortality [14]. The role of thymocyte selection-associated HMG box protein (TOX) on the differentiation of human NK cells has been studied by Yun et al. [15] and in WO2012/046940. Vong et al. (2014) disclose that another member of the thymocyte selection-associated HMG box protein family, i.e. TOX2, is required in normal maturation of human NK cells and directly relates to T-BET expression [16]. CAR-T cells overexpressing T-BET are disclosed in WO2017/190100 and by Gacerez and Sentman [17].
Here, we reveal a method to accelerate human NK cell maturation from umbilical cord blood HSC in vitro, using retroviral constitutive overexpression constructs of either T-BET or EOMES. Whereas control transduced HSC require a culture period of 14 to 21 days to differentiate into mature functional NK cells, NK cells already appear on day 3 of culture with T-BET- or EOMES-transduced HSC. These early arising NK cells have a fully mature phenotype and are also highly functional regarding specific cytotoxicity and IFN-γ production. Importantly, the NK cells also display enhanced ADCC activity. This accelerated NK cell differentiation and maturation of NK cells with enhanced ADCC activity upon T-BET or EOMES transduction of HSC can provide a novel tool to optimize the NK cell-based adoptive cell therapies.
In a first aspect, the present invention provides an ex vivo method of differentiating hematopoietic stem cells (HSC) into mature natural killer (NK) cells, said method comprising the steps of:
whereby said mature NK cells are obtainable from day 3, in particular from day 4 or 5, after the start of step d).
In a specific embodiment of the present invention, said mature NK cells are at least of stage 4, in particular at stage 4 and stage 5 NK cells. At least from 5 days after transfection or transduction, stage 4 NK cells are present and/or can be obtained. At least from 9 days after transfection or transduction, stage 5 NK cells are present and/or can be obtained.
In another particular embodiment, said medium of step b) is complete Iscove's Modified Dulbecco's Medium (IMDM medium), in particular comprising about 1 to 20% fetal calf serum (FCS).
In yet a further embodiment of the present invention, said TPO is present at a concentration from about 1 ng/ml to about 100 ng/ml; preferably about 20 ng/ml.
In a still further embodiment, said SCF is present at a concentration from about 5 ng/ml to about 500 ng/ml; preferably about 100 ng/ml.
In another embodiment, said FLT3-L is present at a concentration from about 5 ng/ml to about 500 ng/ml; preferably about 100 ng/ml.
In yet a further embodiment of the invention, said medium of step d) further comprises a cytokine selected from the list comprising FLT3-L, SCF, IL-3 or IL-7.
In another particular embodiment, said IL-2 and/or IL-15 is present at a concentration from about 0.5 ng/ml to about 50 ng/ml; preferably about 10 ng/ml.
In a further embodiment, step d) of the method of the present invention is a co-culturing step using an (inactivated) feeder cell line, in particular a stromal cell line, such as e.g. using EL08.1D2 cells or OP9 cells.
In a further embodiment of the method of the present invention, in step c) said cells are transduced with a (retroviral) vector comprising a nucleic acid encoding said at least one transcription factor.
In a further aspect, the present invention provides HSC cells which are characterized in that they have been transfected and/or transduced with at least one transcription factor selected from the list comprising: T-Box expressed in T cells (T-BET), Eomesodermin (EOMES) or a combination of T-BET and EOMES.
The present invention also provides differentiated NK cells obtained using the method according to this invention; more in particular differentiated NK cells whereby CD16 expression of said NK cells is increased compared to non-transfected or non-transduced control cells, or to control transfected or control transduced cells.
The present invention also provides the differentiated NK cells as disclosed herein for use in inducing antibody-dependent cellular cytotoxicity in a subject having cancer.
Cord blood-derived HSC are transduced with cDNA encoding the human transcription factors T-BET or EOMES and are cultured in vitro in the NK cell differentiation culture. T-BET and EOMES overexpression in HSC leads to a drastic acceleration of NK cell maturation and the NK cells display increased CD16 (FcγRIII)-expression and antibody-dependent cellular cytotoxicity (ADCC).
As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The term “about” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/−20% or less, preferably +1-10% or less, more preferably +/−5% or less, of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed. Whereas the terms “one or more” or “at least one”, such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.
In a first aspect, the present invention provides a method of differentiating hematopoietic stem cells (HSC) into mature natural killer (NK) cells, said method comprising the steps of:
whereby said mature NK cells are obtainable from day 3 after the start of step d).
In a further aspect, the present invention provides a method of differentiating hematopoietic stem cells (HSC) into mature natural killer (NK) cells, said method comprising the steps of:
In a specific embodiment, human HSC are purified from cord blood and precultured for 2 days in the presence of FLT3L, SCF and TPO to induce proliferation, which enhances the transduction efficiency. Thereafter, cells are transduced with retroviral supernatant of the LZRS virus, containing the encoding cDNA of either TBET and EOMES. The viral construct also contains the EGFP reporter gene, that enables selection of the transduced cells by flow cytometric sorting 1-2 days after transduction. The retroviral transduction results in integration in the DNA of the host cell and in constitutive overexpression (significant) higher expression of the encoded protein as compared to the control transduced cells (displayed as mean fluorescence intensity (MFI)); or when the basal level of protein expression is exceeded) of the encoded protein, as measured by flow cytometric analysis. The negative control vector only contains EGFP. The transduced cells are then cultured on the EL08-1D2 stromal cell line, in the presence of FLT3L, SCF, IL-3, IL-7 and IL-15. In this condition, NK cell differentiation starting from HSC is enabled.
In the context of the present invention, the term “hematopoietic stem cells (HSCs)” is meant to be stem cells which give rise to blood cells during the process called hematopoiesis. The HSC of the present invention may be obtained/isolated from any suitable sample, such as for example from umbilical cord blood as further described in the examples part, or alternatively from placenta, placental blood, placental perfusate, peripheral blood, bone marrow, thymus, spleen, or liver. Enrichment of the cell population for HSCs may for example be done by cell sorting on the basis of CD34 expression, since CD34 is known to be a HSC marker. Hematopoietic cells used in the methods provided herein can be obtained from a single individual, e.g., from a single placenta, or from a plurality of individuals, e.g., can be pooled. Where the hematopoietic cells are obtained/isolated from a plurality of individuals and pooled, the hematopoietic cells may be obtained from the same tissue source. Thus, in various embodiments, the pooled hematopoietic cells are all from placenta, e.g., placental perfusate, all from placental blood, all from umbilical cord blood, all from peripheral blood, and the like.
In the context of the present invention, the term “differentiating” is meant to be a process in which a cell changes from one cell type to another. By using the method of the present invention, HSCs can be changed into mature natural killer cells, during such differentiation process. Specifically, production of NK cells by the present method comprises expanding a population of hematopoietic stem cells. During cell expansion, a plurality of hematopoietic stem cells within the hematopoietic cell population differentiate into NK cells.
“Natural killer cells” or “NK cells” are a type of cytotoxic lymphocytes which are critical to the innate immune system. In the human body, NK cells for example provide rapid response to viral-infected cells, and respond to tumor formation. Differentiation of NK cells in vitro is a complex process regulated by transcription factors, and often a very time consuming process as well. In addition, CD16 expression of in vitro differentiated NK cells is relatively low, resulting in low antibody-dependent cellular cytotoxic (ADCC) capacity. The method of the present invention provides a solution to these problems in that mature NK cells can be obtained much more rapidly compared to prior art known differentiation methods (e.g. after about 3-7 days vs 14-21 days of culture; in particular after 3, 4, 5, 6 or 7 days of culture; or in the alternative after 5, 6, 7, 8 or 9 days after transfection or transduction of the cells e.g. as in step (c) described herein). In addition, the thus obtained NK cells display about 2-10 fold, in particular about 2-5 fold, more in particular about 2.5- to 4.5-fold higher CD16 expression (as compared to control cells), resulting in increased ADCC activity. The thus obtained NK cells are thus highly suitable in human medicine, such as in anti-cancer therapy or NK cell-based adoptive cell therapies.
Hence, the present invention also provides differentiated NK cells whereby CD16 expression of said NK cells is increased compared to non-transfected or non-transduced control cells, or to control transfected or control transduced cells. The present invention also provides differentiated NK cells as defined herein, for use in inducing antibody-dependent cellular cytotoxicity in a subject having cancer.
In the context of the present invention, “thrombopoetin (TPO)” is a protein, which is also known as megakaryocyte growth and development factor. In the human body, it is produced by the liver and kidney and regulates the production of platelets. In a specific embodiment of the present invention, said TPO is present in the medium (such as e.g. used in step b) at a concentration from about 1 ng/ml to about 100 ng/ml; more specifically, from about 5 ng/ml to about 50 ng/ml; more in particular from about 10 ng/ml to about 30 ng/ml; in particular about 15 ng/ml, about 20 ng/ml or about 25 ng/ml.
In the context of the present invention, “stem cell factor (SCF)”, also known as KIT-ligand, is a cytokine that plays an important role in hematopoiesis. In the present context, SCF contributes to self-renewal and maintenance of HSCs. In a specific embodiment of the present invention, said SCF is present in the medium (such as e.g. used in step b) at a concentration from about 5 ng/ml to about 500 ng/ml; more specifically, from about 50 ng/ml to about 200 ng/ml; more in particular from about 90 ng/ml to about 110 ng/ml; in particular about 90 ng/ml, about 100 ng/ml or about 110 ng/ml. SCF may also be used as an additional interleukin in the medium used in the culturing step d), where it may then be present at a concentration from about 1 ng/ml to about 100 ng/ml; more specifically, from about 5 ng/ml to about 50 ng/ml; more in particular from about 10 ng/ml to about 30 ng/ml; in particular about 15 ng/ml, about 20 ng/ml or about 25 ng/ml.
In the context of the present invention, FLT3-ligand (FLT-3-L), also known as FMS-like tyrosine kinase 3 ligand, is an endogenous small molecule that functions as a cytokine and growth factor that increases the number of immune cells by activating the hematopoietic progenitors. In a specific embodiment of the present invention, said FLT3-L is present in the medium (such as e.g. used in step b) at a concentration from about 5 ng/ml to about 500 ng/ml; more specifically, from about 50 ng/ml to about 200 ng/ml; more in particular from about 90 ng/ml to about 110 ng/ml; in particular about 90 ng/ml, about 100 ng/ml or about 110 ng/ml. FLT3-L may also be used as an additional interleukin in the medium used in the culturing step d), where it may then be present at a concentration from about 1 ng/ml to about 50 ng/ml; more specifically, from about 5 ng/ml to about 25 ng/ml; more in particular from about 5 ng/ml to about 15 ng/ml; in particular about 5 ng/ml, about 10 ng/ml or about 15 ng/ml.
In the context of the present invention, the term “transfecting or transfection” is meant to be a process for deliberately introducing naked or purified nucleic acids, such as vectors (DNA or RNA) or mRNA molecules into eukaryotic cells. The term “transducing or transduction” is meant to be a type of transfection process using virus-mediated gene transfer, e.g. by using a retroviral or lentiviral vector. In the context of the present invention, any suitable method for transfection/transduction of HSC cells may be used, such as electroporation, calcium phosphate transfection or RetroNectin-mediated transduction, as further detailed in the examples herein after.
Key to the current invention, is the transfection or transduction of T-BET and/or Eomesodermin (EOMES) transcription factors in HSCs, which leads to a significant reduction in time of the differentiation process into mature NK cells, and which also leads to increased CD16 expression in the thus obtained NK cells, resulting in increased ADCC.
T-BET (or ‘T-Box expressed in T cells’) is a transcription factor involved in the regulation of developmental processes, more specifically it regulates the development of naive T lymphocytes. As detailed in the examples part, it was surprisingly found that overexpression of T-BET in HSCs (after transfection/transduction) resulted in a significant increase in the absolute number of mature stage 4 and stage 5 NK cells already 3 days after the culturing step d). Human T-BET protein and nucleic acid sequences included herein are any homolog or artificial sequence that is substantially identical, i.e. at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding T-BET sequence identified by NCBI Accession number NM_013351.1 (incorporated herein by reference) (SEQ ID NO:1 for the nucleic acid sequence). T-BET as used herein encompasses also natural variants of the aforementioned specific T-BET protein. Such variants have at least the same essential biological and immunological properties as the specific T-BET protein.
EOMES (or eomesodermin) is a transcription factor involved in the regulation of developmental processes of vertebrates, more specifically it controls regulation of neural stem cells as well as other related cells. As detailed in the examples part, it was surprisingly found that overexpression of EOMES (after transfection/transduction) resulted in a significant increase in the absolute number of mature stage 4 and stage 5 NK cells already 3 days after the culturing step d). In addition, the thus obtained NK cells displayed increased ADCC activity.
In said context, the NK cells of the present invention, and more specific the NK cells overexpressing EOMES, are of particular interest for use in combination with therapeutic antibodies. NK cell (adoptive) therapy can thus be combined with injection of a monoclonal antibody specifically recognizing a tumor antigen. Such antibodies are often used in cancer immunotherapy. By combining NK cells and tumor antigen-specific antibody therapies, the tumor cells are efficiently targeted by the NK cells, leading to a better outcome.
In one embodiment, the invention provides the mature NK cells of the invention, characterized by high expression of CD16, in combination with an antibody, in particular a monoclonal antibody. The enhanced expression of CD16 of ex vivo differentiated NK cells might be utilized in therapeutic settings combining the cytotoxic activity of NK cells with therapeutic antibodies against e.g. malignant cells.
Human EOMES protein and nucleic acid sequences included herein are any homolog or artificial sequence that is substantially identical, i.e. at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding EOMES sequence identified by NCBI Accession number NM_001278182.1 (incorporated herein by reference) (SEQ ID NO:2 for the nucleic acid sequence). EOMES as used herein encompasses also natural variants of the aforementioned specific EOMES protein. Such variants have at least the same essential biological and immunological properties as the specific EOMES protein.
The present invention not only discloses the use of a single transcription factor selected from T-BET or EOMES, it also encompasses the combined use of both transcription factors. As the gene targets of T-BET and EOMES are (partially) different, it may be advantageous to combine both transcription factors as this might result in a synergistic effect.
The transcription factors of the present invention may be transfected/transduced in the cells as provided herein using any suitable method. The methods used for transfection or transduction are generally known to the skilled person and are not limiting to the present invention. In a specific embodiment, said cells are transduced with a viral vector, in particular a retroviral vector, comprising a nucleic acid encoding said at least one transcription factor. Alternatively, said cord blood HSC can be transfected with mRNA encoding these transcription factors. This will result in transient TBET and EOMES protein transcription, which, given the relatively short half-life of mRNA, will be lost after a short period of time.
Another approach is to generate an inducible retroviral vector, such as by using a construct generating a fusion protein between the transcription factor of interest and a mutant estrogen receptor (ERT2) in the retroviral vector (e.g. LZRS). The fusion protein is followed by a 2A-sequence and the enhanced green fluorescent protein (eGFP) reporter gene, which allows discrimination of transduced from untransduced cells. Upon retroviral transduction, CD34+Lineage−(CD3/14/19/56) eGFP+cord blood HSC can be sorted and put in differentiation culture to study the impact of the transduced transcription factor on NK cell development. The transduced transcription factor/ERT2 fusion protein is constitutively expressed but it remains cytosolic, and thus inactive, by binding to heat shock proteins. The addition of tamoxifen dissociates the heat shock proteins, translocates the transcription factor to the nucleus, and thus activates the transcription factor. Transcription factors can be activated from the start of the culture and this activation can be stopped thereafter at any time point by removing tamoxifen from the culture medium.
After the transcription/transduction step, the cells obtained therefrom are cultured in a medium containing at least one cytokine. In a particular embodiment, said cytokine is interleukin-3 (IL-3), interleukine-7 (IL-7), interleukin-2 (IL-2) and/or interleukin-15 (IL-15). In a preferred embodiment, the at least one cytokine is IL-15.
In the context of the present invention, “interleukin-3” (IL-3) is an interleukin that stimulates differentiation of HSC towards myeloid precursors. In addition to IL-7, it stimulates the differentiation of HSC towards lymphoid precursors. In a specific embodiment of the present invention, said IL-3 is present in the medium (such as e.g. of step d) at a concentration from about 5 ng/ml to about 500 ng/ml; more specifically, from about 0.5 ng/ml to about 50 ng/ml; more in particular from about 1 ng/ml to about 20 ng/ml; in particular about 5 ng/ml, about 10 ng/ml or about 15 ng/ml; alternatively from about 0.5 ng/ml to about 25 ng/ml; more specifically, from about 1 ng/ml to about 15 ng/ml; more in particular from about 1 ng/ml to about 10 ng/ml; in particular about 10 ng/ml, about 5 ng/ml or about 15 ng/ml.
In the context of the present invention, “interleukin-7” (IL-7) is an interleukin that stimulates differentiation of HSC towards lymphoid precursors. Furthermore, IL-7 plays an important role in regulating survival and expansion of mature NK cells. In a specific embodiment of the present invention, said IL-7 is present in the medium (such as e.g. of step d) at a concentration from about 5 ng/ml to about 500 ng/ml; more specifically, from about 0.5 ng/ml to about 50 ng/ml; more in particular from about 1 ng/ml to about 20 ng/ml; in particular about 5 ng/ml, about 10 ng/ml or about 15 ng/ml; alternatively from about 1 ng/ml to about 100 ng/ml; more specifically, from about 5 ng/ml to about 50 ng/ml; more in particular from about 10 ng/ml to about 30 ng/ml; in particular about 15 ng/ml, about 20 ng/ml or about 25 ng/ml.
In the context of the present invention, “IL-2” or “interleukin-2” is a type of cytokine signaling molecule in the immune system which regulates the activities of white blood cells that are responsible for immunity, in forming part of the body's natural response against microbial infections. In a specific embodiment of the present invention, said IL-2 is present in the medium (such as e.g. of step d) at a concentration from about 5 ng/ml to about 500 ng/ml; more specifically, from about 0.5 ng/ml to about 50 ng/ml; more in particular from about 1 ng/ml to about 20 ng/ml; in particular about 5 ng/ml, about 10 ng/ml or about 15 ng/ml.
In the context of the present invention, “IL-15” or “interleukin-15” is a type of cytokine with structural similarity to IL-2. IL-15 is secreted by mononuclear phagocytes following infection by viruses and it induces cell proliferation of natural killer cells. In a specific embodiment of the present invention, said IL-15 is present in the medium (such as e.g. of step d) at a concentration from about 5 ng/ml to about 500 ng/ml; more specifically, from about 0.5 ng/ml to about 50 ng/ml; more in particular from about 1 ng/ml to about 20 ng/ml; in particular about 5 ng/ml, about 10 ng/ml or about 15 ng/ml; alternatively from about 1 ng/ml to about 50 ng/ml; more specifically, from about 5 ng/ml to about 25 ng/ml; more in particular from about 5 ng/ml to about 15 ng/ml; in particular about 5 ng/ml, about 10 ng/ml or about 15 ng/ml.
In the context of the present invention, the stage (e.g. maturity) of the NK cells of the present invention is determined by evaluation of phenotypic NK cell markers (CD56, CD94, CD16) present on the cell surface of the NK cells by methods generally known, in particular by means of flow cytometric analysis. From the moment a stage 4 or stage 5 NK cell is present in the culture, these cells are considered as the mature NK cell population (e.g. at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50% or more of the cells in the culture have the respective phenotypic NK cell markers). Stage 4 and stage 5 NK cells are determined by a CD56+CD94+CD16− and a CD56+CD94+CD16+ phenotype, respectively.
In a preferred embodiment, the “mature” NK cells are at least of stage 4, in particular stage 4 and stage 5, more in particular stage 5.
The method and medium used for the culturing (such as e.g. in step b or d) may be any suitable method and medium for culturing isolated HSCs. In particular said medium is IMDM medium (Iscove's Modified Dulbecco's Medium). Optionally said medium comprises about 1% to 20% serum (such as e.g. about 5%, 10%, 15%), in particular fetal calf serum or human AB serum. The medium of step d) may further contain a cytokine selected from the list consisting of: FLT3-L, SCF, IL-3, IL-7 and IL-15.
Furthermore, the culturing step d) may be a co-culturing step using any suitable co-culturing cell line or feeder cell line, such as for example an inactivated stromal cell line; more specifically EL08.1D2 cells (i.e. a murine fetal liver stromal cell line) or OP9 cells (i.e. a mouse bone marrow stromal cell line). We found that NK cells differentiated from HSC on the EL08.1D2 feeder cells express higher levels of KIRs and CD16 compared to NK cells differentiated on OP9 feeder cells, indicating increased NK cell maturation.
In a further aspect, the present invention also provides HSCs cells or NK cells which are characterized in that they are or have been transfected and/or transduced with at least one transcription factor selected from the list comprising: T-Box expressed in T cells (T-BET) and Eomesodermin (EOMES); or a combination thereof; in particular EOMES. Further enclosed are HSCs or NK cells transduced with a retroviral vector (e.g. the LZRS virus) containing the cDNA encoding T-BET and/or EOMES. In a particular embodiment, the invention provides HSCs transfected and/or transduced with EOMES, such as e.g. HSCs transduced with a retroviral vector (e.g. the LZRS virus) containing the cDNA encoding EOMES.
Finally, the present invention provides differentiated NK cells obtained using the methods of the present invention.
The invention also includes methods and uses of said NK cells in medical applications, such as e.g. immunotherapy and/or cancer treatment.
The following examples are set forth below to illustrate the methods, compositions, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods, compositions, and results.
1. Material and Methods
Isolation of CD34+ HSC from Umbilical Cord Blood
Umbilical cord blood (UCB) was obtained from the Cord Blood Bank, Ghent University Hospital, Ghent, Belgium. Cord blood usage in this study was approved by the Ethics Committee of the Faculty of Medicine and Health Sciences and informed consent was obtained in accordance with the Declaration of Helsinki. Mononuclear cells were obtained by Lymphoprep density gradient centrifugation. CD34+ HSC were subsequently enriched from the mononuclear cells using Magnetic Activated Cell Sorting (MACS; Direct CD34+ HSC MicroBead Kit, Miltenyi Biotech Leiden, The Netherlands) according to the manufacturer's guidelines. Purity of the CD34+ HSC was determined by labelling the cells with anti-CD34 antibody conjugated with phycoerythrine (PE). Purity of >90% was confirmed by a LSRII Flow Cytometer (BD Biosciences, San Jose, Calif., U.S.A). Freshly isolated CD34+ HSC were frozen in fetal calf serum (FCS)+10% DMSO and stored in liquid nitrogen until usage.
Retrovirus Production of Overexpression Vectors
Molecular Cloning of Overexpression Constructs
Human T-BET and EOMES cDNA was purchased from Source BioScience (Nottingham, UK; T-BET cDNA: IRATp970D0558D sequence is identical to NM_013351.1; EOMES cDNA: IRAKp961A1269Q sequence is identical to NM_001278182.1). Restriction sites for BamHI and Xho-I were added to the cDNA by PCR using Phusion® High Fidelity PCR (New England Biolabs Inc; Ipswich, Mass., U.S.A) with self-designed primers:
Human ID2 and TOX cDNA were purchased from OriGene Technologies (Rockville, Md., U.S.A; ID2 cDNA: SC118791, sequence identical to NM_002166.4; TOX cDNA: SC114879, sequence identical to NM_014729.2). Restriction sites for BamHI, EcoRI and NgoMIV were added to the cDNA as described above. Self-designed primers:
cDNA encoding human ETS-1 p51 or p27 was subcloned from the pLEXhEts1p51HAtag and pCDNA3hEts1p27 vector, respectively (kindly provided by L. A. Garrett-Sinha, State University of New York, Buffalo, N.Y., U.S.A., and [21]).
The cDNA of the different transcription factors was ligated into the LZRS-IRES-eGFP retroviral vector (original LZRS plasmid: T M Kinsella, G P Nolan (1996) [18]). The empty LZRS-IRES-eGFP vector was used as control. Viral vectors were sequenced (GATC Biotech, Ebersberg, Germany) to confirm correct DNA sequence of the constructs.
Retrovirus Production
The control, T-BET, EOMES, TOX, ID2 and ETS-1 retroviral constructs were transfected into Phoenix A cells using the Calcium Phosphate transfection kit (Invitrogen, Carlsbad, Calif., U.S.A) and maintained in Iscove's Modified Dulbecco's medium (IMDM) containing 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine (Life technologies, Carlsbad, Calif., U.S.A) and 2 μg/ml puromycin. Retrovirus was harvested on day 2, day 6 and day 14 after transfection and stored at −80° C. until usage.
NK Cell Differentiation Culture In Vitro
Culture of EL08.1D2 Cells
The murine embryonic liver cell line EL08.1D2 was maintained in 50% Myelocult M5300 medium (Stem Cell Technologies, Grenoble, France), 35% α-MEM, 15% FCS, supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine and 10 μM β-mercaptoethanol on 0.1% gelatin-coated plates at 33° C. EL08.1D2 cells were inactivated by adding 10 μg/ml mitomycin C to the culture medium during 2-3 hours. Cell proliferation of these cells is thereby completely blocked. Thereafter, cells were thoroughly rinsed before harvesting using trypsin-EDTA. Cells were plated at a density of 50,000 cells per well on a 0.1% gelatin-coated tissue culture-treated 24-well plate at least 24 h before adding HSC or before transfer of the differentiated NK cells/ILC3 on day 14 and day 21 of culture.
Retro Viral Transduction of HSC and NK Cell Differentiation
Isolated cord blood-derived CD34+ HSC were cultured in complete IMDM containing 10% FCS (all from Life Technologies) and supplemented with thrombopoietin (TPO) (20 ng/ml), stem cell factor (SCF) (100 ng/ml) (all from Peprotech) and FMS-like tyrosine kinase 3 ligand (FLT3-L) (100 ng/ml, R&D Systems) from day −4 to day −2. Subsequently, these cells were harvested, transferred to RetroNectin (Takara Bio, Saint-Germain-en-Laye, France)-coated plates and viral supernatant was added. Additional cytokines were added to keep the concentrations constant after virus addition. The plates were centrifuged at 950 g and 32° C. during 90 min. At day 0, lineage−(CD3/CD14/CD19/CD56) CD34+eGFP+ HSC were sorted using a FACS ARIA III cell sorter (BD Biosciences, San Jose, Calif., U.S.A.). Sorted HSC were co-cultured with mitomycin-treated EL08.1D2 cells in Dulbecco's modified Eagle medium plus Ham's F-12 medium (2:1 ratio), supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 10 mM sodium pyruvate (all from Life Technologies), 20% of heat-inactivated human AB serum (Merck, Darmstadt, Germany), 24 μM β-mercaptoethanol, 20 μg/mL ascorbic acid and 50 ng/mL sodium selenite (all from Sigma-Aldrich). The following cytokines were added: IL-3 (5 ng/mL, first week only), IL-7 (20 ng/mL), IL-15 (10 ng/mL) (all from R&D Systems), SCF (20 ng/mL), and Flt3-L (10 ng/mL). Alternatively, to test the necessity of IL-15 in NK cell differentiation upon T-BET and EOMES transduction, IL-15 was not included in the cytokine mix. Culture medium was refreshed on day 7 by addition of the same volume of fresh medium with cytokines. At day 14 the non-adherent cells were harvested and transferred to new mitomycin-treated EL08.1D2 feeder cells.
Flow Cytometry
NK cell differentiation co-cultures were examined at different time points using flow cytometry (LSRII flow cytometer, BD Biosciences). Data were analyzed with FACSDiva Version 6.1.2 Software (BD Biosciences) and/or FlowJo_V10 (Ashland, Oreg., U.S.A).
Functional Assay
IFN-γ & TNF-α Production
For intracellular IFN-γ and TNF-α detection by flow cytometry, 105 cells from day 21 T-BET and EOMES overexpression cultures, or from day 21 control transduced cells, were stimulated with 50 ng/ml phorbol myristate acetate (PMA) and 1 μg/ml ionomycin (both from Sigma Aldrich, Sant Louis, Mo., U.S.A); or with 10 ng/ml IL-12 (PeproTech, London, U.K.) and 10 ng/ml IL-18 (R&D Systems, MN, U.S.A.), with or without 10 ng/ml IL-15 (Miltenyi Biotec, Leiden, The Netherlands) for 24 h. For the last 4 h, brefeldin A (BD GolgiPlug, 1/1000, BD Biosciences) was added. Thereafter, NK cell marker surface staining was performed, followed by fixation and permeabilisation using the Cytofix/Cytoperm Kit (BD Biosciences) and IFN-γ/TNF-α staining. The presence of intracellular IFN-γ or TNF-α was analyzed by flow cytometry on the gated NK cells.
Cytotoxicity Assays
To determine cell specific killing, 51Chromium release assays were performed. Therefore, 106 K562 target cells were labeled with 100 μCi Na251CrO4 (Perkin Elmer, Waltham, Mass., U.S.A) for 1.5 h at 37° C. eGFP+CD45+CD11a+CD56+CD94+ NK cells were sorted from day 21 T-BET and EOMES overexpression cultures, or from control-transduced cultures, and were added in a serial dilution to 103 51Cr-labeled K562 cells per well in a V-bottomed 96-well plate. Effector cells were added to the targets cells in triplicate. After 4 h, the supernatant was harvested and radioactivity was measured using a Luminescence counter (Wallac Microbeta Trilux, Perkin Elmer). The percentage of specific lysis was calculated using the formula: [(experimental release−spontaneous release)/(maximal release−spontaneous release)]×100.
ADCC against Raji, a CD20-expressing human Burkitt's lymphoma cell line, was measured in triplicates using the 51Chromium release assay as described above. The target cells were added to the effector cells at an effector:target ratio of 1:1 in medium containing either 0 or 10 μg/ml Rituximab (anti-CD20 antibody) (Hoffmann-La Roche, Basel, Switzerland, kindly provided by the pharmacy of Ghent University Hospital, Belgium) and incubated for 4 h. Specific lysis was calculated using the formula as described above.
CD107a Degranulation Assay
For analysis of CD107a expression on the cell membrane, that is a measure of degranulation, 105 cells from day 21 T-BET or EOMES overexpression cultures and from control transduced cells were added to 105 K562 or Raji targets cells, with 0 or 10 μg/ml Rituximab, and co-cultured for 2 h. Thereafter, the cells were harvested and stained for NK cell surface markers and CD107a. CD107a degranulation in the gated NK cells was analyzed using flow cytometry.
Cytospins
For microscopic evaluation of the cell morphology, eGFP+CD45+CD11a+CD56+CD94+ NK cells were sorted from day 3 or day 7 T-BET and EOMES overexpression cultures, or from day 19 control-transduced cultures. Cytospins were made (Shandon Cytospin™ 4, Thermo Scientific, Cheshire, UK), Wright-Giemsa stained and microscopically evaluated. The percentage of cells containing cytotoxic granules was counted manually.
Library Prep and RNA Sequencing
After RNA extraction (RNeasy micro kit, Qiagen, Hilden, Germany), the concentration and quality of the total extracted RNA was checked by using the ‘Quant-it ribogreen RNA assay’ (Life Technologies, Grand Island, N.Y., U.S.A) and the RNA 6000 nano chip (Agilent Technologies, Santa Clara, Calif., U.S.A), respectively. Subsequently, 70 ng of RNA was used to perform an Illumina sequencing library preparation using the QuantSeq 3′ mRNA-Seq Library Prep Kits (Lexogen, Vienna, Austria) according to manufacturer's protocol. Libraries were quantified by qPCR, according to Illumina's protocol ‘Sequencing Library qPCR Quantification protocol guide’, version February 2011. A High sensitivity DNA chip (Agilent Technologies, Santa Clara, Calif., U.S.A.) was used to control the library's size distribution and quality. Sequencing was performed on a high throughput Illumina NextSeq 500 flow cell generating 75 bp single reads. Per sample, on average 5.3×106±1.7×105 reads were generated. First, these reads were trimmed using cutadapt version 1.11 to remove the “QuantSEQ FWD” adaptor sequence. The trimmed reads were mapped against the Homo sapiens GRCh38.90 reference genome using STAR version 2.5.3a. The RSEM software version 1.2.31 was used to generated the count tables.
To explore if the samples from different treatment groups clustered together and to detect outlier samples, a Principal Component Analysis (PCA) on rlog transformed counts was performed using the R statistical computing software. No outliers among the samples were detected. Differential gene expression analysis was performed using edgeR, whereby HSC upon T-BET or EOMES overexpression were compared to control HSC. Differential expressed genes were tested with edgeR exact Test. Genes with an FDR <0.05 were considered significantly differential.
GSEA was performed using the GSEA software tool v2.2.2 of the Broad Institute [19, 20]. The ‘GSEAPreranked’ module was run using standard parameters and 1000 permutations.
2. Results
Accelerated Human NK Cell Development Upon T-BET and EOMES Overexpression
To investigate the regulatory role of transcription factors T-BET and EOMES in human NK cell development, overexpression constructs of both T-BET and EOMES were made, whereby human cDNA of T-BET or EOMES was cloned separately into the LZRS-IRES-eGFP retroviral vector. These overexpression constructs were transduced on day −2 in human umbilical cord blood-derived CD34+ HSC, in parallel to an empty control vector. At day 0, transduced HSC were sorted as lineage−(CD3/CD14/CD19/CD56) CD34+eGFP+ cells that were subsequently differentiated in the NK/ILC3 culture. From day 0, overexpression of T-BET and EOMES in eGFP+ cells was confirmed at regular time points at the protein level by flow cytometry, showing that overexpression was maintained throughout the culture period (
Currently, in vitro-generated NK cells used in NK cell immunotherapy are usually cultured in the absence of stromal feeder cells. To test whether accelerated NK cell differentiation upon T-BET or EOMES overexpression in HSC is also possible in a feeder-free system, transduced HSC were cultured in the NK cell/ILC3 differentiation culture, in the absence of EL08.1D2 feeder cells. The results show that, similar to NK cell cultures with stromal feeder cells, stage 4 NK cells were already present from day 3 of culture with both T-BET and EOMES overexpression cultures, whereas NK cells only became detectable on day 14 in control-cultures (
Because T-BET and EOMES overexpression in HSC led to extreme acceleration of NK cell differentiation, we reasoned that T-BET or EOMES overexpression might overrule the need for IL-15 in the culture medium. IL-15 is an important cytokine for NK cell development and differentiation through IL-2Rβ signaling in NK cell precursors. Results of cultures without IL-15 in the cytokine mix showed that, as control transduced HSC,-also T-BET- or EOMES-transduced HSC could not develop into NK cells on day 3. Even on day 14 of the culture period, no NK cells developed upon T-BET or EOMES overexpression, nor with control transduced cells (
Early Arising NK Cells Upon T-BET and EOMES Overexpression Express a Mature NK Cell Phenotype
In order to further characterize the early arising NK cells upon T-BET and EOMES transduction of HSC, their phenotype was analyzed by flow cytometry using a panel of mature NK cell markers. As differentiating NK cells gradually express activating NK cell receptors, NKG2D and NKp46 expression was evaluated. NKG2D was expressed by NK cells from day 3 of the EOMES overexpression cultures, at a level comparable to day 14 control transduced NK cells (
Perforin and granzyme B proteins are known to be contained in the cytotoxic granules of NK cells. We therefore performed microscopic analysis of sorted NK cells from day 3 and day 7 overexpression cultures, and from day 19 control cultures. The results show that NK cells from day 3 and day 7 T-BET and EOMES overexpression cultures had multiple cytotoxic granules in their cytoplasm, at equal numbers compared to day 19 control NK cells (
The Early Arising NK Cells Upon T-BET and EOMES Overexpression are Functionally Mature
The most important function of mature NK cells is killing of malignant and virus-infected cells. Because the early arising NK cells, upon T-BET or EOMES overexpression in HSC, express both perforin and granzyme B and contain cytoplasmic granules, we reasoned that they also have cytotoxic potential. Therefore, cytotoxic assays were performed with the human NK cell susceptible K562 cell line as target cells. The results show that NK cells from day 21 T-BET and EOMES overexpression cultures mediated comparable cytotoxicity as day 21 control NK cells (
Another important function of mature NK cells is the production of pro-inflammatory cytokines, including IFN-γ and TNF-α, whereby they are able to influence other immune cells. Therefore, we stimulated day 21 NK cells from both overexpression and control cultures with PMA and ionomycin or with a combination of IL-12, IL-18 with or without IL-15. IFN-γ production of T-BET and EOMES overexpressing versus control NK cells was comparable after stimulation with PMA/ionomycin, and was higher upon IL-12/IL-18 or IL-12/IL-18/IL-15 stimulation (
EOMES-Overexpressing NK Cells have Increased ADCC Activity
Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism whereby the target cell is lysed due to the presence of bound antibodies to the target cell surface that cross-link activating Fc-receptors on the cell surface of the effector cells. CD16 (FcγRIII) is the main activating Fc-receptor widely expressed on NK cells and induces killing by ADCC. Significantly more CD16+ NK cells, both in percentages as well as in absolute cell numbers, were obtained in T-BET and EOMES overexpression cultures as compared to control cultures (
With regard to the therapeutic potential of the T-BET and/or EOMES overexpressing NK cells, we therefore tested their ADCC capacity. For this purpose, the CD20-expressing human Burkitt's lymphoma cell line Raji was used as target in the presence or absence of Rituximab (RTX), a humanized monoclonal anti-CD20 antibody that is used in cancer immunotherapy. The results show that both T-BET and EOMES overexpressing as well as control NK cells displayed ADCC, but the ADCC capacity of EOMES-overexpressing NK cells was significantly higher as compared to control NK cells (
Transcriptome Profiling of T-BET and EOMES Overexpressing HSC Displays Activation of NK Cell Specific Genes.
In order to obtain a mechanistic insight into the accelerated differentiation and maturation of NK cells from T-BET or EOMES transduced versus control transduced HSC, their transcriptome was determined by RNA-sequencing. In T-BET and EOMES overexpressing HSC, 572 and 1427 differentially expressed genes (false discovery rate (FDR)<0.05), respectively, were identified.
HSC overexpressing T-BET or EOMES both showed higher expression of transcription factors that have a proven role in murine and/or human NK cell differentiation, including HELIOS (IKZF2), IRF8 and TOX. Higher expression of ETS-1 and RUNX2 was only present upon EOMES overexpression, while HOBIT (ZNF683) was only higher expressed in HSC overexpressing T-BET. In addition to transcription factors, also perforin (PRF1), granzyme B (GZMB) and IL2RB were higher expressed in EOMES-transduced HSC. As expected, CD34 expression was downregulated in both T-BET and EOMES overexpressing HSC (
The above tables provide an overview of the top 10 upregulated and downregulated genes in T-BET or EOMES transduced HSCs vs control. The genes listed therein are suitable for further differentiating the cells of the present invention from non-transduced/non-transfected or control-transduced/control-transfected ones.
Importantly, gene set enrichment analysis (GSEA) further revealed that T-BET or EOMES transduced HSC both have increased expression of a large set of mature CD56dim NK cell specific genes (
Overexpression of ID2, TOX or ETS-1 in Human HSC does not Accelerate NK Cell Differentiation.
Several transcription factors have been shown to play crucial roles in NK cell lineage specification, differentiation and/or maturation. Both ETS proto-oncogene 1 (ETS-1) and Inhibitor of DNA binding 2 (ID2) have been shown to specify early stages of NK cell development and are key regulators of NK cell lineage specification in mice [7]. Moreover, ETS-1 deficiency in human HSC results in decreased NK cell differentiation in vitro, revealing a critical role for ETS-1 in human NK cell development [22]. The lack of mature NK cells was reported in mice that are deficient in Thymocyte Selection Associated High Mobility Group Box (TOX) [7]. This defect was also seen in human in vitro NK cell cultures, whereby the mature NK cell population decreases [15].
To analyze whether the accelerated NK cell differentiation and maturation observed with T-BET or EOMES overexpression also occurs with overexpression of other transcription factors involved in NK cell differentiation, we tested the effect of overexpression of ID2, TOX and ETS-1. ID2 overexpression in HSC did not result in significant differences in NK cell maturation in comparison to control transduced cells (
We additionally overexpressed two isoforms of ETS-1 were overexpressed: p27, a dominant-negative isoform that inhibits signaling of endogenous ETS-1, and p51, the full-length isoform. Whereas p27 overexpression inhibited NK cell differentiation, showing a critical role for ETS-1 in this process, overexpression of the functionally active p51 isoform did not increase NK cell differentiation (
| Number | Date | Country | Kind |
|---|---|---|---|
| 18198021.0 | Oct 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/076459 | 9/30/2019 | WO | 00 |
