Natural killer cells

Abstract

The present invention relates to expanded natural killer (NK) cell populations, methods for their preparation and therapeutic applications thereof. More specifically, the invention relates to increasing the number of CD16 + NK cells in a population of expanded NK cells without the need for exogenous gene expression.

Description

Field of the Invention

[0001] The present invention relates to expanded natural killer (NK) cell populations, methods for their preparation, and therapeutic applications. More specifically, the present invention relates to increasing CD16 expression in expanded NK cell populations without the need for exogenous gene expression. + The number of NK cells. Background of the Invention

[0003] There is growing interest in natural killer (NK) cells because of their cytotoxic properties against cancer cells, pathogen-infected cells, and other damaged cells. NK cells are innate lymphocytes (ILCs), specifically large granular cytotoxic lymphocytes, that bridge the innate and adaptive aspects of the immune response. They comprise 10% to 15% of circulating lymphocytes in peripheral blood. NK cells also exhibit the highest levels of cytotoxic activity in the immune system. Therefore, alterations in NK cell function or number can affect the immune system's ability to fight infection and cancer. For example, a large study in Japan showed that reduced NK cell levels were associated with a significantly increased incidence of cancer in a cohort of people over 40 years old.

[0004] Similar to B cells and T cells, these NK cells are derived from common lymphoid progenitor cells (CLPs), which in turn are derived from hematopoietic stem cells (HSCs). However, NK cells differ from B cells and T cells in that they lack specific cell surface antigen receptors. Because of this, NK cells can kill cancer cells and pathogen-infected cells without prior sensitization, making them part of the innate immune response. They also play a key role in tumor immune surveillance and can directly influence the adaptive immune response.

[0005] Activation of NK cells causes them to release perforin and cytoplasmic granules containing granzymes. 2 + In the presence of NK cells, perforins polymerize to form pores in target cells. Granzymes can enter target cells through these pores, causing DNA fragmentation and cell apoptosis. NK cells can also secrete cytokines that trigger the actions of other immune cells involved in adaptive immunity.

[0006] Many research groups have been dedicated to developing methods to increase the number of endogenous NK cells in patients. One approach involves administering cytokines that are crucial for NK cell development. Administration of interleukin-2 (IL-2) and interleukin-15 (IL-15) is expected to promote NK cell development. IL-2 promotes NK cell proliferation and cytotoxicity, while IL-15 promotes NK cell development and expansion. However, in vivo studies have shown that even at very high doses, these cytokines only minimally expand NK cells and shorten their half-lives. Furthermore, the administered cytokines often lead to systemic toxicity due to inappropriate activation of the immune response and induction of NK cell apoptosis. Furthermore, even when previous approaches have been able to increase NK cell expansion, the resulting expanded NK cells often have limited functional activity, such as low levels of interferon gamma (IFNγ) production and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0007] Therefore, it is difficult to produce NK cells in large quantities using traditional methods and technologies, and it is even more difficult to produce fully functional NK cells with high cytotoxicity. Currently, there are no drugs that can be used to selectively increase the number of NK cells, especially no drugs that can be used to mass-produce mature, active NK cells. Therefore, whether it is to produce large quantities of functional NK cells for treatment and research in vitro or in vivo, new NK cell production methods need to be developed. Summary of the Invention

[0008] Natural killer (NK) cells play a key role in the immune system, destroying cancer cells, pathogen-infected cells, or damaged cells. Enhancing the number or function of NK cells is expected to increase their ability to kill these cells. Existing treatments, such as adoptive transfer of NK cells and cytokine-boosting therapy of endogenous NK cells, have not been very successful in terms of efficacy.

[0009] NK cells differentiate from hematopoietic stem cells in the bone marrow and are distributed in lymphoid and non-lymphoid tissues, including lymph nodes, spleen, peripheral blood, lungs, and liver. Specific cytokines and transcription factors are required to drive the development of hematopoietic stem cells into NK cells. To encourage hematopoietic stem cells to differentiate into NK cells, each cytokine and transcription factor must be present at a precise time and concentration. However, the precise hierarchy of cytokines and transcription factors that regulate NK cell maturation remains unclear.

[0010] The inventors of the present invention have previously shown that inhibiting the action of REV-ERB can increase the production of NK cells. In particular, the inventors have demonstrated that inhibiting the action of REV-ERB (e.g., using the REV-ERB antagonist SR8278) increases the expression of E4bp4, which in turn increases the production of NK cells. The inventors of the present invention have also previously shown that the absence of Notch signaling impedes the production of NK cells, and that the complete lack of NK cell development in E4bp4- / - progenitor cells can be completely rescued by short-term exposure to Notch peptide ligands, particularly Delta-like ligand 4 (DLL4). Furthermore, the inventors have shown that combining these two independent mechanisms (use of Notch ligands and inhibition of REV-ERB) results in a surprisingly effective method for enhancing the production of NK cells.

[0011] However, there is still a need to increase the production of large numbers of fully functional NK cells that can be used for in vivo therapy. The inventors have now developed a new NK cell expansion method that surprisingly increases the CD16 + The proportion of NK cells. In particular, the inventors surprisingly found that a production method including a unique pre-differentiation culture stage can produce NK cells with increased CD16 expression. In addition, the inventors have also demonstrated that extending the duration of this pre-differentiation stage beyond an upper limit will have a negative impact on the expression of CD16. Therefore, there is a favorable time window for pre-differentiation because it will increase the expression of CD16 during this period, but beyond this time window, this advantage will decrease or disappear. Without being limited by theory, it is believed that incorporating a certain pre-differentiation stage according to the present invention will affect the expression of CD34 + The transcriptome of hematopoietic progenitor cells (HPCs) favors CD56 +

[0012] Epigenetic changes in CD16 expression in NK cells.

[0013] Since CD16 + NK cells are crucial for antibody-dependent cell-mediated cytotoxicity (ADCC), and the methods of the present invention advantageously enable the production of expanded NK cell populations with improved function. A further advantage is that the present invention allows the production of CD16 T cells without the need to introduce exogenous genetic elements into NK cells. + NK cells, which provides a further significant benefit when these NK cells are used clinically. Therefore, increasing CD16 in the expanded NK cell population + The in vitro method for quantifying NK cell numbers and the expanded NK cell populations prepared by this method have significant advantages in that they can expand NK cells from hematopoietic progenitor cells (HPCs), which minimizes cell exhaustion and produces a large number of functional CD16+ NK cells.

[0014] Therefore, the present invention provides a method for producing and expanding CD16 in vitro. + A method for producing a natural killer (NK) cell population comprises the following steps: a) culturing a sample containing hematopoietic progenitor cells (HPCs) obtained from an individual in a culture medium that does not induce HPC differentiation for about 2 days to about 8 days to produce a pre-differentiated HPC population; and b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells.

[0015] The present invention also provides a method for increasing CD16 in the expanded NK cell population. + An in vitro method for quantifying NK cell numbers comprises the following steps: a) culturing a sample containing hematopoietic progenitor cells (HPCs) obtained from an individual in a culture medium that does not induce HPC differentiation for about 2 days to about 8 days to produce a pre-differentiated HPC population; and b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells.

[0016] In step (a), the HPCs may be cultured for about 2 days to about 6 days, optionally for about 4 days to about 6 days.

[0017] During at least part of step (a), the HPCs may be cultured in the presence of a Notch ligand.

[0018] During at least part of step (a), the HPCs may be cultured in a vessel coated with a Notch ligand; and / or (ii) the Notch ligand may be Delta-like ligand 4 (DLL4), or a fragment thereof that retains DLL4 function. Preferably, in step (a), the HPCs are cultured in the absence of the Notch ligand for about one day and then cultured in the presence of the Notch ligand for the remainder of step (a).

[0019] In step (a), the HPCs may be cultured in the presence of a compound that inhibits the action of REV-ERB; and / or in step (b), the pre-differentiated HPC population may be cultured in the presence of a compound that inhibits the action of REV-ERB.

[0020] The compound may:

[0021] (i) Increased E4bp4 expression by reducing REV-ERB activity;

[0022] (ii) reducing the activity of REV-ERB-α and / or REV-ERB-β, preferably reducing the activity of REV-

[0023] ERB-β activity;

[0024] (iii) reducing the activity of REV-ERB-α and REV-ERB-β;

[0025] (iv) is a REV-ERB antagonist, preferably an antagonist of REV-ERB-α and REV-ERB-β;

[0026] (v) selected from small molecules, proteolysis targeting chimeras (PROTAC) agents, double-stranded RNA

[0027] (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), micro

[0028] RNA, antisense RNA, aptamer, antibody, ribozyme, peptide or peptidomimetic, preferably a small molecule; and / or

[0029] or

[0030] (vi) is SR8278 or GSK1362.

[0031]

[0032] The culture medium that does not induce HPC differentiation in step (a) and / or the culture medium that induces HPC differentiation into NK cells in step (b) may not contain IL-3. Preferably, the culture medium that induces HPC differentiation into NK cells in step (b) does not contain IL-3.

[0033] The culture medium in step (a) may contain at least one of Flt3 ligand (Flt3L), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, IL-6, thrombopoietin (TPO) and / or stem cell factor (SCF), preferably Flt3L, GM-CSF, IL-3, IL-6, TPO and SCF. The culture medium in step (b) may contain IL-7, Flt3L, IL-15 and / or SCF, preferably IL-7, Flt3L, IL-15 and SCF.

[0034] Step (a) and / or step (b) may be performed in the absence of stromal support cells. Preferably, both step (a) and step (b) are performed in the absence of stromal support cells.

[0035] HPC samples can be obtained from bone marrow, umbilical cord blood, and / or peripheral blood.

[0036] CD16 produced by the corresponding method omitting step (a) + Compared with the proportion of NK cells, CD16 + The proportion of NK cells can be increased. The expanded NK cell population can contain at least 10% CD16+ NK cells, preferably at least 15% CD16 + NK cells, more preferably at least 20% CD16 + NK cells, even more preferably at least 30% CD16 + NK cells. The expanded NK cell population may exhibit at least 30% higher antibody-dependent cell-mediated cytotoxicity (ADCC), preferably at least 50% higher ADCC, compared to NK cells produced by a corresponding method omitting step (a).

[0037] The method of the present invention may not comprise the (further) step of introducing exogenous nucleic acid into HPCs and / or NK cells.

[0038] The present invention further provides an expanded CD16 + NK cell population, where at least 10% of the NK cells are CD16 + NK cells, preferably at least 15% of NK cells are CD16 + NK cells, more preferably at least 20% of the NK cells are CD16 + NK cells, even more preferably at least 30% of the NK cells are CD16 + NK cells.

[0039] The present invention provides amplified CD16 obtained by the method of the present invention. + NK cell population, where at least 10% of the NK cells are CD16 + NK cells, preferably at least 15% of NK cells are CD16 + NK cells, more preferably at least 20% of the NK cells are CD16 + NK cells, even more preferably at least 30% of the NK cells are CD16 + NK cells.

[0040] CD16 of the present invention + NK cells may not contain exogenous nucleic acids.

[0041] The expanded NK cell population may exhibit at least 30% higher ADCC, preferably at least 50% higher ADCC, more preferably at least 70% higher ADCC, compared to NK cells produced by a corresponding method omitting step (a).

[0042] The present invention further provides a composition comprising the expanded NK cells of the present invention and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0043] The present invention also provides the amplified CD16 + NK cell populations or compositions for use in therapeutic methods.

[0044] The method of treatment can be a method of treating a disease or condition selected from cancer, infectious disease (acute or chronic), autoimmune disease or female infertility or pregnancy-related disease. The method of treatment can be a method of treating viral infection, bacterial infection, protozoan infection, fungal infection and / or helminth infection.

[0045] The present invention also provides the expanded CD16 + NK cell populations or compositions used in combination with antibody-mediated immunotherapy. The compound can be administered before, simultaneously with, or after administration of the antibody-mediated immunotherapy.

[0046] BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 : NK cell developmental pathway. NK cells differentiate from hematopoietic stem cells (HSCs). From HSCs, NK cells develop into common lymphoid progenitors (CLPs), NK precursors (NKPs), immature NK cells (iNKs), mature NK cells (mNKs), and ultimately conventional NK cells (cNKs) that circulate in the blood. Below the schematic diagram of the pathway are the cytokines and transcription factors required for NK cell development. IL-15 is one of the main cytokines required for NK cell development, while E4bp4 is a key transcription factor that guides the developmental process. The others are transcription factors required for the transitions at each stage shown in the diagram. Eomes, Id2, and T-bet are additional transcription factors that play an important role in completing the NK cell maturation process.

[0048] Figure 2: (A) Experimental timeline and flow cytometry data demonstrating increased CD16 expression when specific pre-differentiation steps are added prior to differentiation. Flow cytometry data showing cell surface CD56 and CD16 levels after 20 days of culture under NK cell differentiation conditions. (B) Human CD56 generated after 20 days of differentiation on stromal cells. + CD16 - and CD56 + CD16 + Absolute number of NK cells. D0, D-2, D-4, and D-6 refer to the time the cells spent in the pre-differentiation stage before being placed on stromal cells. (C) Graph showing the relationship between the number of NK cells generated at different differentiation times on stromal cells and the number of days of pre-differentiation.

[0049] Figure 3 : Experimental timeline and flow cytometry data indicate that addition of DLL4 further increases CD16 expression when combined with specific pre-differentiation steps.

[0050] Figure 4: Graph showing the changes in CD56 over time in the presence or absence of DLL4 and at different lengths of the pre-differentiation step. + The percentage and absolute number of NK cells (A) and CD56 + CD16 +

[0051] Percentage and absolute number of NK cells (B). ● = presence of DLL4; ▲ = absence of DLL4

[0052] Figure 5 : Graph showing the effect of IL-3 on CD56 expression over time when hematopoietic progenitor cells (HPCs) were cultured in the presence / absence of DLL4 and / or in the presence / absence of EL08 stromal cells. + CD16 + Effect of NK cell percentage.

[0053] Figure 6 : Flow cytometry data showed that increasing the duration of the pre-differentiation step from 4 to 14 days significantly reduced the amount of CD56 + CD45 + The number of NK cells and the efficiency of the generated CD56 + CD16 + The number of NK cells decreased to zero.

[0054] Figure 7 : Flow cytometry data showed that the CD56 + CD45 + NK cell populations after (upper left) a 4-day predifferentiation step; (upper right) a 4-day predifferentiation step + REV-ERB inhibitor; (lower left) a 4-day predifferentiation step + DLL4; and (lower right) a 4-day predifferentiation step + REV-ERB inhibitor + DLL4. Both REV-ERB inhibitor and DLL4 increased CD56 compared to the 4-day predifferentiation step alone. + CD45 + Combining REV-ERB inhibitors and DLL4 with a 4-day predifferentiation step resulted in a decrease in the number of CD56 + CD45 + The number of NK cells increased the most.

[0055] Figure 8 : Flow cytometry data showed that the use of anti-CD158 antibodies in the CD56 cells generated by the method of the present invention (using a 6-day pre-differentiation period and a 23-day differentiation period) + CD16 + NK cells and CD56 + CD16 -The expression of killer cell immunoglobulin-like receptors (KIR) (KIR2DL1, KIR2DS1, KIR2DS3, KIR2DS5) was detected on NK cells. Detailed Description of the Invention

[0057] definition

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present disclosure belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology (20th edition, John Wiley & Sons, New York, 1994), and Hale and Malham, HarperCollins Dictionary of Biology (Harper Permanent Press, New York, 1991) provide a general dictionary of many terms used in the present disclosure for technical personnel. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definition.

[0059] It should be understood that the present invention is not limited to the specific methods, protocols, reagents, etc. described herein, and these may vary. In particular, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed embodiments.

[0060] The description of the embodiments of the present disclosure is not intended to be exhaustive, nor is it intended to limit the disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications can be made within the scope of the present disclosure, as will be appreciated by those skilled in the relevant art. For example, although the method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of the present disclosure provided herein may be appropriately applied to other procedures or methods. The various embodiments described herein may be combined to provide further embodiments. If necessary, various aspects of the present disclosure may be modified to adopt the compositions, functions, and concepts of the above-mentioned references and applications to provide further embodiments of the present disclosure. In addition, due to considerations of biological functional equivalence, some changes may be made to the protein structure without affecting the type or extent of the biological or chemical action. These and other changes may be made to the present disclosure based on the detailed description. All of these modifications are intended to be included within the scope of the appended claims.

[0061] Unless otherwise indicated, any nucleic acid sequence is written from left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxyl orientation, respectively.

[0062] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0063] As used herein, when used with a verb, the term "capable of" encompasses or indicates the action of the corresponding verb. For example, "capable of interacting" also refers to interacting, "capable of cleaving" also refers to cleaving, "capable of binding" also refers to binding, and "capable of specifically targeting..." also refers to specific targeting.

[0064] As used herein, the term "CD16 + ” refers to cells that are CD16 positive and / or highly express CD16, i.e., cells that are positive for CD16 and / or highly express CD16.

[0065] Numerical ranges include the values ​​that define the range. When a numerical range is provided, it is understood that between the upper and lower limits of the range, each intermediate value to the tenths of the lower limit unit is also specifically disclosed unless the context clearly dictates otherwise. Each smaller range between any stated value or intermediate value in the described range and any other stated value or intermediate value in the described range is included in this disclosure. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range, and in the case of any specific exclusions within the described range, each range in which either limit, both limits do not include, or both limits are included in the smaller range is also included in this disclosure. When the described range includes one or two limits, the range excluding the included one or both limits is also included in this disclosure.

[0066] Amino acids are referred to herein by their names, three-letter abbreviations, or single-letter abbreviations.

[0067] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein and refer to a series of amino acid residues that are interconnected by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are generally used to refer to relatively large polypeptides, while the term "peptide" is generally used to refer to smaller polypeptides, but there is overlap in the usage of these terms in the art. In this article, when referring to gene products and fragments thereof, the terms "protein" and "polypeptide" are used interchangeably. Therefore, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologues, orthologues, paralogues, fragments, and other equivalents, variants, fragments, and analogs described above. In the present disclosure and claims, conventional single-letter and three-letter codes for amino acid residues can be used. The three-letter codes for amino acids are defined according to the IUPAC / IUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence.

[0068] It is contemplated that minor variations in the amino acid sequences of the invention are within the scope of the invention, provided that the variations in the amino acid sequence maintain at least 60%, at least 70%, more preferably at least 80%, at least 85%, at least 90%, at least 95%, and most preferably at least 97% or at least 99% sequence identity to an amino acid sequence of the invention as defined anywhere herein, or a fragment thereof. The term "homology" as used herein refers to identity. Thus, variants or analogs of the amino acid sequences of the invention may differ in sequence by substitutions (usually conservative substitutions), deletions, or insertions. Proteins containing such variations are referred to herein as variants.

[0069] The proteins of the present invention may include variants in which amino acid residues from one species are substituted at conserved or non-conserved positions with corresponding residues from another species. Variants of the protein molecules disclosed herein can be generated and used in the present invention. Drawing on the experience of computational chemistry in applying multivariate data analysis techniques to structure / property-activity relationships [e.g., see Wald et al., Multivariate Data Analysis in Chemistry, in Chemometrics: Mathematics and Statistics in Chemistry (ed. B. Kowalski); D. Redl Verlag GmbH, Dordrecht, The Netherlands, 1984 (ISBN 90-277-1846-6)], well-known mathematical techniques, such as statistical regression, pattern recognition, and classification, can be used to derive quantitative activity-property relationships for proteins [e.g., see Norman et al., Applied Regression Analysis, Wiley-Transscience; 3rd ed. (April 1998), ISBN: 0471170828; Kandel, Abraham et al., Computer-Assisted Inference in Cluster Analysis, Prentice-Hall, (May 11, 1995), ISBN: 013341884 7; Krzanowski, Wojtek, Principles of Multivariate Analysis: A User's Perspective (Oxford Statistical Science Series, No. 22 (Paperback)), Oxford University Press; (December 2000), ISBN: 0198507089; Witten, Ian H. et al., Data Mining: Practical Machine Learning Tools and Techniques with a Java Implementation, Morgan Kaufman Publishers; (October 11, 1999), ISBN: 1558605525; Denison, David G.T. (ed.), et al., Bayesian Methods for Nonlinear Classification and Regression (Wiley Probability and Statistics Series), John Wiley & Sons; (July 2002), ISBN: 0471490369; Goss, Arup K. et al., Combinatorial Library Design and Evaluation: Principles, Software, Tools, and Applications in Drug Discovery, ISBN: 0-8247-0487-8]. Protein properties can be inferred from empirical and theoretical models of protein sequence, function, and three-dimensional structure (e.g., analysis of likely contacting residues or calculated physicochemical properties), and these properties can be considered individually or in combination.

[0070] The amino acid residues at non-conserved positions can be substituted with conservative or non-conservative residues. In particular, conservative amino acid substitutions are contemplated.

[0071] "Conservative amino acid substitution" refers to the replacement of an amino acid residue by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the amino acid substitution is considered conservative. Variants comprising conservative modifications in the proteins of the present invention do not exclude other forms of variants, such as polymorphic variants, interspecies homologs, and alleles.

[0072] "Non-conservative amino acid substitutions" include the following: (i) a residue with a positive side chain (e.g., arginine, histidine, or lysine) is substituted by or is replaced by an electronegative residue (e.g., glutamic acid or aspartic acid); (ii) a hydrophilic residue (e.g., serine or threonine) is substituted by or is replaced by a hydrophobic residue (e.g., alanine, leucine, isoleucine, phenylalanine, or valine); (iii) cysteine ​​or proline is substituted by or is replaced by any other residue; or (iv) a residue with a bulky, hydrophobic or aromatic side chain (e.g., valine, histidine, isoleucine, or tryptophan) is substituted by or is replaced by a residue with a smaller side chain (e.g., alanine or serine), or by a residue without a side chain (e.g., glycine).

[0073] The range of "insertions" or "deletions" is generally about 1, 2, or 3 amino acids. The permissible variation can be determined experimentally by systematically introducing amino acid insertions or deletions into the protein using recombinant DNA technology and measuring the activity of the resulting recombinant variants. For the skilled artisan, this does not require more than routine experimentation.

[0074] A "fragment" of a polypeptide typically comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or more, of the original polypeptide.

[0075] As used herein, the terms "polynucleotide," "nucleic acid," and "nucleic acid sequence" refer to any molecule, preferably a polymeric molecule, comprising units of ribonucleic acid, deoxyribonucleic acid, or their analogs. A nucleic acid can be single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including small interfering RNA (siRNA), small hairpin RNA (shRNA), and antisense oligonucleotides. The terms "transgene" and "gene" are also used interchangeably, and both terms encompass fragments or variants thereof that encode a target protein.

[0076] The polynucleotides of the present invention include nucleic acid sequences that have been isolated from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.

[0077] Polynucleotide of the present invention can be prepared by any method known in the art.For example, polynucleotide can be produced in a large number by replicating in a suitable host cell.The natural or synthetic DNA fragment of the required segment of encoding will be incorporated into the recombinant nucleic acid construct, normally a DNA construct, and this construct can import prokaryotic or eukaryotic cells and replicate therein.Usually, the DNA construct will be suitable for autonomous replication in a unicellular host (such as yeast or bacterium), but can also be intended to import the genome of cultivated insects, mammals, plants or other eukaryotic cell lines and be integrated therein.

[0078] The polynucleotides of the present invention can also be prepared by chemical synthesis, for example by the phosphoramidite method or the triester method, and can be performed on a commercial automated oligonucleotide synthesizer. Double-stranded fragments can be obtained from single-stranded products of chemical synthesis by synthesizing complementary strands and annealing the strands together under appropriate conditions, or by adding complementary strands using a DNA polymerase with an appropriate primer sequence.

[0079] When applied to nucleic acid sequences, in the context of the present invention, the term "isolated" means that the polynucleotide sequence has been removed from its natural genetic environment and is therefore free of other extraneous or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions, such as promoters and terminators) and is in a form suitable for use in genetically engineered protein production systems. Such isolated molecules are those that have been separated from their natural environment.

[0080] Given the degeneracy of the genetic code, there may be considerable sequence variation between the polynucleotides of the present invention. The degenerate codons encompassing all possible codons for a given amino acid are shown below:

[0081]

[0082]

[0083] Those skilled in the art will appreciate that there is a certain degree of flexibility in determining degenerate codons that represent all possible codons encoding each amino acid. For example, some polynucleotides encompassed by a degenerate sequence may encode variant amino acid sequences, but those skilled in the art can readily identify such variant sequences by reference to the amino acid sequences of the present invention.

[0084] A "variant" nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or a fragment thereof). A nucleic acid sequence or fragment thereof is "substantially homologous" (or "substantially identical") to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions), the sequence and another nucleic acid (or its complementary strand) share nucleotide sequence identity over at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the nucleotide bases. Methods for determining nucleic acid sequence homology are known in the art.

[0085] Alternatively, a "variant" nucleic acid sequence is substantially homologous (or substantially identical) to a reference sequence (or fragment thereof) if the "variant" nucleic acid sequence is capable of hybridizing to the reference sequence (or fragment thereof) under stringent (e.g., highly stringent) hybridization conditions. As readily understood by those skilled in the art, nucleic acid sequence hybridization is also affected by conditions such as salt concentration (e.g., sodium chloride), temperature, or organic solvent, in addition to the base composition and length of the complementary chains and the number of nucleotide base mismatches between the hybridizing nucleic acids. Stringent temperature conditions are preferably employed, typically including temperatures exceeding 30° C., typically exceeding 37° C., and preferably exceeding 45° C. Stringent salt concentration conditions will typically be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. The pH value is typically between 7.0 and 8.3. The combination of parameters is much more important than any single parameter.

[0086] The method for determining nucleotide sequence identity percentage is known in the art. For example, when assessing nucleotide sequence identity, the sequence with a determined number of continuous nucleotides can be compared with the nucleotide sequence (having the same number of continuous nucleotides) from the corresponding portion of nucleotide sequence of the present invention. Known tools for determining nucleotide sequence identity percentage in this area include the basic local comparison search tool for nucleotides (Nucleotide BLAST, as described below).

[0087] It is understood by those of ordinary skill in the art that different species exhibit "codon preference". As used herein, the term "codon preference" refers to the codons that are most frequently used in the cells of a particular species, and therefore tends to select one or more representatives of the possible codons that encode each amino acid. For example, the amino acid threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT, but ACC is the most commonly used codon in mammalian host cells; in other species, different codons may be preferred. The preferred codons of a particular host cell species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. For example, introducing a preferred codon sequence into recombinant DNA can increase protein production by making the translation of the protein more efficient in a specific cell type or species. Therefore, according to the present invention, any nucleic acid sequence, in addition to the gag-pol gene, can be codon-optimized for expression in a host cell or target cell. In particular, the vector genome (or corresponding plasmid), REV gene (or corresponding plasmid), fusion protein (F) gene (or corresponding plasmid) and / or hemagglutinin-neuraminidase (HN) gene (or corresponding plasmid), or any combination thereof can be codon optimized.

[0088] " fragment " of the polynucleotide of interest comprises a series of continuous nucleotides from the full-length polynucleotide sequence. For example, " fragment " of the polynucleotide of interest can comprise (or be made up of ... forming) at least 30 continuous nucleotides (for example, at least 35, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 continuous nucleic acid residues of the polynucleotide from the polynucleotide sequence. A fragment can comprise at least one antigenic determinant and / or can encode at least one antigenic epitope of the corresponding polypeptide of interest. Typically, fragments defined herein retain the same function as the full-length polynucleotide.

[0089] The terms "increase," "increase," "enhance," or "activate" are all used herein to mean an increase in a statistically significant amount. The terms "increase," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, up to and including a 100% increase, or any increase between 10% and 100% compared to a reference level, or at least about a 2-fold increase, or at least about a 3-fold increase, or at least about a 4-fold increase, or at least about a 5-fold increase, or at least about a 10-fold increase, or any multiple between 2-fold and 10-fold or more compared to a reference level. In the context of yield or titer, "increase" refers to an observable or statistically significant increase at that level.

[0090] The terms "reduce," "lower," "reduction," or "inhibit" are all used herein to refer to a reduction in a statistically significant amount. The terms "reduce," "reduction," or "reduce" or "inhibit" generally refer to a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. As used herein, "reduce" or "inhibit" encompasses complete inhibition or reduction compared to a reference level. "Complete inhibition" refers to 100% inhibition (i.e., elimination) compared to a reference level.

[0091] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a Notch ligand" includes a plurality of such substances and reference to "the Notch ligand" includes reference to one or more Notch ligands and equivalents thereof known to those skilled in the art, and so forth. Furthermore, the use of the term "including" as well as other forms, such as "comprising" and "comprising," is not limiting.

[0092] "About" generally refers to an acceptable degree of error for the amount being measured, given the nature or precision of the measurement. An exemplary degree of error is within 20%, typically within 10%, and more typically within 5% of a given value or range of values. Preferably, "about" herein is understood to mean plus or minus (±) 5%, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%, based on the numerical value being used.

[0093] The term "consisting of" refers to the compositions, methods, and respective components described herein, excluding any elements not enumerated in this description of the invention.

[0094] As used herein, the term "consisting essentially of" refers to those elements required for a given invention. The term permits the presence of elements that do not materially affect the basic and novel characteristics or functional characteristics of the invention (i.e., inactive or non-immunogenic components).

[0095] Embodiments described herein as “comprising” one or more features may also be considered disclosures of corresponding embodiments “consisting of” and / or “consisting essentially of” these features.

[0096] Concentrations, amounts, volumes, percentages and other numerical values ​​may be presented herein in a range format. It should also be understood that this range format is used for convenience and brevity only and should be interpreted flexibly to include not only the values ​​explicitly listed as the limits of the range, but also all individual values ​​or sub-ranges contained within the range, as if each value and sub-range were explicitly listed.

[0097] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to a mammalian subject for whom diagnosis, prognosis, disease monitoring, treatment, therapy, and / or therapy optimization is desired. The mammal can be, but is not limited to, a human, non-human primate, mouse, rat, dog, cat, horse, or cow. In a preferred embodiment, the individual, subject, or patient is a human. An "individual" can be an adult, adolescent, or infant. An "individual" can be male or female.

[0098] A "subject in need thereof" in need of treatment for a particular disorder may be an individual suffering from, diagnosed with, or at risk of suffering from the disorder.

[0099] The subject may be a person who has been previously diagnosed or determined to have or is suffering from a condition in need of treatment or one or more complications associated with the condition, and optionally, has been treated for a condition as defined herein or one or more complications associated with the condition. Alternatively, the subject may be a person who has not been previously diagnosed with a condition as defined herein or one or more complications associated with the condition. For example, the individual may be a person who exhibits one or more risk factors for a condition, or a person who exhibits one or more complications associated with the condition, or a subject who does not exhibit risk factors.

[0100] As used herein, the term "healthy individual" refers to an individual or group of individuals who are in a healthy state, e.g., an individual who does not exhibit any symptoms of a disease, has not been diagnosed with the disease, and / or is unlikely to develop the disease (e.g., cancer or any other disease described herein). Preferably, the healthy individual is not taking drugs that affect cancer and has not been diagnosed with any other disease. One or more healthy individuals may have a similar sex, age, and / or body mass index (BMI) as the test individual. Normal expression levels in healthy individuals, as well as significant deviations from these normal levels, can be determined using standard statistical methods used in medicine.

[0101] The terms "control" and "reference population" are used interchangeably herein.

[0102] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopeia.

[0103] Other definitions of terms may appear throughout the specification. Before describing exemplary embodiments in greater detail, it should be understood that the present disclosure is not limited to the specific embodiments described and, therefore, may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the present disclosure is limited solely by the appended claims.

[0104] The publications discussed herein are presented solely for purposes of illustrating their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art for the purposes of the appended claims. All references cited in this specification are hereby incorporated by reference for their entire disclosures and for any disclosures specifically mentioned in this specification.

[0105] Disclosures relating to various methods of the invention are intended to be equally applicable to other methods, therapeutic uses or approaches, and vice versa.

[0106] Natural killer cells

[0107] Natural killer (NK) cells exhibit the highest level of cytotoxic activity in the immune system. NK cells are similar to B and T cells but lack specific cell surface antigen receptors. Instead, NK cells possess activating and inhibitory receptors that recognize specific motifs.

[0108] NK cells circulate in the blood and peripheral lymphoid organs, such as lymph nodes and the spleen. They can be activated by cytokines or upon encountering target cells. Target cell recognition and elimination are based on a balance between inhibitory and activating signals. Activating signals are generated by the binding of activating receptors (such as NKG2D, NKp46, and NKp30) to ligands, which are present not only on cancer cells, pathogen-infected cells, and damaged cells, but also on healthy cells. On the other hand, inhibitory signals are generated when inhibitory receptors on NK cells (such as killer cell immunoglobulin-like receptors (KIRs) and CD94 / NKG2A) bind to major histocompatibility complex (MHC) class I molecules, which are normally present on all healthy cells. The absence or significant downregulation of MHC class I molecules on target cells makes them ideal targets for NK cells. This allows NK cells to distinguish between target cells and healthy cells. For NK cells to recognize and kill target cells, the overall activation signal must be greater than the inhibitory signal.

[0109] NK cells can recognize and kill cancer cells, pathogen-infected cells, and damaged cells without prior sensitization, making them part of the innate immune response. For example, NK cells can respond early to viral infections and take effect before T cells kill infected cells. NK cells can kill target cells within minutes. NK cells also secrete cytokines and "arm" other parts of the immune system. For example, NK cells can promote the effector function of T cells and enhance antibody-mediated cytotoxicity (ADCC).

[0110] NK cells are Figure 1The pathway shown is differentiated from hematopoietic stem cells (HSC). In more detail, NK cells develop into common lymphocyte progenitor cells (CLP), pre-NK precursor cells (pre-NKP), NK precursor cells (NKP), immature NK cells (iNK), mature NK cells (mNK) from HSC, and eventually develop into conventional NK cells (cNK) circulating in the blood. Although these terms are derived from the NK cell development process of mice, there is also a corresponding pathway for the development of human NK cells. For example, HSC can pass through multiple precursor cell stages (stages 1, 2 and 3) before developing into mature NK cells (stages 4 and 5). For consistency, the terms HSC, CLP, pre-NKP, NKP, iNK, mNK, cNK and NK cells are used herein. However, in the context of the present invention, these terms can be used interchangeably with the 1 to 5 stages in the human nomenclature. Figure 1 Below the diagram of the developmental pathway are the cytokines and transcription factors essential for NK cell development. Interleukin-15 (IL-15) is one of the primary cytokines required for NK cell development. Other extrinsic factors, such as specific stromal cells, are also essential for NK cell development and maturation.

[0111] According to the present invention, hematopoietic progenitor cells (HPCs) are a heterogeneous population that includes multipotent progenitor cells, such as HSCs, CLPs, and NKPs. HPCs are referred to as lineage-negative cells because they have not yet determined their developmental direction. Therefore, in the context of the present invention, unless otherwise explicitly stated, HSCs, CLPs, and NKPs are all HPCs, and reference to HPCs refers to any one of HSCs, CLPs, and / or CLPs, or any combination thereof.

[0112] Because NK cells are crucial in immune responses, multiple clinical trials have tested their efficacy in adoptive transfer protocols. Typically, this involves allogeneic transfer, in which NK cells are isolated and expanded from healthy donors. However, MHC class I molecules on target cells are only partially downregulated, and the KIR genotypes of the donor and recipient may be similar. Therefore, even if NK cells infused into the recipient are from a different individual, they may not attack target cells if their KIR recognizes MHC class I molecules. Therefore, it is crucial to screen the KIR genotype of the NK cell donor; the donor's KIR allele polymorphisms must match those of the recipient to ensure that they can recognize and kill target cells. Furthermore, expanded NK cell products have been found to have lower clinical success rates than expected and to have a weaker ability to kill cancerous or infected cells.

[0113] NK cells can be defined based on their marker expression, function / activity, or a combination of the two. These definitions are standard in the art, and there are known methods to assess marker expression and / or NK cell activity. Therefore, those skilled in the art can easily classify a cell as an NK cell using standard methods and definitions.

[0114] For example, mature NK cells (mNK) and conventional NK cells (cNK) can be identified by their expression of the surface markers CD16 (FcγRIII) and / or CD56, which are usually expressed simultaneously in humans and in some mouse strains as NK1.1 or NK1.2. NKp46 is another marker for mNK and cNK cells that is expressed in humans and several mouse strains. Therefore, NKp46 can be used as a marker for NK cells alone or in combination with CD16 and / or CD56 (in humans), or alone or in combination with NK1.1 or NK1.2 (in mice). According to the present invention, other examples of markers that can be used to identify / define NK cells include Ly49, natural cytotoxicity receptor (NCR), CD94, NKG2, killer cell immunoglobulin-like receptor (KIR) and / or leukocyte inhibitory receptor (ILT or LIR), or any combination thereof, including use in combination with CD16 and / or CD56 (in humans) or NK1.1 / NK1.2 (in mice). Mature NK cells (i.e., mNK and cNK cells) according to the present invention are CD56 + and CD45 + Yes, it may also be CD16 + As used herein, the term "mature human NK cells" includes NK cells with high CD56 expression (stage 4) and low CD56 expression (stage 5), both of which are CD56 + Mature NK cells can also be defined by the absence of certain markers, such as CD34, and the lymphocyte markers CD3 and / or CD19. Thus, the mature NK cells of the present invention can be CD56 + 、CD45 + 、CD16 + 、CD3 - and / or CD19 - , or any combination thereof, such as CD56 + 、CD45 + 、CD16 + 、CD3 - and CD19 - .

[0115] In addition to having increased CD16 expression as described herein, the (mature) NK cells of the present invention are typically at least 80% CD56 +or high expression of CD56, for example, at least 81% are CD56 + Or CD56 high expression, 85% are CD56 + or CD56 high expression, at least 86% are CD56 + or CD56 high expression, at least 87% CD56 + or CD56 high expression, at least 88% are CD56 + or CD56 high expression, at least 89% CD56 + or CD56 high expression, at least 90% are CD56 + or CD56 high expression, at least 91% CD56 + or CD56 high expression, at least 92% CD56 + or CD56 high expression, at least 93% CD56 + or CD56 high expression, at least 94% CD56 + or CD56 high expression, at least 95% are CD56 + or CD56 high expression, at least 96% CD56 + or CD56 high expression, at least 97% CD56 + or CD56 high expression, at least 98% CD56 + or CD56 high expression, at least 99% CD56 + or high expression of CD56, or higher, until 100% is CD56 + or CD56 high expression.

[0116] In addition to having increased CD16 expression as described herein, at least 50% of the NK cells in the expanded NK cell populations (typically mature NK cells) provided herein may be CD56 + or high expression of CD56, for example, at least 60% of the NK cells in the population may be CD56 + Or CD56 high expression, at least 70% of NK cells can be CD56 + Or CD56 high expression, at least 80% of NK cells can be CD56 + or high expression of CD56, or at least 90% of NK cells can be CD56 + Or CD56 high expression, or higher. Generally, in the expanded NK cell population provided by the present invention (usually mature NK cells), at least 80% of the NK cells are CD56 + Or CD56 high expression. For example, at least 80% of the NK cells in the population can be CD56 + Or CD56 high expression, at least 81% of NK cells can be CD56+ Or CD56 high expression, at least 85% of NK cells can be CD56 + Or CD56 high expression, at least 80% of NK cells can be CD56 + Or CD56 high expression, at least 86% of NK cells can be CD56 + Or CD56 high expression, at least 87% of NK cells can be CD56 + Or CD56 high expression, at least 88% of NK cells can be CD56 + Or CD56 high expression, at least 89% of NK cells can be CD56 + Or CD56 high expression, at least 90% of NK cells can be CD56 + Or CD56 high expression, at least 91% of NK cells can be CD56 + Or CD56 high expression, at least 92% of NK cells can be CD56 + Or CD56 high expression, at least 93% of NK cells can be CD56 + Or CD56 high expression, at least 94% of NK cells can be CD56 + Or CD56 high expression, at least 95% of NK cells can be CD56 + Or CD56 high expression, at least 96% of NK cells can be CD56 + Or CD56 high expression, at least 97% of NK cells can be CD56 + Or CD56 high expression, at least 98% of NK cells can be CD56 + Or CD56 high expression, at least 99% of NK cells can be CD56 + or CD56-high expression, or higher, until 100% of the NK cells in the population are CD56 + or CD56 high expression.

[0117] By the method of the present invention, such a high ratio of CD56 expression can be achieved without any purification and / or concentration step, or before any purification and / or concentration step is performed. Alternatively, such a high ratio of CD56 expression can also be achieved by the method of the present invention after purification and / or concentration. Typically, in order to achieve such a high ratio of CD56 expression in the NK cell population amplified according to the present invention, the number of purification and / or concentration steps and / or the number of purification and / or concentration techniques used are lower than the number required for the prior art methods. As a non-limiting example, a negative selection step can be used to purify the amplified NK cell population produced by the method of the present invention.

[0118] Typically, the NK cell populations thus amplified can be obtained by the methods of the present invention. Standard techniques can be used to determine the expression of CD56, examples of which are known in the art, and those of ordinary skill in the art can routinely select suitable techniques. Non-limiting examples of suitable techniques include flow cytometry, cell imaging, and enzyme-linked immunosorbent assay (ELISA).

[0119] In addition, or as another approach, the NK cells of the present invention, or the expanded NK cell populations of the present invention (typically mature NK cells), can express one or more killer cell immunoglobulin-like receptors (KIRs), in particular one or more of KIR2DL1, KIR2DS1, KIR2DS3 and / or KIR2DS5, i.e., KIR2DL1 + or KIR2DL1 high expression; KIR2DS1 + or KIR2DS1 high expression; KIR2DS3 + or KIR2DS3 high expression; and / or KIR2DS5 + The expression level of one or more of KIR2DL1, KIR2DS1, KIR2DS3, and / or KIR2DS5 can be increased compared to the level of the same KIR expressed by a suitable reference or control NK cell or NK cell population (e.g., a reference or control NK cell or NK cell population produced by a control method described herein). For example, the expression level of one or more of KIR2DL1, KIR2DS1, KIR2DS3, and / or KIR2DS5 can be increased compared to the level of the same KIR expressed by a suitable reference or control NK cell or NK cell population (e.g., a reference or control NK cell or NK cell population produced by a control method described herein). + CD16 - Compared with NK cells, the CD56 + CD16 + KIR expression in NK cells may increase the expression of KIR2DL1, KIR2DS1, KIR2DS3, and / or KIR2DS5.

[0120] Alternatively, or as a supplement, the NK cells of the present invention, or the amplified NK cell populations of the present invention (typically mature NK cells) may not express KIR2DL4, or may express KIR2DL4 at a low level, i.e., they may be KIR2DL4- or KIR2DL4-lowly expressed. Compared to the level of KIR2DL4 expressed by a suitable reference or control NK cell or NK cell population (such as a reference or control NK cell or NK cell population produced by the control method described herein), the expression level of KIR2DL4 may be reduced. Without being limited by theory, it is believed that high levels of KIR2DL4 expression are disadvantageous because KIR2DL4 is an inhibitory receptor that reduces the function of NK cells when it is bound to the ligand HLA-G. For example, in ovarian cancer, some tumors overexpress HLA-G to inhibit the function of NK cells in killing cancer cells.

[0121] Additionally, or alternatively, NK cells can be identified / defined based on their activity. For example, NK cells can be identified / defined by the presence of cytolytic granules in their cytoplasm, by their ability to secrete antimicrobial molecules such as α-defensins, and / or by their ability to secrete cytokines such as tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), interferon-γ (IFN-γ), and transforming growth factor-β (TGF-β).

[0122] Unless otherwise specified herein, reference to NK cells includes reference to iNK, mNK, and cNK cells. HSCs, CLP cells, and NKPs will generally be referred to by their names.

[0123] As described herein, one advantage of the present invention is that, compared to conventional NK cell production methods, the NK cells it provides have higher CD16 expression and: (i) do not contain exogenous nucleic acids; and / or (ii) have not been extensively purified and / or concentrated, because extensive purification and / or concentration may damage the purified and / or concentrated NK cells. Alternatively, as a supplement, the NK cells of the present invention may have further phenotypic differences compared to NK cells produced by prior art methods, such as expressing one or more characteristic markers, or having a characteristic marker spectrum as described above. These differences are the result of the method of the present invention, and therefore, the NK cells produced by the method of the present invention differ from the NK cells produced by the prior art methods in key features.

[0124] CD16 and antibody-dependent cell-mediated cytotoxicity (ADCC)

[0125] CD16 (FcγRIII) is an Fc receptor expressed on the surface of natural killer (NK) cells, neutrophils, monocytes, macrophages, and some T cells. NK cells express CD16A (FcγRIIIA). The human CD16A protein (UniProt accession number P08637, sequence version 2, accessed May 26, 2022) is expressed on NK cells. The terms CD16, CD16A, FcγRIII, and FcγRIIIA are used interchangeably herein.

[0126] CD16 transmits signals by binding to the common gamma chain of Fc receptors, which has an immunoreceptor tyrosine-based activation motif (ITAM). The signal transmitted by CD16 mediates the ADCC effect of NK cells. During the ADCC process, the Fc region of the antibody bound to the surface of the target cell is recognized and bound by CD16 on the surface of the NK cell. The cross-linking of CD16 and the Fc region of the antibody activates NK cells, causing the NK cells to release cytotoxic factors (also known as degranulation), thereby causing the target cells to die through apoptosis. Therefore, compared with NK cells that do not express or express low levels of CD16, NK cells with increased CD16 expression exhibit a stronger ADCC effect.

[0127] The present inventors have developed a method for expanding NK cells in vitro, which can also increase the CD16 + The number of NK cells.

[0128] Therefore, the present invention provides mature NK cells, in particular expanded NK cell populations (typically mature NK cells), which are CD56 + 、CD45 + 、CD3 - and / or CD19 - (e.g. CD56 + 、CD45 + 、CD3 - and CD19 - ), and compared with the control expanded NK cell population, its CD16 + The control expanded NK cell population can be a population produced by any conventional NK cell expansion method, or a population produced by a method corresponding to the method described herein but not including the pre-differentiation step (step (a)).

[0129] Thus, the present invention provides an expanded NK cell population wherein at least 10% of the NK cells in the population are CD16 + NK cells, at least 15% of which are CD16 + NK cells, at least 20% of which are CD16 + NK cells, at least 25% of which are CD16 + NK cells, at least 30% of which are CD16 + NK cells, at least 35% of which are CD16 + NK cells, at least 40% of which are CD16 + NK cells, at least 45% of which are CD16 + NK cells, at least 50% of which are CD16 +NK cells, at least 60% of which are CD16 + NK cells, at least 70% of NK cells are CD16 + NK cells, at least 80% of NK cells are CD16 + NK cells, until 100% of the NK cells in the population are CD16 + NK cells. Preferably, at least 15% of the NK cells are CD16 + NK cells, more preferably at least 20% of the NK cells are CD16 + NK cells, more preferably at least 25% of the NK cells are CD16 + NK cells, even more preferably at least 30% of the NK cells are CD16 + NK cells, and even more preferably at least 40% of the NK cells are CD16 + NK cells. Such amplified NK cell population can generally be obtained by the method of the present invention. Standard techniques can be used to determine the expression of CD16, examples of which are known in the art, and those of ordinary skill in the art can routinely select appropriate techniques. Non-limiting examples of suitable techniques include flow cytometry, cell imaging, and enzyme-linked immunosorbent assay (ELISA).

[0130] CD16 of the present invention + One advantage of NK cells is that they exhibit increased CD16 expression without the need for the introduction of exogenous nucleic acids (e.g., by transduction or transfection). This is in contrast to conventional methods of expanding NK cells with increased CD16 expression, which require the expression of exogenous CD16 transgenes to achieve even minimal increases in CD16 expression. Alternatively, and in addition, the CD16 of the present invention + Another advantage of NK cells is that CD16 can be obtained without or with less purification and / or concentration than conventionally produced expanded NK cell populations. + Thus, a conventionally produced expanded NK cell population is distinct from the population of the present invention, even if the NK cells in the population are treated to increase CD16 expression, and / or the conventionally produced NK cell population is treated to select for and / or enrich for CD16. + NK cells, because: (i) CD16 + NK cells are genetically modified and contain exogenous nucleic acids, which is a disadvantage from a clinical / Good Manufacturing Practice (GMP) perspective; and / or (ii) when CD16 + When NK cells are extensively purified and / or concentrated, this may cause damage to the purified and / or concentrated NK cells.

[0131] Therefore, the present invention provides a method comprising an increased number of CD16 + NK cell population (also referred to herein as CD16 + NK cell population), of which CD16 + NK cells have not been exposed to exogenous nucleic acid (typically nucleic acid encoding CD16). + The NK cell population of NK cells may comprise CD16 cells that do not contain exogenous nucleic acid encoding CD16. + NK cells. In view of the potential clinical application value of the NK cells of the present invention, the NK cells of the present invention contain an increased number of CD16 + The NK cell population may comprise CD16 cells that do not contain any exogenous nucleic acid. + NK cells. The term "exogenous nucleic acid" includes naked nucleic acid (e.g., plasmid), or viral vectors (e.g., adeno-associated virus or lentiviral vectors) for introducing nucleic acids of interest (e.g., nucleic acids encoding CD16) into NK cells. Alternatively, as a supplement, the present invention provides a method comprising increasing amounts of CD16 + NK cell population (also referred to herein as CD16 + NK cell population), wherein CD16 generated by conventional methods (including methods using exogenous nucleic acid encoding CD16) + Compared with NK cells, CD16 + The NK cells are not purified and / or concentrated or are only minimally purified and / or concentrated.

[0132] As described in this article, CD16 + NK cells are more efficient in mediating ADCC because it is the binding of CD16 to the Fc portion of the antibody that triggers the degranulation of NK cells, which in turn triggers ADCC. + The present invention provides an expanded NK cell population with increased NK cell number, and provides a CD16 +NK cells and amplified NK cell colonies. The enhancing of this ADCC can be compared with suitable reference or control NK cells or NK cell colonies. A control method can be any standard method known in the art for producing NK cell colonies. For example, a control method can use traditional adoptive transfer technology, rather than the method of the present invention. An example of suitable control NK cells or NK cell colonies is the NK cells or NK cell colonies produced by a corresponding method, in which hematopoietic progenitor cells (HPC) are not cultured in a culture medium that does not induce HPC to NK cell differentiation, or wherein HPC is cultured in a culture medium that does not induce HPC to NK cell differentiation less than 2 days. In other words, an example of suitable control NK cells or NK cell colonies is the NK cells or NK cell colonies produced by a corresponding method that omits step (a) described herein. The NK cells and NK cell colonies produced by this control / standard method can be used as control cells and colonies as described herein.

[0133] The expanded NK cell population according to the present invention or the CD16 in said population is compared to a suitable reference or control NK cell or NK cell population. + NK cells can exhibit at least a 25% increase in ADCC, for example, at least a 30% increase in ADCC, at least a 40% increase in ADCC, at least a 50% increase in ADCC, at least a 70% increase in ADCC, or at least an 80% increase in ADCC, preferably at least a 50% increase in ADCC, more preferably at least a 60% increase in ADCC, and further preferably at least a 70% increase in ADCC.

[0134] Expanded NK cell population

[0135] As disclosed herein, the present invention provides methods for producing a method comprising an increased number of CD16 + Methods for expanding NK cell populations (interchangeably referred to herein as expanding CD16 + NK cell population, CD16 + NK cell population, NK cell population or expanded NK cell population). In this article, the CD16 + Any disclosure related to NK cells can also be applied to the CD16 + NK cell population.

[0136] Therefore, the present invention provides a CD16 + NK cell population. Generally, the CD16 +In some embodiments, the NK cell colony comprises immature NK cells (iNK), mature NK cells (mNK) and / or conventional NK cells (cNK), or a combination thereof. The colony can comprise hematopoietic progenitor cells (HPC), such as hematopoietic stem cells (HSC), common lymphoid progenitor cells (CLP) and / or NK precursor cells (NKP), or a combination thereof, although relative to the quantity of NK cells, the quantity of these cells is generally less, because in the colony, the great majority of these HPCs have been differentiated into NK cells. The colony can comprise other immune and / or non-immune cells. Equally, relative to the NK cell population present in the colony, the quantity of any such cell is generally less.

[0137] As a non-limiting example, the CD16 + At least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more, up to 100% of the cells in the NK cell population can be NK cells. + At least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% of the cells in a NK cell population are NK cells.

[0138] CD16 of the present invention + At least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more, up to 100% of the cells in the NK cell population are mature NK cells (i.e., mNK cells and / or cNK cells). Preferably, the CD16 + At least 80%, more preferably at least 85%, even more preferably at least 90%, still even more preferably at least 95%, and still even more preferably at least 98% or more of the cells in a NK cell population are mature NK cells.

[0139] The number of hematopoietic progenitor cells (including hematopoietic stem cells, common lymphoid progenitor cells and / or NK progenitor cells) can be less than CD16 + 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of the total number of cells in the NK cell population. Typically, the number of hematopoietic progenitor cells (including hematopoietic stem cells, common lymphoid progenitor cells and / or NK precursor cells) is less than CD16 +20%, preferably less than 15%, more preferably less than 10%, even more preferably less than 5%, and still even more preferably less than 2% or less of the total number of cells in the NK cell population.

[0140] Other immune and / or non-immune cells may be less abundant than CD16 + 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of the total number of cells in the NK cell population. Typically, other immune and / or non-immune cells are less numerous than CD16 + 20%, preferably less than 15%, more preferably less than 10%, even more preferably less than 5%, and still even more preferably less than 2% or less of the total number of cells in the NK cell population.

[0141] As described herein, the CD16 NK cell populations generated by the methods of the present invention are significantly higher than those generated by conventional methods. + NK cell populations have several advantages. In particular, the method of the present invention is able to generate CD16 + In addition, compared with the population obtained by traditional methods, the population of the present invention has more CD16 + And therefore the proportion of functional (preferably fully functional) NK cells is higher, whereas in the population obtained by traditional methods, a large number of NK cells are "exhausted".

[0142] As used herein, in the context of NK cells, the term "exhaustion" refers to that NK cells or amplified NK cell colonies have lost at least some of their effector functions, such as cytotoxic function, cytokine production and / or ADCC function. Therefore, the NK cells exhausted or the NK cell colonies expanded may show impaired viability, impaired cytotoxic function, altered or impaired cytokine production and / or impaired ADCC function. For example, the NK cells exhausted or the NK cell colonies exhausted may show at least a 50% reduction in one of their effector functions. For example, cytokine secretion is at least reduced by 50%, ADCC is at least reduced by 50% and / or cytotoxic activity is at least reduced by 50%. These numerical values ​​can be quantified relative to any suitable control defined herein. Any suitable technique can be used to determine the effector function, thereby quantifying it and determining the degree of reduction therein. Suitable techniques are known in the art. Alternatively, as a supplement, the NK cells exhausted may show changes in marker expression, such as increased expression of one or more inhibitory receptors (as described herein) and / or reduced expression of one or more activating receptors (as described herein). Increased expression of NKG2A and / or Tim3 can be used as a marker of NK cell exhaustion. Likewise, the expression of these markers can be quantified relative to any suitable control as defined herein.

[0143] In contrast, in the context of NK cells, the terms "functional" and "fully functional" refer to NK cells or expanded NK cell populations that have all the expected effector functions in response to a given immune challenge. Thus, (fully) functional NK cells or expanded NK cell populations will typically exhibit cytotoxic function, cytokine production and / or ADCC, as observed when NK cells are activated in vivo in response to an immune challenge, and will typically exhibit enhanced viability compared to NK cells produced using traditional methods. Alternatively, in addition, (fully) functional NK cells may exhibit altered marker expression, such as increased expression of one or more activating receptors (as described herein) and / or decreased expression of one or more inhibitory receptors (as described herein). As a non-limiting example, a functional (mature) human NK cell may be CD56 + and / or CD45 + , preferably CD56 + and CD45 + .

[0144] Furthermore, as described herein, the (fully) functional NK cells of the present invention are CD16 + and compared to any suitable control, the CD16 + NK cell populations contain increased numbers of CD16 + NK cells.

[0145] Alternatively, or in addition to detecting and / or quantifying the expression of CD16 to determine the function of NK cells, degranulation assays can also be used to determine the cytotoxicity of NK cells incubated with "target cells". Degranulation assays involve analyzing the expression of CD107a in the NK cell population. The amount of CD107a correlates with cytokine secretion and NK cell-mediated target cell lysis. NK cells can also be analyzed for expression of interferon-gamma (IFN-gamma), which is the main cytokine secreted when functional NK cells are activated. Functional NK cells should express similar or higher levels of CD107a and IFN-gamma compared to controls.

[0146] In addition, or as a supplement, flow cytometry can also be used to quantify the cell death in the target cell population, thereby determining the cytotoxicity of NK cells. The target cell population can optionally be pre-labeled with a fluorescent marker. Using flow cytometry to quantify the cell death in the target cell population may be a preferred method for determining the cytotoxicity of NK cells.

[0147] CD16 produced by the method of the present invention + CD16 in NK cell population + Any increase in NK cell number / function can be compared to the NK cell number / function of an NK cell population obtained by a control method described herein.

[0148] Compared with the traditionally prepared NK cell population, the CD16 + NK cell populations contained significantly fewer exhausted NK cells and / or CD16 - NK cells, but contain a higher proportion of fully functional CD16 + NK cells, which advantageously allows a smaller number of cells to be used to treat a patient.

[0149] As described herein, the methods of the present invention produce NK cell populations with a higher proportion of (fully) functional NK cells than conventional methods that produce large "exhausted" NK cell populations. +In the NK cell population, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more, up to 100% of the NK cells in the expanded NK cell population of the present invention are (fully) functional. According to any definition herein (such as marker and / or effector function definition), typically at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 98% or more of the NK cells in the expanded NK cell population of the present invention are fully functional. The function of the NK cells of the present invention is preferably associated with the expression of CD16. As a non-limiting example, in a CD16 + At least 40% of the NK cells in the NK cell population are likely to be functional and at least 40% of the NK cells are CD16 + Alternatively, the proportion of functional NK cells may be higher than that of CD16 + As a non-limiting example, in a CD16 + In the NK cell population, at least 60% of the NK cells may be functional and at least 40% of the NK cells are CD16 + Or, CD16 + The proportion of NK cells may be higher than the proportion of functional NK cells. As a non-limiting example, in a CD16 + At least 40% of the NK cells in the NK cell population are likely to be functional and at least 60% of the NK cells are CD16 + .

[0150] The expanded NK cell population of the present invention can be prepared by any of the methods disclosed herein. + NK cell populations are prepared by the in vitro methods disclosed herein.

[0151] Notch ligands

[0152] The Notch signaling pathway is primarily associated with promoting T cell development and inhibiting concurrent B cell development. Mammals have four Notch receptors—Notch1, Notch2, Notch3, and Notch4—all of which are single-pass, heterodimeric transmembrane proteins. Mammals have two classical Notch ligands: Delta-type and Jagged-type, collectively referred to as DSL ligands. There are three Delta-like ligands (DLLs): DLL1, DLL3, and DLL4, and two Jagged (JAG) ligands: JAG1 and JAG2. DLL and JAG ligands typically contain the following domains: a module (MNNL) domain at the N-terminus of the Notch ligand, a Delta / Jagged / Lag-2 (DSL) domain, and epidermal growth factor (EGF) repeats. DLL3 contains six EGF repeats. DLL1 and DLL4 contain eight EGF repeats. JAG1 and JAG2 contain 16 EGF repeats. In addition, there are numerous non-classical ligands that may be membrane-bound or secreted.

[0153] Unless otherwise expressly stated herein, the Notch ligand referred to herein refers to any Notch ligand, such as a ligand of Notch1, Notch2, Notch3 and / or Notch 4, preferably at least a ligand of Notch1. The protein sequence of human Notch1 is shown in SEQ ID NO: 10 (GenBank Accession No. CR457221, Version CR457221.1), and the corresponding cDNA sequence is shown in SEQ ID NO: 9 (GenBank Accession No. CR457221, Version CR457221.1). Generally, the Notch ligand used in the present invention is a classical Notch ligand. In some preferred embodiments, the Notch ligand is a DLL, more preferably DLL4. The protein sequence of human DLL4 is shown in SEQ ID NO: 8

[0154] (GenBank Accession No. AF253468, Version AF253468.1), the corresponding mRNA sequence is shown in SEQ ID NO: 7 (GenBank Accession No. AF253468, Version AF253468.1).

[0155] The Notch ligands mentioned herein also include fragments thereof, provided that the fragment retains the Notch binding and activation activity of the Notch ligand from which it is derived. The inventors have previously described Notch ligand fragments suitable for use in the present invention (see WO2018 / 178666, which is incorporated herein by reference in its entirety, particularly pages 15 and 16 and the Examples section). Preferred examples of Notch ligand fragments include Notch ligand (N-EGF1) and Notch ligand (N-EGF2), such as DLL4 (N-EGF1) and DLL4 (N-EGF2), particularly DLL4.

[0156] Alternatively, in addition, Notch ligands, fragments thereof, or molecules that mimic the effects (e.g., functions / activities) of Notch ligands (e.g., DLL4) may comprise modifications, such as amino acid mutations, that alter (usually increase) the affinity of the ligand / fragment / mimetic for its Notch receptor. Techniques for identifying such modifications are known in the art. For example, yeast surface display technology can be used to identify amino acids that increase the affinity of Notch ligands / fragments / mimetic. Similarly, the inventors have previously described such modifications (see WO2018 / 178666, which is incorporated herein by reference in its entirety, particularly page 16). In some preferred embodiments, the DLL4 ligands, fragments thereof, or mimics of the present invention comprise amino acid substitutions G28S, F107L, and L206P, more preferably G28S, F107L, N118I, I143F, H194Y, L206P, and / or K215E.

[0157] As a further non-limiting example, a functional fragment of DLL4 comprises at least residues 65 to 114 and residues 179 to 219 of full-length DLL4, preferably maintaining the correct conformation to interact with Notch ligands.

[0158] In addition, the present invention encompasses the use of molecules (also referred to herein as mimics) that mimic the effects (e.g., activity / function) of Notch ligands. For example, the present invention encompasses the use of peptides, cyclic peptides, peptoids, and peptidomimetics that can mimic the effects of desired Notch ligands (e.g., DLL4). Peptidomimetics may have advantages over natural peptides in terms of stability and bioavailability. Peptidomimetics can modify the main chain or side chain of the parent peptide to achieve a specific biological function. Examples of peptidomimetics include, but are not limited to, peptoids and β-peptides, as well as peptides incorporating D-amino acids.

[0159] Methods for preparing synthetic peptides and peptidomimetics (such as peptoids), as well as sequences of classical and non-classical Notch ligands, are known in the art. Therefore, it is routine for those skilled in the art to use known techniques and prepare suitable molecules that mimic the effects of the desired Notch ligand based on known Notch ligand sequences. As a non-limiting example, peptidomimetics can be designed to interact with key residues of Notch (such as Notch1) known to be involved in binding to DLL4, such as residues 415 (E415), 418 (L418), 420 (A420), 421 (N421), 422 (P422), 424 (E424), 425 (H425), 436 (F436), 447 (P447), 448 (R448), 450 (E450), 452 (D452), 469 (D469), 477 (I477), 480 (P480), or any combination thereof.

[0160] The methods of the present invention may include the use of any Notch ligand or fragment thereof that can increase NK cell production, or a molecule that mimics its effects. Notch ligands or fragments thereof can be used in the methods of the present invention, wherein the methods do not include the use of compounds described herein that inhibit REV-ERB activity.

[0161] Notch ligands or fragments thereof can be used in the methods of the present invention, which also include the use of a compound that inhibits REV-ERB activity, or a compound that causes a change in the post-translational modification of E4bp4 and thereby increases E4bp4 activity as disclosed herein. In particular, Notch ligands or fragments thereof can be used in the methods of the present invention, which also include the use of a compound that inhibits REV-ERB activity, or a compound that causes a change in the post-translational modification of E4bp4 and thereby increases E4bp4 activity, wherein the Notch ligands or fragments thereof have a synergistic effect with the compounds of the present invention disclosed herein that inhibit REV-ERB activity, or with the compounds that cause a change in the post-translational modification of E4bp4 and thereby increases E4bp4 activity.

[0162] The inventors have previously shown that E4bp4 directly binds to the regulatory region of the Notch1 gene in vivo, thereby enhancing Notch's transcriptional regulation, and that Notch1 expression is significantly reduced in E4bp4 knockout (E4bp4- / -) mice. Building on this, the inventors discovered that short-term exposure of Notch ligands to mouse hematopoietic stem cells (HSCs) and very early progenitor cells promotes the development of NK cells, even in the absence of the key transcription factor E4bp4. Furthermore, the inventors have shown that the Notch ligand Delta-like ligand 4 (DLL4) is particularly effective in stimulating NK cell expansion.

[0163] Therefore, the present invention relates to increasing CD16 expression in an expanded NK cell population by exposing hematopoietic progenitor cells (HPCs) to Notch ligands in a method. + The number of NK cells or the expansion of CD16 + NK cells, the method includes the step of culturing HPCs in a medium that does not induce HPC differentiation, in particular, a medium that does not induce HPC differentiation into NK cells. The method may include exposing HPCs to Notch ligands as part of the step of culturing HPCs in a medium that does not induce HPC differentiation (in particular, does not induce HPC differentiation into NK cells). The method may also include using a compound that inhibits the action of REV-ERB as described herein. In the method, REV-ERB is inhibited

[0164] The compound can be used before HPC differentiates into NK cells, or during the HPC differentiation into NK cells.

[0165] Preferably, the compound that inhibits REV-ERB can be used before HPCs differentiate into NK cells.

[0166] In the in vitro or ex vivo methods of the present invention, exposing the HPCs to the Notch ligand may include a step of culturing the HPCs in the presence of the Notch ligand, such as step (a) of the methods described herein. For in vivo methods, this may include administering the compound together with the Notch ligand. In a preferred embodiment, the Notch ligand is DLL4, or a fragment or variant thereof that retains DLL4 function. All disclosures herein relating to the use of Notch ligands also apply without reservation to the preferred Notch ligand DLL4 and its fragments / variants / mimics.

[0167] Variant Notch ligands and / or fragments / mimics thereof can be used according to the present invention. The variant Notch ligands / fragments / mimics of the present invention generally retain at least the activity of the corresponding Notch ligands / fragments / mimics of the present invention. Thus, for example, the variant DLL4 ligands or fragments thereof of the present invention retain the ability of the corresponding DLL4 molecule to bind Notch1 and / or enhance NK cell production. In some embodiments, the variant DLL4 ligands / fragments / mimics have higher activity than the corresponding unmodified DLL4 ligands / fragments / mimics. The inventors have previously described such variants (see WO2018 / 178666, which is incorporated herein by reference in its entirety, particularly pages 17 and 18). As a non-limiting example, a Notch ligand / fragment / mimetics / variant (e.g., a DLL4 ligand / fragment / mimetics / variant) may bind to Notch1 with a dissociation constant (KD) value of less than 1 micromolar (μM), less than 900 nanomolar (nM), less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM or less, preferably less than 500 nM, less than 400 nM, less than 300 nM or less. In some embodiments, the variant Notch ligand / fragment / mimetics (e.g., variant DLL4 ligand / fragment / mimetics) can increase the number of NK cells or result in an increase in NK cell production by at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 3-fold or more compared to the corresponding unmodified Notch ligand / fragment / mimetics. A variant Notch ligand / fragment / mimetics (e.g., a variant DLL4 ligand / fragment / mimetics) can increase the number of NK cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 300% or more compared to the corresponding unmodified DLL4 ligand / fragment / mimetics.

[0168] The Notch ligands / fragments / mimics of the present invention may be labeled (or tagged). Any suitable label may be used. Suitable labels are known in the art.

[0169] E4bp4

[0170] E4bp4 (also known as Nfil3) is a basic leucine zipper protein transcription factor that is involved in the regulation of interleukin-3 (IL-3) expression and in coordinating the circadian clock. The mRNA sequence of the human E4bp4 gene is shown in SEQ ID NO: 1 (Genbank accession number X64318, version X64318.1), and the corresponding amino acid sequence is shown in SEQ ID NO: 2. Figure 1 As shown, E4bp4 is expressed in common lymphoid progenitor cells (CLP) and plays a key role in the generation of natural killer cells (NK cells) from hematopoietic stem cell progenitors. Mice in which the E4bp4 gene is knocked out have no functional NK cells, but the number of T cells and B cells is normal. In contrast, in vitro, overexpression of E4bp4 in hematopoietic stem cells (HSC) increases the production of NK cells. Therefore, E4bp4 is a lineage-committing factor that controls the development of hematopoietic stem cells into NK precursor cells (NKP) ( Figure 1 The key function of E4bp4 in NK cells is specific to the early stages of the developmental pathway, as specific knockout of E4bp4 in peripheral mature NK cells (mNK) did not affect the number of NK cells or the response to cytomegalovirus infection. In addition, E4bp4 also regulates other transcription factors that are critical for NK cell development, such as Id2 and Eomes.

[0171] Although interleukin-7 (IL-7) and interleukin-15 (IL-15) have been shown to regulate E4bp4 expression, overall, little is known about how extrinsic or intrinsic stimuli affect E4bp4. Transcription factors like E4bp4 can be difficult targets due to their structure and function. For example, they often lack enzymatic activity or cofactor binding sites. However, the present inventors have previously demonstrated that E4bp4 expression can be increased using a compound that inhibits REV-ERB activity (see WO2018 / 158587, particularly the Examples section therein, which is incorporated herein by reference in its entirety). Furthermore, the present inventors have demonstrated that increasing E4bp4 expression using a REV-ERB inhibitor leads to an increase in the number of NK cells. Without being bound by theory, REV-ERB binds to porphyrin heme, and it is this property that is believed to make REV-ERB a druggable target (see below). Thus, the inventors have previously shown that by targeting REV-ERB and inhibiting its activity, it is possible to increase the expression of E4bp4, thereby increasing the number of NK cells. Therefore, compounds that inhibit the action of REV-ERB and thereby increase E4bp4 expression and NK cell numbers can be used in the methods of the present invention to increase CD16 + The number of NK cells.

[0172] Increased E4BP4 expression

[0173] As described herein, the present invention provides a method for preparing and expanding CD16 + Method for increasing CD16 in expanded NK cell population + Methods for increasing the number of NK cells, as well as therapeutic methods and uses for increasing the number of NK cells in patients in need thereof. As disclosed herein, the methods and uses may involve the use of compounds that inhibit the action of REV-ERB. Typically, the compounds act by increasing the expression of E4bp4.

[0174] The increase in E4bp4 expression can be measured relative to a control. Thus, the expression of E4bp4 in a hematopoietic progenitor cell (HPC) sample, an expanded NK cell population, or a sample obtained from an individual / patient to be treated according to the present invention can be compared to the expression of E4bp4 in a control. Expression can be quantified based on gene and / or protein expression and can be compared to the expression of a control (e.g., a housekeeping gene or protein). The actual amount of E4bp4 gene, mRNA transcript, and / or protein, such as the mass, molar amount, concentration, or molar concentration of the E4bp4 gene, mRNA transcript, and / or protein, or the number of mRNA molecules per cell in an HPC sample, an expanded NK cell population, or a sample obtained from an individual / patient to be treated according to the present invention can be assessed and compared and compared to the corresponding value of the control. Alternatively, an HPC sample, an expanded CD16 + The expression of the E4bp4 gene and / or protein in a population of NK cells or in a sample obtained from an individual / patient to be treated according to the present invention is compared to the expression of a control without quantifying the mass, molar amount, concentration or molar concentration of one or more genes and / or proteins.

[0175] Controls can be as described herein. In the context of E4bp4 expression, a control can be an equivalent population or sample that does not achieve increased E4bp4 expression. As a non-limiting example, in the case where an individual / patient receives a compound that inhibits REV-ERB activity to increase E4bp4 expression, a suitable control can be a different individual who has not received the compound, or the same individual before receiving the compound. Conventional methods for in vitro expansion of NK cells, including known methods, can be considered control methods according to the present invention.

[0176] In the context of the present invention, reference to increasing the expression of E4bp4 is understood to mean that the expression of E4bp4 is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200% compared to a control. Typically, the expression of E4bp4 is increased by at least 50%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% or more compared to a control.

[0177] Reference to increasing the expression of E4bp4 is understood to mean that the expression of E4bp4 is increased by at least 1.5-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or more, relative to a control. Typically, the expression of the E4bp4 gene is increased by at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, or more, relative to a control. Typically, the expression of the E4bp4 protein is increased by at least 2-fold, at least 3-fold, preferably at least 5-fold, and more preferably at least 6-fold, or more, relative to a control.

[0178] According to the present invention, the expression of the E4bp4 gene and / or protein can be determined by quantitative and / or qualitative analysis. Generally, gene expression can be expressed as mRNA levels.

[0179] According to the present invention, the expression level of the E4bp4 gene and / or protein includes the mass of the E4bp4 mRNA transcript and / or protein, the molar amount of the E4bp4 gene, mRNA transcript and / or protein, the concentration of the E4bp4 gene and / or protein, and the molar concentration of the E4bp4 gene and / or protein. The expression level can be expressed in any suitable unit. For example, the concentration of the E4bp4 gene and / or protein can be expressed in picograms per milliliter (pg / ml), nanograms per milliliter (ng / ml), or micrograms per milliliter (μg / ml).

[0180] According to the present invention, the expression level of the E4bp4 gene and / or protein can be measured directly or indirectly.

[0181] Any suitable technique can be used to determine the relative expression of the E4bp4 gene and / or protein according to the present invention relative to a control. Suitable standard techniques are known in the art, such as Western blotting, enzyme-linked immunosorbent assay (ELISA), and reverse transcription-real-time quantitative polymerase chain reaction (RT-qPCR).

[0182] Compared to a control, the expression level of the E4bp4 gene and / or protein can be increased for at least 6 hours, at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, or at least 1 week. Preferably, the expression level of the E4bp4 gene and / or protein is increased for at least 12 to 72 hours. Typically, this is assessed relative to the last administration of a compound that inhibits REV-ERB activity.

[0183] Compared to a control, the expression level of the E4bp4 gene and / or protein can be increased when the cultured NK cell precursors are passaged at least once, twice, three times, four times, five times, ten times, 20 times, 30 times, 40 times, or more. The expression level of the E4bp4 gene and / or protein can change indefinitely.

[0184] REV-ERB

[0185] REV-ERB proteins are members of the nuclear receptor family of intracellular transcription factors. The mRNA sequence of the human REV-ERBα gene (Nr1d1) is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4 (Genbank Accession No. NM_021724, Version NM_021724.4). The mRNA sequence of the human REV-ERBβ gene (Nr1d2) is shown in SEQ ID NO:5 (Genbank Accession No. AB307693, Version AB307693.1), and the corresponding amino acid sequence is shown in SEQ ID NO:6. REV-ERB regulates the circadian clock and is also involved in the regulation of cartilage breakdown.

[0186] The inventors have previously demonstrated that inhibition of REV-ERB activity is sufficient to cause a significant increase in E4bp4 expression, which in turn leads to NK cell expansion and thus an increase in the number of NK cells (see WO2018 / 158587, in particular the Examples section therein, which is incorporated herein by reference in its entirety). Inhibition of REV-ERB activity can increase the number of NK cells, and the NK cells produced are generally (fully) functional as defined herein. The effects of REV-ERB inhibition are mediated in an E4pb4-dependent manner. Without being bound by theory, it is believed that inhibition of REV-ERB activity leads to an increase in E4bp4 expression (the expression of E4bp4 is normally inhibited by REV-ERB), and that E4bp4 acts to stimulate the production of NK cells (e.g. Figure 1 In particular, the present inventors have previously demonstrated that a class of small molecules, especially SR8278 (1,2,3,4-tetrahydro-2-[[5-(methylthio)-2-thienyl]carbonyl]-3-isoquinolinecarboxylic acid ethyl ester, CAS No.: 1254944-66-5), can bind to the porphyrin heme portion of REV-ERB, thereby inhibiting the activity of REV-ERB and increasing the number of NK cells. The present inventors have also previously developed new small molecules that can effectively inhibit REV-ERB, especially REV-ERB inhibitors that are more effective than SR8278. These improved REV-ERB antagonists are described in WO2020 / 002911, in particular the Examples section thereof, which are incorporated herein by reference in their entirety. Specifically, Compound 11 and Compound 7 in WO2020 / 002911 are included within the scope of the present invention.

[0187] Inhibition of REV-ERB activity

[0188] In some embodiments, the present invention relates to the use of compounds to inhibit the effects of REV-ERB, i.e., compounds that inhibit the activity of REV-ERB. The activity of REV-REB can be inhibited by any suitable means. Suitable standard techniques are known in the art. Inhibition can occur by any suitable mechanism, for example, depending on the properties of the compound used (see below), such as steric interference or inhibition of any direct or indirect interaction of REV-ERB. In the context of the present invention, a REV-ERB inhibitor (interchangeably referred to herein as a REV-ERB antagonist) refers to any compound that inhibits, reduces, suppresses or eliminates the effect of REV-ERB, whether partially or completely.

[0189] The reduction in REV-ERB activity can be measured relative to a control. +The activity of REV-ERB in a population of NK cells or in a sample obtained from an individual / patient to be treated according to the present invention is compared to the activity of REV-ERB in a control. The activity can be quantified using any suitable method, such as binding of REV-ERB to the E4bp4 gene, or based on E4bp4 expression as defined herein. Any suitable technique or method can be used to quantify REV-ERB activity. Suitable techniques are known in the art, such as luciferase assays for quantifying reporter gene expression.

[0190] The control can be as described herein. In the context of REV-ERB activity, the control can be an equivalent population or sample to which a REV-ERB inhibitory compound has not been added, such as a sample obtained from a different individual who has not received the compound, or a sample from the same individual before receiving the compound. Conventional methods for in vitro expansion of NK cells, including known methods, can be considered control methods according to the present invention.

[0191] In the context of the present invention, reference to inhibition of REV-ERB activity is to be understood as a reduction in REV-ERB activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, up to complete (100%) inhibition of REV-ERB activity, as compared to a control. Typically, REV-ERB activity is reduced by at least 50%, preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, even more preferably by at least 95% or more, as compared to a control.

[0192] The activity of REV-ERB can be determined by quantitative and / or qualitative assays and can be measured directly or indirectly.

[0193] Any suitable technique can be used to determine the activity of REV-ERB relative to a control. Suitable standard techniques are known in the art, for example by quantifying the expression of E4bp4 and / or luciferase assays.

[0194] The activity of REV-ERB can be inhibited for at least 6 hours, at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, or at least 1 week compared to a control. Preferably, the activity of REV-ERB is reduced for at least 12 to 72 hours. Typically, this is assessed relative to the last administration of a compound that inhibits REV-ERB activity.

[0195] The activity of REV-ERB can be inhibited when the cells are passaged at least once, at least twice, at least three times, at least four times, at least five times, at least ten times, at least 20 times, at least 30 times, at least 40 times, or more (whether in vivo, in vitro, or in vitro culture) compared to a control. The activity of REV-ERB can be inhibited, and / or the expression level of the E4bp4 gene and / or protein can be altered indefinitely.

[0196] In the context of the present invention, any reference to inhibiting REV-ERB activity is to be understood as inhibiting the activity of REV-ERBα and / or REV-ERBβ. In preferred embodiments, the activity of both REV-ERBα and REV-ERBβ is inhibited. Thus, the present invention relates to compounds that inhibit REV-ERB activity, including compounds that inhibit the activity of REV-ERBα (i.e., REV-ERBα inhibitors, also known as REV-ERBα antagonists) and / or compounds that inhibit the activity of REV-ERBβ (i.e., REV-ERBβ inhibitors, also known as REV-ERBβ antagonists). In preferred embodiments, the present invention relates to compounds that inhibit the activity of both REV-ERBα and REV-ERBβ (i.e., REV-ERBα and REV-ERBβ inhibitors, also known as REV-ERBα and REV-ERBβ antagonists).

[0197] REV-ERB antagonists / inhibitors

[0198] The REV-ERB inhibitory compounds of the present invention may be specific for REV-ERB. Specificity should be understood as meaning that the compound binds to REV-ERBα and / or REV-ERBβ without significantly cross-reacting with any other molecule, particularly any other protein. For example, a modulator specific for REV-ERBα and / or REV-ERBβ may not significantly cross-react with human neutrophil elastase. Cross-reactivity can be assessed by any suitable method. A REV-ERBα and / or REV-ERBβ inhibitor is considered to have significant cross-reactivity with a molecule other than REV-ERBα and / or REV-ERBβ if the inhibitor binds to that molecule at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% of the strength with which the inhibitor binds to that molecule. Inhibitors specific for REV-ERBα and / or REV-ERBβ may bind to other molecules (e.g., human neutrophil elastase) with less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% of their binding strength to REV-ERBα and / or REV-ERBβ. Preferably, the inhibitor binds to other molecules with less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1% of its binding strength to REV-ERBα and / or REV-ERBβ.

[0199] The REV-ERB inhibitory compounds of the present invention may have off-target effects. Off-target effects refer to activity produced on targets other than REV-ERB. Generally, if the activity of a compound on non-REV-ERB targets is not obvious compared to its activity on REV-ERB, then the compound with off-target effects is also within the scope of the present invention. Whether the off-target effect is obvious may depend on the intended use of the compound. As a non-limiting example, for compounds used in the in vitro methods disclosed herein, a compound that may have off-target effects on the central nervous system may not have obvious off-target effects, but for the in vivo therapeutic indications disclosed herein, its off-target effects may be significant (depending on the extent of the off-target effect). The presence and extent of any potential off-target effects can be easily assessed using standard methods known in the art.

[0200] According to the present invention, any suitable inhibitor can be used, such as small molecules, proteolysis targeting chimera (PROTAC) agents, double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (microRNA), antisense (single-stranded) RNA, peptides and peptidomimetics, antibodies, aptamers and ribozymes. Preferred inhibitors include small molecules and PROTAC agents.

[0201] Small molecules

[0202] As described herein, small molecules can be used to inhibit REV-ERB activity. As defined herein, small molecules are low molecular weight compounds, typically organic compounds. Generally, small molecules have a maximum molecular weight of 900 Daltons (Da), which allows them to diffuse rapidly across cell membranes. In some embodiments, the maximum molecular weight of a small molecule is 500 Da. Typically, a small molecule is about 1 nanometer (nm) in size.

[0203] According to the present invention, small molecules may be able to inhibit the activity of REV-ERB by binding to the porphyrin heme portion of REV-ERB. Thus, in some preferred embodiments, the compound that inhibits the action of REV-ERB according to the present invention is a compound that binds to the porphyrin heme portion of REV-ERB, thereby inhibiting the activity of REV-ERB. Alternatively, the small molecule may also act through a different mechanism, for example, by binding to a non-heme portion of REV-ERB. Standard techniques for producing small molecules are known in the art, and these small molecules can then be readily tested for their inhibitory activity against REV-ERB as described herein.

[0204]

[0205] Structure of porphyrin heme

[0206] The present invention encompasses the use of small molecule REV-ERB antagonists as described in WO2018 / 158587 and WO2018 / 178666 (both of which are incorporated herein by reference in their entirety), as well as variants of such small molecule REV-ERB antagonists that retain the inhibitory function of the small molecule REV-ERB antagonists from which they are derived. Non-limiting examples include SR8278,

[0207] GSK1362 and 4-[[[1-(2-fluorophenyl)cyclopentyl]amino]methyl]-2-[(4-methylpiperazin-1-yl)methyl]phenol (also referred to herein as ARN5187), ethyl 2-(5-methylfuran-2-carbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate, 4-((4-chlorobenzyl)((5-nitrothiophen-2-yl)methyl)amino)-N-phenyl Piperidine-1-carboxamide, 4-(((1-(4-fluorophenyl)cyclopentyl)amino)methyl)-2-((4-methylpiperazin-1-yl)methyl)phenol, 1-(2-fluorophenyl)-N-(3-((1-methylpiperidin-4-yl)methyl)benzyl)cyclopentane-1-amine and 1-(4-fluorophenyl)-N-(3-((1-methylpiperidin-4-yl)methyl)benzyl)cyclopentane-1-amine. Preferably, the present invention encompasses the use of improved REV-ERB antagonists as described in WO2020 / 002911, in particular the Examples section thereof, which are incorporated herein by reference in their entirety. Specifically, the present invention encompasses the use of Compound 11 and Compound 7 in WO2020 / 002911. Compound 11 and Compound 7 in WO2020 / 002911 have the following structures:

[0208]

[0209] Proteolysis-Targeting Chimera (PROTAC) Reagents

[0210] As described herein, proteolysis targeting chimeras (also referred to as PROTACs or PROTAC reagents) can be used to inhibit the activity of REV-ERB. PROTAC is a heterobifunctional small molecule that can simultaneously bind to a target protein and a ubiquitin ligase, thereby causing the target protein to be ubiquitinated and degraded. In more detail, a PROTAC reagent typically comprises a ligand for a target protein (in the present invention, REV-ERB) and a ligand for an E3 ligase recognition domain. By using this PROTAC, the E3 ligase is recruited to REV-ERB bound to PROTAC, inducing ubiquitin to be transferred from the E3 ligase complex to the target protein (in the present invention, REV-ERB). Once PROTAC induces a sufficient degree of ubiquitination of the target protein, it is recognized and degraded by the proteasome.

[0211] As a non-limiting example, a PROTAC reagent can be prepared by coupling a ligand of an E3 ligase to a small molecule inhibitor described herein (preferably SR8278) via a linker. In a preferred embodiment, the PROTAC reagent comprises a ligand for the E3 RING-type Cullin ligase Hippel-Lindau protein (VHL) or casein ligase (part of the CRL4 E3RING-type Cullin ligase complex), which is connected to the small molecule inhibitor of the present invention via a linker. In some particularly preferred embodiments, the PROTAC reagent comprises a ligand for the E3 RING-type Cullin ligase Hippel-Lindau protein (VHL), which is connected to SR8278 via a linker. In other particularly preferred embodiments, the PROTAC reagent comprises a casein ligase (part of the CRL4 E3 RING-type Cullin ligase complex) and SR8278, which are connected via a linker.

[0212] Due to its mechanism of action, PROTAC reagents only require any ligand for the target protein. In the absence of a linker and E3 ligase ligand, the functional pharmacological properties of the ligand are not important. Therefore, in some embodiments, the REV-ERB inhibitory PROTAC reagent of the present invention may include a small molecule REV-ERB agonist as a ligand, such as GSK4112 (1,1-dimethylethyl N-[(4-chlorophenyl)methyl]-N-[(5-nitro-2-thienyl)methyl]glycine ester, SR6452).

[0213] double-stranded RNA

[0214] As described herein, double-stranded RNA (dsRNA) molecules can be used to inhibit the activity of REV-ERB. dsRNA molecules can be used for RNA interference (RNAi) to inhibit the activity of REV-ERB.

[0215] Using known techniques and based on knowledge of the REV-ERB sequence, dsRNA molecules can be designed to antagonize REV-ERB by targeting the corresponding RNA sequence based on sequence homology. Such dsRNAs are typically small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), or microRNAs (miRNAs). The sequence of such dsRNAs will contain a portion corresponding to a portion of the mRNA encoding REV-ERB. This portion is typically 100% complementary to the target portion in the mRNA transcribed from the REV-ERB gene, but lower levels of complementarity (e.g., 90% or higher or 95% or higher) can also be used. Typically, the percentage of complementarity is determined over a continuous length of nucleic acid residues. For example, a dsRNA molecule of the invention can have at least 80% complementarity to a target portion of an mRNA transcribed from a REV-ERB gene when measured over at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or more nucleic acid residues, up to a dsRNA molecule that has at least 80% complementarity to an mRNA transcribed from a REV-ERB gene of the invention over its entire length.

[0216] In a preferred embodiment, dsRNA is shRNA. shRNA can be delivered to natural killer (NK) cell precursors by any appropriate means. Suitable techniques known in the art include using plasmids, viruses, and bacterial vectors to deliver shRNA. Typically, shRNA is delivered using a viral vector delivery system. In a preferred embodiment, the viral vector is a lentiviral vector.

[0217] Generally speaking, once shRNA is delivered to NK precursor cells, it is transcribed and processed in the nucleus. The resulting precursor shRNA is exported from the nucleus, then processed by Dicer and loaded into the RNA-induced silencing complex (RISC). The positive (passenger) strand is degraded. The antisense (guide) strand guides RISC to an mRNA with a complementary sequence. In the case of complete complementarity, RISC cuts the mRNA. In the case of incomplete complementarity, RISC inhibits the translation of the mRNA. In both cases, shRNA leads to target gene silencing.

[0218] The variant sequence can have at least 80% sequence identity with the shRNA sequence of the invention when measured over any suitable sequence length. Typically, percent sequence identity is determined over a length of contiguous nucleic acid or amino acid residues. For example, a variant sequence of the invention can have at least 80% sequence identity with a sequence of the invention when measured over at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or more nucleic acid or amino acid residues.

[0219] For example, a variant shRNA molecule of the invention can have at least 80% sequence identity to an shRNA molecule of the invention when measured over at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 or more nucleic acid residues, up to a variant shRNA molecule having at least 80% sequence identity to an shRNA molecule of the invention over its entire length.

[0220] antisense RNA

[0221] As described herein, single-stranded DNA (ssDNA) molecules, also known as antisense RNA, can be used to inhibit the activity of REV-ERB.

[0222] Using known techniques and based on knowledge of the REV-ERB gene sequence, antisense RNA molecules can be designed to antagonize the REV-ERB gene by targeting the corresponding RNA based on sequence homology. The sequence of such antisense RNA will contain a portion corresponding to a portion of the mRNA transcribed from the REV-ERB gene. This portion will typically be 100% complementary to the target portion of the transcribed mRNA, but lower complementarity levels (e.g., 90% or greater or 95% or greater) can also be used.

[0223] Aptamer

[0224] Aptamers are typically nucleic acid molecules that bind to specific target molecules. Aptamers can be engineered entirely in vitro, are easily prepared by chemical synthesis, have good storage properties, and rarely induce immunogenicity in therapeutic applications. These properties make them particularly useful in the pharmaceutical and therapeutic fields.

[0225] As used herein, "aptamer" generally refers to a single-stranded or double-stranded oligonucleotide, or a mixture of such oligonucleotides, wherein the oligonucleotide or mixture is capable of specifically binding to a target. Oligonucleotide aptamers will be discussed herein, but those skilled in the art will appreciate that other aptamers, such as peptide aptamers, having equivalent binding properties may also be used.

[0226] In general, aptamers can comprise oligonucleotides that are at least 5, at least 10, or at least 15 nucleotides in length. Aptamers can comprise sequences that are up to 40, up to 60, or up to 100 or more nucleotides in length. For example, aptamers can be 5 to 100 nucleotides, 10 to 40 nucleotides, or 15 to 40 nucleotides in length. Where possible, aptamers of shorter length are preferred because they are generally less susceptible to interference from other molecules or substances.

[0227] Aptamers can be generated using conventional methods, such as the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) program. SELEX is a method for in vitro evolution of nucleic acid molecules that have highly specific binding to a target molecule. For example, it is described in US 5,654,151, US 5,503,978, US 5,567,588 and WO 96 / 38579, the entire contents of which are incorporated herein by reference.

[0228] The SELEX method involves selecting nucleic acid aptamers, particularly single-stranded nucleic acids, that can bind to a desired target from a collection of oligonucleotides. A collection of single-stranded nucleic acids (e.g., DNA, RNA, or variants thereof) is contacted with the target under conditions favorable for binding, nucleic acids bound to the target are separated from unbound nucleic acids in the mixture, the nucleic acid-target complex is dissociated, and those nucleic acids bound to the target are amplified to produce a collection or library enriched in nucleic acids with the desired binding activity. This series of steps is then repeated as needed to produce a library of nucleic acids (aptamers) with specific binding affinity for the relevant target.

[0229] Peptoids

[0230] Peptidomimetics are compounds that mimic natural peptides or proteins, possessing the ability to interact with biological targets and produce the same biological effects. Compared to natural peptides, peptidomimetics may offer advantages in terms of stability and bioavailability. Peptidomimetics can modify the backbone or side chains of the parent peptide to achieve biological function. Examples of peptidomimetics include, but are not limited to, peptoids and β-peptides, as well as peptides containing D-amino acids.

[0231] Antibody

[0232] As described herein, antibodies can be used to inhibit the activity of REV-ERB.

[0233] As used herein, the term "antibody" includes the use of monoclonal or polyclonal antibodies, as well as antigen-binding fragments of monoclonal or polyclonal antibodies, or peptides that specifically bind to REV-ERB. The antibody can be Fab, F(ab')2, Fv, scFv, Fd, or dAb.

[0234] Post-translational modifications of E4bp4

[0235] The present inventors have previously shown that altering the post-translational modification of E4bp4 can increase the activity of E4bp4 (see WO2018 / 178666, which is incorporated herein by reference in its entirety, particularly pages 33 to 36 and Examples 1 to 5). Furthermore, increasing the activity of E4bp4 by altering the post-translational modification leads to an increase in the number of NK cells (as defined herein).

[0236] Thus, the methods of the present invention may include contacting a sample containing hematopoietic progenitor cells (HPCs) obtained from an individual / patient with a compound that alters the post-translational modification of E4bp4, thereby increasing E4bp4 activity. Thus, the compounds described herein that alter the post-translational modification of E4bp4 may be used in conjunction with the methods of the present invention. This combination may also be further combined with the use of Notch ligands (e.g., DLL4) and / or compounds described herein that inhibit REV-ERB activity.

[0237] Similarly, according to the present invention, a compound that can change or affect the post-translational modification of E4bp4 can be used to increase the production of NK cells in a patient, wherein the compound can increase the activity of E4bp4, or be used in a therapeutic method of increasing the number of NK cells in a patient in need thereof, in combination with the method disclosed herein for increasing the number of CD16+ NK cells by comprising a pre-differentiation step (a), the method disclosed herein for increasing the number of CD16+ NK cells by increasing E4bp4 expression by reducing REV-ERB activity, and / or the method disclosed herein for increasing the number of CD16+ NK cells by culturing HPCs in the presence of Notch ligands.

[0238] Any disclosure herein regarding methods for increasing the number of NK cells, methods for expanding CD16+ NK cells (optionally in the context of compounds that inhibit REV-ERB action and / or Notch ligands), expanded NK cell populations produced by the methods, and therapeutic indications associated with the compounds and populations is particularly applicable to the disclosed methods for increasing the number of CD16+ NK cells by increasing E4bp4 activity. As a non-limiting example, the feeder cell layer, growth factors and / or other culture conditions, and the disease to be treated may be the same as other aspects disclosed herein (e.g., REV-ERB inhibition and / or Notch ligands) in terms of post-translational modification. REV-ERB inhibitor compounds, Notch ligands and / or E4bp4 post-translational modifiers can be used simultaneously, separately or sequentially. When a compound that alters the post-translational modification of E4bp4 is used in combination with a compound that inhibits REV-ERB action, the sample is typically contacted with the REV-ERB inhibitory compound before contacting it with the post-translational modifier. If a Notch ligand is also used, the E4bp4 post-translational modifier is typically administered after the REV-ERB inhibitory compound and the Notch ligand; preferably, the REV-ERB inhibitory compound is administered together, or more preferably, before the Notch ligand (as described herein).

[0239] Types of post-translational modifications

[0240] The methods encompass changes in any post-translational modification that results in an increase in E4bp4 activity. Non-limiting examples of post-translational modifications include phosphorylation, SUMOylation, addition of hydrophobic groups (e.g., myristoylation, palmitoylation), addition of cofactors, addition of small chemical groups (e.g., acylation, alkylation, amidation, glycosylation), glycosylation, carbamylation, carbonylation, chemical modification (e.g., deamidation), and / or structural changes. Typically, changes in post-translational modifications according to the present invention result in a wild-type (unmodified)

[0241] Reduced phosphorylation of one or more phosphorylation sites in E4bp4 and / or reduced SUMOylation of one or more SUMOylation sites in wild-type (unmodified) E4bp4, or a combination thereof. As previously shown by the inventors (see WO2018 / 178666, the entirety of which is incorporated herein by reference, particularly pages 33 to 36 and Examples 1 to 5), wild-type (unmodified) E4bp4 is typically SUMOylated at one or more of residues K10, K116, K219, K337, and / or K394, or residues corresponding thereto, or any combination thereof. Typically, wild-type (unmodified) E4bp4 is SUMOylated at least at residue K219 (or a corresponding residue). Alternatively or additionally, wild-type (unmodified) E4bp4 is typically phosphorylated at residues S286, S301, and S454, or residues corresponding thereto, or any combination thereof. Thus, in some embodiments, a compound that alters a post-translational modification of E4bp4 reduces, inhibits, or eliminates SUMOylation at residue K219 (or a corresponding residue), and / or reduces, inhibits, or eliminates phosphorylation at residues S286, S301, and S454 (or a corresponding residue), or any combination thereof. Thus, according to the present invention, a compound can be used to (a) reduce SUMOylation at one or more of residues K10, K116, K219, K337, and / or K394 of E4bp4, or residues corresponding thereto, or any combination thereof; and / or reduce phosphorylation at one or more of residues S286, S301, and / or S454, or residues corresponding thereto, or any combination thereof.

[0242] Any compound that can alter or affect the post-translational modification of E4bp4 and that can increase the activity of E4bp4 can be used according to the present invention. In some embodiments, the compound inhibits, reduces or eliminates the phosphorylation and / or SUMOylation that occurs in wild-type (unmodified) E4bp4. Any suitable kinase inhibitor can be used to inhibit, reduce or eliminate the phosphorylation of E4bp4. Suitable kinase inhibitors are known in the art and can be routinely selected by those skilled in the art. For example, based on the current understanding of the kinases that phosphorylate E4bp4, it may be appropriate to use inhibitors of phosphoinositide-dependent protein kinase-1 (PDK1) and / or casein kinase 1 epsilon (CK1 epsilon). Non-limiting examples of suitable kinase inhibitors include 4-(4-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl)benzamide (D4476) and 4,5,6,7-tetrabromo-2-azabenzimidazole, 4,5,6,7-tetrabromobenzotriazole (TBB).

[0243] Increased E4BP4 activity

[0244] An increase in E4bp4 activity (e.g., achieved by post-translational modification of E4bp4) can be measured relative to a control. Thus, the activity of E4bp4 in a HPC sample to be treated according to the present invention, an expanded NK cell population, or a sample taken from an individual / patient can be compared to the activity of E4bp4 in a control. The activity can be quantified in any suitable manner, for example, by increasing the expression of any downstream target of E4bp4. Any suitable technique or method can be used to quantify the activity of E4bp4. Suitable techniques are known in the art, such as luciferase assays for quantifying reporter gene expression.

[0245] Typically, the control is an equivalent population or sample that has not been treated according to the present invention. For example, in the case of using a compound to change or affect the post-translational modification of E4bp4, the corresponding control can be a population or sample to which no compound has been added to change or affect the post-translational modification of E4bp4. As another example, in the case of using a compound to inhibit the action of REV-ERB, the corresponding control can be a population or sample to which no compound has been added to inhibit the action of REV-ERB. As yet another example, in the case of using a compound to inhibit the action of REV-ERB and using a compound to change or affect the post-translational modification of E4bp4, the corresponding control can be a population or sample to which no compound has been added to inhibit the action of REV-ERB or change or affect the post-translational modification of E4bp4.

[0246] The control may be as described herein. With respect to E4bp4 activity, the control may be an equivalent population or sample that has not achieved an increase in E4bp4 activity. As a non-limiting example, in the case where an individual / patient is treated with a compound that increases E4bp4 activity, a suitable control may be a different individual who has not been administered the compound or the same individual before administration of the compound. Conventional methods for in vitro expansion of NK cells, including known methods, may be considered control methods according to the present invention.

[0247] In the context of the present invention, reference to increasing E4bp4 activity is understood to mean that the activity of E4bp4 is increased by at least 1.5-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold or more relative to a control. Typically, E4bp4 activity is increased by at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold or more relative to a control. E4bp4 activity can be measured indirectly by determining an increase in the number of (CD16+) NK cells. Thus, the number of (CD16+) NK cells can be increased by at least 1.5-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold or more relative to a control. Typically,

[0248] The number of (CD16+) NK cells is increased by at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold or more compared to the control.

[0249] The activity of E4bp4 can be determined by quantitative and / or qualitative analysis and can be measured directly or indirectly. Any suitable technique can be used to determine the activity of E4bp4 relative to a control. Suitable standard techniques are known in the art.

[0250] The activity of E4bp4 can be increased for at least 6 hours, at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, or at least 1 week compared to a control. Preferably, the activity of E4bp4 is increased for at least 12 to 72 hours. Typically, this is assessed relative to the last administration of the compound that post-translationally modifies E4bp4.

[0251] The activity of E4bp4 can be increased over at least one, at least two, at least three, at least four, at least five, at least ten, at least 20, at least 30, at least 40, or more passages of the cultured cells compared to a control. The activity of E4bp4 can be increased indefinitely.

[0252] Methods for the expansion of natural killer cells

[0253] The present invention relates to the generation of natural killer cell populations comprising an increased number of CD16+ natural killer cells. In particular, the inventors have demonstrated that culturing hematopoietic progenitor cells (HPCs) for a specific period of time (a first culture period) in a culture medium that does not induce differentiation unexpectedly results in the generation of expanded natural killer cell populations having an increased number of CD16+ natural killer cells.

[0254] Therefore, the present invention provides a method for producing an expanded CD16+ natural killer cell population, comprising the steps of: (a) culturing a sample comprising hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period to produce a pre-differentiated hematopoietic progenitor cell population;

[0255] (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of the hematopoietic progenitor cells into natural killer cells for a second culture period. Step (b) generally includes differentiation of the hematopoietic progenitor cells in the pre-differentiated hematopoietic progenitor cell population and expansion of the resulting natural killer cells. Therefore, the present invention provides a method for producing an expanded CD16+ natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period to produce a pre-differentiated hematopoietic progenitor cell population; and (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of the hematopoietic progenitor cells into natural killer cells for a second culture period, wherein expansion of the natural killer cells also occurs during the second culture period. The present invention also provides a method for producing an expanded CD16+ natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period to produce a pre-differentiated hematopoietic progenitor cell population; (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for a second culture period; and (c) expanding the cells in vitro to produce an expanded natural killer cell population.

[0256] The present invention further provides a method for increasing the number of CD16+ natural killer cells in an expanded natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period to produce a pre-differentiated hematopoietic progenitor cell population; and (b) culturing the pre-differentiated hematopoietic progenitor cell population in a medium that induces differentiation of the hematopoietic progenitor cells into natural killer cells for a second culture period. Step (b) generally includes differentiating the hematopoietic progenitor cells in the pre-differentiated hematopoietic progenitor cell population and expanding the resulting natural killer cells. Therefore, the present invention provides a method for increasing the number of CD16+ natural killer cells in an expanded natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period to produce a pre-differentiated hematopoietic progenitor cell population; and (b) culturing the pre-differentiated hematopoietic progenitor cell population in a medium that induces differentiation of the hematopoietic progenitor cells into natural killer cells for a second culture period, wherein expansion of the natural killer cells also occurs during the second culture period. The present invention also provides a method for increasing the number of CD16+ natural killer cells in an expanded natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period to produce a pre-differentiated hematopoietic progenitor cell population; (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for a second culture period; and (c) expanding the cells in vitro to produce an expanded natural killer cell population.

[0257] The inventors have surprisingly demonstrated that culturing a population of predifferentiated hematopoietic progenitor cells for a first culture period of about 2 to about 8 days in a medium that does not induce differentiation, prior to differentiating the predifferentiated hematopoietic progenitor cell population into natural killer cells, increases the number of CD16+ natural killer cells in the resulting expanded natural killer cell population. As exemplified herein, the inventors have found that extending this first culture period beyond 8 days reduces the yield of natural killer cells and CD16 expression. Therefore, according to the present invention, the duration of the first culture period (predifferentiation culture phase) is strictly controlled and precisely defined. According to the present invention, the first culture period (predifferentiation culture phase) typically does not exceed 8 days. In other words, step (a) of the method of the present invention typically does not exceed 8 days.

[0258] Therefore, the present invention provides a method for producing an expanded CD16+ natural killer cell population in vitro, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for about 2 days to about 8 days to produce a pre-differentiated hematopoietic progenitor cell population; (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for a second culture period, wherein expansion of the natural killer cells also occurs during the second culture period. The present invention also provides a method for producing an expanded CD16+ natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for about 2 days to about 8 days to produce a pre-differentiated hematopoietic progenitor cell population; and (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for a second culture period; and (c) expanding the cells in vitro to produce an expanded natural killer cell population.

[0259] The present invention further provides a method for increasing the number of CD16+ natural killer cells in an expanded natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for about 2 days to about 8 days to produce a pre-differentiated hematopoietic progenitor cell population; (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for a second culture period, wherein expansion of the natural killer cells also occurs during the second culture period. The present invention also provides a method for increasing the number of CD16+ natural killer cells in an expanded natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for about 2 days to about 8 days to produce a pre-differentiated hematopoietic progenitor cell population; and (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for a second culture period; and (c) expanding the cells in vitro to produce an expanded natural killer cell population.

[0260] The first incubation period is from about 2 days to about 8 days. Therefore, the first incubation period can be 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days or 8 days, or any duration between 2 days to 8 days. Preferably, the first incubation period can be from about 2 days to about 6 days (for example, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days or 6 days, or any duration between 2 days to 6 days, for example, from about 4 days to 6 days). More preferably, the first incubation period can be from about 2 days to about 4 days (for example, 2 days, 2.5 days, 3 days, 3.5 days or 4 days, or any duration between 2 days to 4 days). The first incubation period (pre-differentiation culture stage, i.e. step (a)) is usually no more than 8 days.

[0261] The first culture period is a period of time during which hematopoietic progenitor cells or a sample containing hematopoietic progenitor cells obtained from an individual are cultured in a culture medium that does not induce differentiation of hematopoietic progenitor cells, typically wherein the culture medium does not induce differentiation of hematopoietic progenitor cells into natural killer cells. Any culture medium suitable for culturing hematopoietic progenitor cells can be used, as long as the culture medium does not induce differentiation of hematopoietic progenitor cells, typically does not induce differentiation into natural killer cells. Non-limiting examples of suitable culture media and factors that can be contained in the culture medium are described herein. This step of culturing hematopoietic progenitor cells or a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells, typically wherein the culture medium does not induce differentiation of hematopoietic progenitor cells into natural killer cells, is typically step (a) of the method of the present invention.

[0262] As used herein, the term "pre-differentiated hematopoietic progenitor cell population" is used to define a hematopoietic progenitor cell population obtained by culturing hematopoietic progenitor cells or a sample containing hematopoietic progenitor cells obtained from an individual for a first culture period in a medium that does not induce differentiation of hematopoietic progenitor cells, typically wherein the medium does not induce differentiation of hematopoietic progenitor cells into natural killer cells. The pre-differentiated hematopoietic progenitor cell population may contain other non-hematopoietic progenitor cell types, as long as hematopoietic progenitor cells are the predominant cell type (e.g., at least 50% of the cells are hematopoietic progenitor cells). Typically, the pre-differentiated hematopoietic progenitor cell population contains less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5% or less natural killer cells. Preferably, the pre-differentiated hematopoietic progenitor cell population contains less than 2% natural killer cells, more preferably less than 1% natural killer cells. The number of hematopoietic progenitor cells and / or natural killer cells in a sample comprising hematopoietic progenitor cells and / or a population of predifferentiated hematopoietic progenitor cells can be determined by any suitable technique, common examples of which include techniques such as fluorescence activated cell sorting (FACS) or flow cytometry.

[0263] After culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period (e.g., about 2 days to about 8 days, such as about 2 days to about 6 days, about 4 days to about 6 days, or about 2 days to about 4 days) to produce a pre-differentiated hematopoietic progenitor cell population, the in vitro method of the present invention includes the step of culturing the (pre-differentiated) hematopoietic progenitor cells or the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of the (pre-differentiated) hematopoietic progenitor cells into natural killer cells for a second culture period.

[0264] The second incubation period can be about 10 days to about 30 days. Therefore, the second incubation period can be 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days or 30 days, or any duration between 10 days to 30 days, for example 24 days to 30 days. Preferably, the second incubation period can be about 15 days to about 25 days (for example 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days or 25 days, or any duration between 15 days to 25 days). More preferably, the second culture period can be about 18 days to about 22 days (e.g., 18 days, 19 days, 20 days, 21 days or 22 days, or any duration between 18 days and 22 days). Most preferably, the second culture period is about 19 days to 21 days, particularly preferably about 20 days.

[0265] Therefore, the present invention provides a method for producing an expanded CD16+ natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period (e.g., about 2 days to about 8 days, such as about 2 days to about 6 days or about 2 days to about 4 days) to produce a pre-differentiated hematopoietic progenitor cell population; and (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for about 15 days to about 30 days (e.g., about 18 days to about 22 days, for example, about 20 days). Step (b) generally includes differentiation of the hematopoietic progenitor cells in the pre-differentiated hematopoietic progenitor cell population and expansion of the resulting natural killer cells. Therefore, the present invention provides a method for producing an expanded CD16+ natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period (e.g., about 2 days to about 8 days, such as about 2 days to about 6 days, about 4 days to 6 days, or about 2 days to about 4 days) to produce a pre-differentiated hematopoietic progenitor cell population; (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for about 15 days to about 30 days (e.g., about 18 days to about 22 days, for example, about 20 days), wherein expansion of the natural killer cells also occurs in a second culture period. The present invention also provides a method for producing an expanded CD16+ natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period (e.g., about 2 days to about 8 days, such as about 2 days to about 6 days, about 4 days to 6 days, or about 2 days to about 4 days) to produce a pre-differentiated hematopoietic progenitor cell population; and (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for about 15 days to about 30 days (e.g., about 18 days to about 22 days, for example, about 20 days); and (c) expanding the cells in vitro to produce an expanded natural killer cell population.

[0266] The present invention further provides a method for increasing the number of CD16+ natural killer cells in an expanded natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period (e.g., from about 2 days to about 8 days, such as from about 2 days to about 6 days or from about 2 days to about 4 days) to produce a pre-differentiated hematopoietic progenitor cell population; and (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for about 15 days to about 30 days (e.g., from about 18 days to about 22 days, for example, about 20 days). Step (b) generally includes differentiation of the hematopoietic progenitor cells in the pre-differentiated hematopoietic progenitor cell population and expansion of the resulting natural killer cells. Therefore, the present invention provides a method for increasing the number of CD16+ natural killer cells in an expanded natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period (e.g., about 2 days to about 8 days, such as about 2 days to about 6 days, about 4 days to 6 days, or about 2 days to about 4 days) to produce a pre-differentiated hematopoietic progenitor cell population; (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for about 15 days to about 30 days (e.g., about 18 days to about 22 days, for example, about 20 days), wherein expansion of the natural killer cells also occurs in a second culture period. The present invention also provides a method for increasing the number of CD16+ natural killer cells in an expanded natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period (e.g., about 2 days to about 8 days, such as about 2 days to about 6 days, about 4 days to 6 days, or about 2 days to about 4 days) to produce a pre-differentiated hematopoietic progenitor cell population; (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for about 15 days to about 30 days (e.g., about 18 days to about 22 days, for example, about 20 days); and (c) expanding the cells in vitro to produce an expanded natural killer cell population.

[0267] The second culture period is a period of time during which the (pre-differentiation) hematopoietic progenitor cells or a pre-differentiation hematopoietic progenitor cell population is cultured in a culture medium that induces the differentiation of (pre-differentiation) hematopoietic progenitor cells, typically wherein the culture medium induces the differentiation of (pre-differentiation) hematopoietic progenitor cells to natural killer cells. Any culture medium suitable for culturing pre-differentiation hematopoietic progenitor cells can be used, as long as the culture medium induces the differentiation of (pre-differentiation) hematopoietic progenitor cells, typically inducing differentiation to natural killer cells. Non-limiting examples of suitable culture media and factors that can be included in the culture medium are described herein. This step of culturing the (pre-differentiation) hematopoietic progenitor cells or a pre-differentiation hematopoietic progenitor cell population in a culture medium that induces the differentiation of (pre-differentiation) hematopoietic progenitor cells, typically wherein the culture medium induces the differentiation of (pre-differentiation) hematopoietic progenitor cells to natural killer cells, is typically step (b) of the method of the present invention.

[0268] The methods of the present invention generally further comprise expanding (CD16+) natural killer cells. By expansion, it is meant increasing the number of (CD16+) natural killer cells. Increasing the number of (CD16+) natural killer cells can be as described herein.

[0269] The expansion of (CD16+) natural killer cells can occur simultaneously with (i.e., simultaneously with) the differentiation of (pre-differentiated) hematopoietic progenitor cells into natural killer cells. In other words, steps (b) and (c) can be simultaneous, such that the second culture period includes differentiation and expansion as described herein. Therefore, the present invention provides a method for producing an expanded CD16+ natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period (e.g., from about 2 days to about 8 days, such as from about 2 days to about 6 days, from about 4 days to 6 days, or from about 2 days to about 4 days) to produce a pre-differentiated hematopoietic progenitor cell population; and (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for a second culture period (e.g., from about 15 days to about 30 days, such as from about 18 days to about 22 days, for example, about 20 days), and expanding the cells in vitro to produce an expanded natural killer cell population. The present invention also provides a method for increasing the number of CD16+ natural killer cells in an expanded natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture period (e.g., about 2 days to about 8 days, such as about 2 days to about 6 days, about 4 days to 6 days, or about 2 days to about 4 days) to produce a pre-differentiated hematopoietic progenitor cell population; and (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for a second culture period (e.g., about 15 days to about 30 days, such as about 18 days to about 22 days, for example, about 20 days), and expanding the cells in vitro to produce an expanded natural killer cell population.

[0270] Alternatively, after culturing (pre-differentiated) hematopoietic progenitor cells (HPCs) or a population of pre-differentiated hematopoietic progenitor cells for a second culture cycle in a medium that induces differentiation of (pre-differentiated) hematopoietic progenitor cells into natural killer cells (NK cells), expansion of (CD16+) natural killer cells may occur. Therefore, the present invention provides a method for producing an expanded CD16+ natural killer cell population in vitro, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a medium that does not induce differentiation of hematopoietic progenitor cells for a first culture cycle (e.g., from about 2 days to about 8 days, such as from about 2 days to about 6 days, from about 4 days to about 6 days, or from about 2 days to about 4 days) to produce a population of pre-differentiated hematopoietic progenitor cells; (b) culturing the pre-differentiated hematopoietic progenitor cell population in a medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for a second culture cycle (e.g., from about 15 days to about 30 days, such as from about 18 days to about 22 days, such as about 20 days); and then (c) expanding the cells in vitro to produce an expanded natural killer cell population. The present invention also provides an in vitro method for increasing the number of CD16+ natural killer cells in an expanded natural killer cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells obtained from an individual in a culture medium that does not induce differentiation of hematopoietic progenitor cells for a first culture cycle (e.g., about 2 days to about 8 days, such as about 2 days to about 6 days, about 4 to 6 days, or about 2 days to about 4 days) to produce a pre-differentiated hematopoietic progenitor cell population; (b) culturing the pre-differentiated hematopoietic progenitor cell population in a culture medium that induces differentiation of hematopoietic progenitor cells into natural killer cells for a second culture cycle (e.g., about 15 days to about 30 days, such as about 18 days to about 22 days, such as about 20 days); and then (c) expanding the cells in vitro to produce an expanded natural killer cell population.

[0271] The expansion of natural killer (NK) cells may include culturing (CD16+) NK cells for about 10 days to about 30 days. As described herein, this can be performed in parallel with the differentiation step (conducted simultaneously) or after the differentiation step. In either case, the expansion period can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days, or any duration between 10 and 30 days, for example, between 24 and 30 days. Preferably, the expansion period can be between about 15 days to about 25 days (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days, or any duration between 15 and 25 days). More preferably, the expansion period can be between about 18 days and about 22 days (e.g., 18, 19, 20, 21 or 22 days, or any length between 18 and 22 days). Most preferably, the expansion period is about 19 to 21 days, particularly preferably about 20 days.

[0272] The expansion phase refers to a period of time during which (CD16+) NK cells are cultured in a culture medium inducing their expansion. Any culture medium suitable for cultivating (CD16+) NK cells can be used, as long as the culture medium can induce the expansion of (CD16+) NK cells. Suitable culture medium and the non-limiting example of the factor that can be included in the culture medium are described herein. The expansion culture medium can be identical with the culture medium used for the second culture phase, i.e., identical with the culture medium inducing (pre-differentiation) hematopoietic progenitor cells (HPCs) to differentiate into NK cells. Typically, expansion involves culturing cells in cytokines and growth factors (such as IL-15) related to NK cell development, and may involve transferring pre-differentiated HPCs or NK cells to a suitable matrix (support) cell layer, such as OP9 or EL08 stromal cells, preferably EL08 cells (such as EL08-ID2 cells), or acellular alternative support layer as described herein, such as extracellular matrix (ECM). The expansion step typically continues to the remainder of the in vitro culture phase (as defined herein). At this stage, culture medium can be frequently replaced as needed to promote the expansion of NK cells. Any REV-ERB inhibitory compound, Notch ligand, and / or compound that alters a post-translational modification of E4bp4 present in the culture medium prior to the culture medium change can be reapplied with fresh culture medium at the same or different concentrations. Preferably, the REV-ERB inhibitory compound, Notch ligand, and / or compound that alters a post-translational modification of E4bp4 used in steps (a) and / or (b) is not present during the amplification phase.

[0273] The present invention relates to a method for expanding (CD16+) NK cell populations and / or increasing the number of (CD16+) NK cells in an expanded NK cell population. The method can be in vitro, in vivo or ex vivo. Typically, the method of the present invention is ex vivo.

[0274] Steps (a) and (b) of the method of the present invention (wherein the expansion is included in step (b)) or steps (a) to (c) (when the expansion is after step (b)) are typically performed sequentially. In other words, a sample containing HPCs is typically first cultured in a culture medium that does not induce HPCs differentiation (toward NK cells) for a first culture period to produce a pre-differentiated HPC population, and then the pre-differentiated HPC population is cultured in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, after which a separate step of in vitro amplification of NK cells may be performed. As described herein, preferably, the NK cell expansion step is performed simultaneously or synchronously with the step of culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, i.e., steps (b) and (c) are simultaneous / synchronous, or at least partially overlap.

[0275] The durations of step (a) and step (b) (where amplification is included in step (b)) or step (a), step (b) and step (c) (when amplification follows step (b)) are independent of each other, and any duration of the above-mentioned step (a) can be used in combination with any duration of the above-mentioned step (b) and / or any duration of the above-mentioned step (c).

[0276] The duration of in vitro method of the present invention can be at least 12 days (comprising step (a), (b) and the amplification step when amplification step is asynchronous with step (b)). As non-limiting example, the duration of in vitro method of the present invention can be at least 14 days, at least 16 days, at least 18 days, at least 22 days, at least 24 days, at least 26 days, at least 28 days, at least 30 days, at least 32 days, at least 34 days, at least 36 days or longer. As further non-limiting example, the duration of in vitro method of the present invention can be between about 12 to about 36 days, for example, between about 12 to about 28 days, between about 12 to about 26 days, between about 12 to about 24 days, between about 12 to about 22 days, between about 12 to about 18 days, between about 12 to about 16 days, or between about 12 to about 14 days.

[0277] In all methods of the present invention, the sample comprising HPCs obtained from an individual / patient can be a sample obtained from bone marrow, cord blood and / or peripheral blood. Therefore, the sample can be a cord blood or peripheral blood sample, or a bone marrow sample or a biopsy specimen. The sample can be obtained from an individual (i.e., patient) who will receive the NK cell colony treatment produced by the inventive method. Alternatively, the sample is obtained from a healthy individual.

[0278] The sample comprising HPCs can be processed before being used in the present method. As a non-limiting example, the sample comprising HPCs can be frozen after being obtained from the subject. Frozen samples usually need to be thawed before being used in the method of the present invention. After thawing, the sample may need to be plated and / or subjected to an initial culture period so that the HPCs can recover from freezing before starting the method of the present invention. This initial culture period can be referred to as a "recovery period". The method of the present invention may include such a recovery period or may not include such a recovery period. If a recovery period exists, its duration can be one week, up to six days, up to five days, up to four days, up to three days, up to two days, up to one day. The recovery period is preferably up to 2 days, i.e., up to 1 day, up to 12 hours, up to 8 hours. If a recovery period is not included in the method of the present invention, step (a) of the method can start on the same day the method starts.

[0279] Step (a) of the method of the present invention can be started on the same day that the method is started (e.g., from the date of thawing and plating HPCs or isolating HPCs from a sample). Step (a) of the method of the present invention can be started within one week, six days, five days, four days, three days, two days, one day, or the same day after the method is started (e.g., from the date of thawing and plating HPCs, from the date of isolating HPCs from a sample, or from the same day of isolating NK cell precursors). The date of isolating HPCs and / or NK cell precursors is typically the same day that the sample is obtained from the patient.

[0280] The step (a) of the inventive method is usually started after the recovery period defined herein. Therefore, the step (a) of the inventive method can be started within a week, six days, five days, four days, three days, two days, one day, 12 hours or 8 hours after the method starts (for example, from the date of thawing and plating HPCs, separating HPCs in the sample, or separating NK cell precursors). Preferably, the step (a) of the inventive method can be started within two days, one day, 12 hours or 8 hours after the method starts, more preferably about 1 day after the method starts.

[0281] According to the present invention, the sample comprising HPCs is any sample from an individual, comprising a sufficient number of HPCs (as described herein) such that amplified (CD16+) NK cell colonies can be obtained by the method of the present invention. Typically, the sample comprises hematopoietic stem cells (HSCs). Preferably, the sample is rich in HSCs, such as cord blood or peripheral blood samples or bone marrow samples or biopsy specimens as described herein.

[0282] The methods of the present invention can increase the number of CD16+ NK cells relative to a control by at least 1.5 times, at least 2 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2.9 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times or more. Typically, the number of NK cells is increased by at least 2 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2.9 times, at least 3 times or more compared to a control.

[0283] Compared with the number of CD16+NK cells in the NK cell population amplified by the control, the method of the present invention can increase the number of CD16+NK cells in the amplified NK cell population. Compared with the number of CD16+NK cells in the NK cell population amplified by the control, the method of the present invention can increase the ratio of CD16+NK cells in the amplified NK cell population. The NK cell population amplified by the control can be as described herein. The NK cell population amplified by the control can be an amplified NK cell population produced by a control (e.g., traditional) method. Generally, the NK cell population amplified by the control can be an amplified NK cell population produced by a method corresponding to the method described herein, but wherein (i) the step of culturing the first culture period of the sample containing HPCs in a culture medium that does not induce HPCs differentiation (i.e., step (a)) is omitted; or (ii) the step of culturing the sample containing HPCs in a culture medium that does not induce HPCs differentiation is carried out for less than about 2 days or more than about 8 days.

[0284] The methods of the present invention can increase the number or proportion of CD16+ NK cells in the expanded NK cell population by at least 1.5 times, at least 2 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2.9 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times or more compared to a control expanded NK cell population or an expanded NK cell population produced by a control method. Typically, the number of NK cells is increased by at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, or at least 2x, 2.5x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or more compared to a control expanded NK cell population or an expanded NK cell population produced by a control method.

[0285] The methods of the present invention produce expanded (CD16+) NK cell populations as described herein. Typically, in the expanded (CD16+) NK cell population produced by the methods of the present invention, at least 10% of the NK cells in the population are CD16+ NK cells, at least 15% of the NK cells in the population are CD16+ NK cells, at least 20% of the NK cells in the population are CD16+ NK cells, at least 25% of the NK cells in the population are CD16+ NK cells, at least 30% of the NK cells in the population are CD16+ NK cells, at least 35% of the NK cells in the population are CD16+ NK cells, at least 40% of the NK cells in the population are CD16+ NK cells, at least 45% of the NK cells in the population are CD16+ NK cells, at least 50% of the NK cells in the population are CD16+ NK cells, at least 60% of the NK cells in the population are CD16+ NK cells, at least 70% of the NK cells in the population are CD16+ NK cells, at least 80% of the NK cells in the population are CD16+ NK cells, and up to 100% of the NK cells in the population are CD16+ NK cells. Preferably, at least 15% of NK cells are CD16+ NK cells, more preferably at least 20% of NK cells are CD16+ NK cells, even more preferably at least 25% of NK cells are CD16+ NK cells, and even more preferably at least 30% of NK cells are CD16+ NK cells.

[0286] The methods of the present invention can increase the number of CD16+ NK cells without introducing exogenous nucleic acids (e.g., by transduction or transfection). This is in contrast to traditional methods of expanding NK cells by increasing CD16 expression, which require the expression of exogenous CD16 transgenes to achieve minimal increases in CD16 expression.

[0287] Therefore, the present invention provides a method for producing expanded CD16+ NK cells, and a method for increasing the number of CD16+ NK cells in an expanded NK cell population, wherein the method does not include the step of contacting HPCs and / or NK cells and / or introducing exogenous nucleic acid (typically a nucleic acid encoding CD16) therein.

[0288] The method of the present invention can accelerate the generation of phenotypically mature NK cells. In other words, the method of the present invention can reduce the time required to obtain a mature NK cell population. Compared with conventional NK cell expansion methods known in the art, the reduction in the running time of this method provides a further advantage.

[0289] The in vitro method of the present invention may include one or more additional steps. As a non-limiting example, the in vitro method of the present invention may include one or more additional initial steps of separating and / or enriching HPCs from a sample (e.g., peripheral blood or cord blood sample). As a further non-limiting example, the in vitro method of the present invention may include an additional, typically final step, to purify the amplified (CD16+) NK cell colony. This ensures a pure cell colony for therapeutic administration as described herein. The purification of the amplified NK cell colony can be carried out by any appropriate method. Standard cell purification methods known in the art, such as cell sorting, include fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS).

[0290] In some methods of the present invention, a REV-ERB inhibitory compound is not used in step (a) and / or step (b). In some methods of the present invention, a REV-ERB inhibitory compound is not used in either step (a) or step (b). Any and all disclosures herein relating to the methods of the present invention also apply without reservation to methods that do not use a REV-ERB inhibitory compound, unless otherwise indicated.

[0291] In some methods of the invention, a Notch ligand (e.g., DLL4) is not used in step (a) and / or step (b). In some methods of the invention, a Notch ligand (e.g., DLL4) is not used in either step (a) or step (b). Any and all disclosures herein relating to the methods of the invention also apply without reservation to methods that do not use a Notch ligand (e.g., DLL4), unless otherwise indicated.

[0292] In some methods of the present invention, neither a REV-ERB inhibitory compound nor a Notch ligand (e.g., DLL4) is used in step (a) and / or step (b). In some methods of the present invention, neither a REV-ERB inhibitory compound nor a Notch ligand (e.g., DLL4) is used in step (a) or step (b). Any and all disclosures herein relating to the methods of the present invention are equally applicable without reservation to methods in which neither a REV-ERB inhibitory compound nor a Notch ligand (e.g., DLL4) is used, unless otherwise indicated. In embodiments in which a REV-ERB inhibitory compound and a Notch ligand (e.g., DLL4) are not used, the step of culturing HPCs in a culture medium that does not contain HPCs differentiation is sufficient to increase the number of CD16+ NK cells, i.e., step (a) is sufficient to increase the number of CD16+ NK cells in the NK cell population expanded by the present invention.

[0293] In some methods of the present invention, including but not limited to methods involving a combination of Notch ligands and REV-ERB inhibitory compounds, the percentage of NK cells in the final cell population may be very high (typically greater than 85%, preferably greater than 90%, more preferably greater than 95%, and may approach 100%). In such cases, the final purification step may be omitted.

[0294] Methods using Notch ligands

[0295] Methods for producing expanded CD16+ natural killer (NK) cell populations and / or methods for increasing the number of CD16+ NK cells in expanded NK cell populations may involve culturing NK cell precursors (hematopoietic progenitor cells, HPCs) in the presence of a Notch ligand (e.g., Delta-like ligand 4, DLL4) as described herein. In the case of using a Notch ligand (e.g., DLL4), a method may include contacting HPCs with a Notch ligand (e.g., DLL4). These methods of the present invention can achieve rapid expansion of NK cells, reduce the time required for culture, thereby reducing the risk of cell exhaustion, and enhance the cytotoxicity of NK cells when they are infused into patients.

[0296] In the methods described herein, HPCs can be cultured in the presence of a Notch ligand (e.g., DLL4) for at least part of the first culture period. Thus, a Notch ligand (e.g., DLL4) can be included in a culture medium that does not induce differentiation of HPCs.

[0297] Therefore, the present invention provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period. Step (b) generally includes differentiation of HPCs in the pre-differentiated HPC population and expansion of the resulting NK cells. Therefore, the present invention provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, wherein expansion of NK cells also occurs in the second culture period. The present invention also provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period; (c) amplifying the cells in vitro to produce an expanded NK cell population. During at least part of the first culture period, HPCs may also be contacted with (cultured in the presence of) a Notch ligand (e.g., DLL4). Therefore, the culture medium that does not induce HPC differentiation may contain a Notch ligand (e.g., DLL4). Therefore, during at least part of step (a) of the method of the present invention, HPCs may be contacted with (cultured in the presence of) a Notch ligand (e.g., DLL4).

[0298] The present invention also provides a method for increasing the number of CD16+NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period. Step (b) generally includes differentiation of HPCs in the pre-differentiated HPC population and expansion of the resulting NK cells. Therefore, the present invention provides a method for increasing the number of CD16+NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, wherein the expansion of NK cells also occurs in the second culture period. The present invention also provides a method for increasing the number of CD16+ NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period; (c) amplifying the cells in vitro to produce an expanded NK cell population. During at least part of the first culture period, HPCs may also be contacted with (cultured in the presence of) a Notch ligand (e.g., DLL4). Therefore, the culture medium that does not induce HPCs differentiation may contain a Notch ligand (e.g., DLL4). Therefore, during at least part of step (a) of the method of the present invention, HPCs may be contacted with (cultured in the presence of) a Notch ligand (e.g., DLL4).

[0299] As described herein, the first culture period is between about 2 days and about 8 days. HPCs can be contacted with a Notch ligand (e.g., DLL4) during at least part of the first culture period, up to the entire first culture period. Thus, HPCs can be contacted with a Notch ligand (e.g., DLL4) during 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, or 8 days of the first culture period, or any duration between 2 days and 8 days. Preferably, HPCs can be contacted with a Notch ligand (e.g., DLL4) during about 2 days to about 6 days (e.g., 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, or 6 days, or any duration between 2 days and 6 days, e.g., about 4 days to 6 days) of the first culture period. More preferably, HPCs can be contacted with the Notch ligand (eg, DLL4) within about 2 days to about 4 days (eg, 2 days, 2.5 days, 3 days, 3.5 days, or 4 days, or any time period between 2 days and 4 days) of the first culture period.

[0300] Most preferably, the first culture period comprises a period of about 0 days to about 2 days, in particular about 1 day, during which the Notch ligand (e.g., DLL4) is absent, and the HPCs are then contacted with the Notch ligand (e.g., DLL4) for the remainder of the first culture period. Thus, the Notch ligand may be added to the culture medium that does not induce differentiation of HPCs after a period of about 0 days to about 2 days, in particular, the Notch ligand may be added to the culture medium that does not induce differentiation of HPCs after about 1 day.

[0301] The methods of the present invention may include a recovery period such that the HPCs are cultured for a recovery period of about 0 to about 2 days before being contacted with a Notch ligand (e.g., DLL4) and cultured for a first culture period. As described herein, the first culture period is between about 2 days and about 8 days. The HPCs may be contacted with a Notch ligand (e.g., DLL4) for at least part of the first culture period, up to the entire first culture period. Thus, HPCs may be contacted with a Notch ligand (e.g., DLL4) for 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, or 8 days of the first culture period, or for any duration between 2 and 8 days. Preferably, HPCs are contacted with a Notch ligand (e.g., DLL4) within about 2 days to about 6 days (e.g., 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, or 6 days, or any length between 2 days and 6 days, e.g., about 4 days to 6 days) of the first culture period. More preferably, HPCs are contacted with a Notch ligand (e.g., DLL4) within about 2 days to about 4 days (e.g., 2 days, 2.5 days, 3 days, 3.5 days, or 4 days, or any length between 2 days and 4 days) of the first culture period. A method may include a recovery period of about 0 days to about 2 days, particularly about 1 day, during which there is no Notch ligand (e.g., DLL4), and then contacting the HPCs with a Notch ligand (e.g., DLL4) for cultivation for the first culture period. Therefore, the Notch ligand may be added to the culture medium that does not induce HPCs differentiation after a period of about 0 days to about 2 days, and in particular, may be added to the culture medium that does not induce HPCs differentiation after about 1 day.

[0302] If the culture medium that does not induce HPCs differentiation is replaced during the first culture period, the culture medium that does not induce HPCs differentiation after replacement may or may not contain Notch ligands (e.g., DLL4), depending on whether the presence of Notch ligands (e.g., DLL4) is required at that time point in the first culture period. As a non-limiting example, if the culture medium that does not induce HPCs differentiation is replaced on day 2, but HPCs are cultured with Notch ligands (e.g., DLL4) only from day 4, then the culture medium that does not induce HPCs differentiation replaced on day 2 will not contain Notch ligands (e.g., DLL4). As a further limiting example, or in addition, if the culture medium that does not induce HPCs differentiation is replaced on day 6, and HPCs are cultured with Notch ligands (e.g., DLL4) from day 4, then the culture medium that does not induce HPCs differentiation replaced on day 6 will contain Notch ligands (e.g., DLL4). Notch ligands (eg, DLL4) may be added to a medium that does not induce HPC differentiation before being used to replace the medium on HPCs, or the medium that does not induce HPC differentiation may be replaced first and then Notch ligands (eg, DLL4) may be added to HPCs.

[0303] Alternatively, or in addition, in the methods described herein, the pre-differentiated HPC population can be cultured in the presence of a Notch ligand (e.g., DLL4) during at least a portion of the second culture period. Thus, a Notch ligand (e.g., DLL4) can be included in the culture medium that induces differentiation of HPCs.

[0304] Accordingly, the present invention provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period. Step (b) generally includes differentiation of HPCs in the pre-differentiated HPC population and expansion of the resulting NK cells. Therefore, the present invention provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, wherein expansion of NK cells also occurs in the second culture period. The present invention also provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period; (c) expanding the cells in vitro to produce an expanded NK cell population. During at least part of the second culture period, the pre-differentiated HPCs may also be contacted with (cultured in the presence of) a Notch ligand (e.g., DLL4). Therefore, the culture medium that induces HPC differentiation may contain a Notch ligand (e.g., DLL4). Therefore, during at least part of step (b) of the method of the present invention, the pre-differentiated HPCs may be contacted with (cultured in the presence of) a Notch ligand (e.g., DLL4). This can serve as an alternative or supplement to the inclusion of a Notch ligand (e.g., DLL4) in step (a) described herein.

[0305] The present invention also provides a method for increasing the number of CD16+NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period. Step (b) generally includes differentiation of HPCs in the pre-differentiated HPC population and expansion of the resulting NK cells. Therefore, the present invention provides a method for increasing the number of CD16+NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, wherein the expansion of NK cells also occurs in the second culture period. The present invention also provides a method for increasing the number of CD16+ NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period; (c) expanding the cells in vitro to produce an expanded NK cell population. During at least part of the second culture period, the pre-differentiated HPCs may also be contacted with (cultured in the presence of) a Notch ligand (e.g., DLL4). Therefore, the culture medium that induces HPC differentiation may contain a Notch ligand (e.g., DLL4). Therefore, during at least part of step (b) of the method of the present invention, the pre-differentiated HPCs may be contacted with (cultured in the presence of) a Notch ligand (e.g., DLL4). This can serve as an alternative or supplement to the inclusion of a Notch ligand (e.g., DLL4) in step (a) described herein.

[0306] As described herein, the second culture period can be between about 10 days and about 30 days. The pre-differentiated HPCs can be contacted with a Notch ligand (e.g., DLL4) during at least a portion of the second culture period, up to the entire second culture period. Thus, pre-differentiated HPCs can be contacted with a Notch ligand (e.g., DLL4) on day 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the second culture period, or any time period between 10 and 30 days. Preferably, the predifferentiated HPCs can be contacted with a Notch ligand (e.g., DLL4) within about 15 days to about 25 days (e.g., 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, or 25 days, or any time period between 15 days and 25 days) or about 24 days to about 30 days (e.g., 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days, or any time period between 24 days and 30 days) of the second culture period. More preferably, the predifferentiated HPCs can be contacted with a Notch ligand (e.g., DLL4) within about 18 days to about 22 days (e.g., 18 days, 19 days, 20 days, 21 days, or 22 days, or any time period between 18 days and 22 days) of the second culture period. Most preferably, the pre-differentiated HPCs are contacted with the Notch ligand (eg, DLL4) during the second culture period of about 19 to 21 days, particularly preferably about 20 days.

[0307] Preferably, the Notch ligand (e.g., DLL4) is present only before inducing HPCs differentiation. In other words, the Notch ligand (e.g., DLL4) is present only during at least part of the first culture period, and preferably is not present during at least part of the second culture period. Therefore, the Notch ligand (e.g., DLL4) is preferably present in a culture medium that does not induce HPCs differentiation, and is not present in a culture medium that induces HPCs differentiation. Prior to step (b), the pre-differentiated HPC population obtained in step (a) of the method of the present invention can be transferred to different culture containers. This reduces the risk of contamination of the Notch ligand in step (b) of the method.

[0308] Typically, the Notch ligand is a Notch ligand as described herein. Preferably, the Notch ligand is DDL4, or a fragment thereof that retains DLL4 function as described herein.

[0309] Notch ligands (such as DLL4) can be present in a solution (for example, in a culture medium) or can be used to coat containers for culturing hematopoietic progenitor cells (HPCs). Notch ligands (such as DLL4) are preferably used to coat containers for culturing HPCs. Any suitable concentration of Notch ligand can be used. As a non-limiting example, in any aspect of the present invention using Notch ligands, the concentration of Notch ligands (such as DLL4) can be from about 1 μg / ml to about 100 μg / ml, from about 1 μg / ml to about 50 μg / ml, from about 1 μg / ml to about 25 μg / ml, from about 1 μg / ml to about 10 μg / ml or less. In some embodiments, the concentration of Notch ligands (such as DLL4) is about 50 μg / ml, about 25 μg / ml, about 20 μg / ml, about 15 μg / ml, about 10 μg / ml, about 5 μg / ml, about 2 μg / ml, preferably about 10 μg / ml, more preferably about 2 μg / ml. The Notch ligands (e.g., DLL4) of the present invention can be directly coated on tissue culture plastic vessels. Alternatively, additional substrates and / or linkers can be used to facilitate attachment of the Notch ligands (e.g., DLL4) to the surface of the culture vessel. Examples of such substrates are known in the art, such as poly-L-lysine.

[0310] As described above, HPCs can be cultured with or without stromal support cells or feeder cells or cell groups thereof. In some preferred embodiments using Notch ligands, the cells are cultured without stromal support cells or cell groups thereof, but may also be cultured with an extracellular matrix (ECM) as described herein.

[0311] The Notch ligand of the present invention can be added to a sample containing HPCs within a week, six days, five days, four days, three days, two days, or one day after the start of the method of the present invention. For example, the date of thawing and plating from HPCs, the date of separating HPCs from the sample, or the same day as the separation of NK cell precursors. The date of separation of HPCs and / or NK cell precursors is usually the same day as the date on which the sample is obtained from the patient. Preferably, the Notch ligand of the present invention is added to the sample within four days after the start of the method of the present invention (for example, counting from the thawing and plating of HPCs or the separation of HPCs from the sample), such as on the day of start (Day 0), or on the first or second day after the start (for example, counting from the thawing and plating of HPCs or the separation of HPCs). Most preferably, the Notch ligand is added to the sample one day after the start of the method of the present invention (for example, counting from the thawing and plating of HPCs or the separation of HPCs). Thus, Notch ligands are typically present from day one after initiation and thereafter (e.g., counting from the time the HPCs are thawed and plated or the HPCs are isolated) until the pre-differentiated HPC population is cultured in a medium that induces the pre-differentiated HPCs to differentiate into NK cells (i.e., from the first day after initiation (e.g., counting from the time the HPCs are thawed and plated or isolated) until the end of step (a) / the beginning of step (b)).

[0312] Cells (e.g., HPCs in step (a)) can be cultured in the presence of a Notch ligand (e.g., DLL4) for at least 6 hours, at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, or 8 days. Typically, the culture time is 1 to 7 days, preferably about 4 days.

[0313] Alternatively, these culture durations can also be measured by the number of cell passages. For example, cells are passed through at least one generation, at least two generations, at least three generations, at least four generations, at least five generations, at least ten generations, at least twenty generations or more (whether in vivo, or in vitro or in vitro culture). Typically, the number of cell passages corresponding to these durations can reach 10 times, that is, the number of passages can be any number between 1 and 10 (1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 passages), preferably 1 time, 2 times or 3 passages.

[0314] The present invention provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population, wherein (i) a Notch ligand (such as DLL4) is added on the first day after the start of the method, or the HPCs are transferred to a culture container coated with the Notch ligand (such as DLL4) on the first day after the start of the method and cultured in contact with the Notch ligand (such as DLL4) for 4 days; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period to differentiate the HPCs in the pre-differentiated HPC population and expand the resulting NK cells. (i) The culture medium that does not induce HPC differentiation does not contain a REV-ERB inhibitory compound; and (ii) the culture medium that induces HPC differentiation into NK cells contains neither a REV-ERB inhibitory compound nor a Notch ligand.

[0315] The present invention also provides a method for increasing the number of CD16+ NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population, wherein (i) a Notch ligand (such as DLL4) is added on the first day after the method is started, or the HPCs are transferred to a culture container coated with the Notch ligand (such as DLL4) on the first day after the method is started and cultured in contact with the Notch ligand (such as DLL4) for 4 days; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period to differentiate the HPCs in the pre-differentiated HPC population and expand the resulting NK cells. (i) The culture medium that does not induce HPC differentiation does not contain a REV-ERB inhibitory compound; and (ii) the culture medium that induces HPC differentiation into NK cells contains neither a REV-ERB inhibitory compound nor a Notch ligand.

[0316] Methods of using compounds that inhibit REV-ERB activity

[0317] As described herein, the methods of the present invention may not include the use of a compound that inhibits REV-ERB activity. However, in some embodiments, in the methods described herein, hematopoietic progenitor cells (HPCs) may be cultured in the presence of a compound that inhibits REV-ERB activity for at least a portion of the first culture period. Thus, a compound that inhibits REV-ERB activity may be included in a culture medium that does not induce differentiation of HPCs.

[0318] Therefore, the present invention provides a method for producing an expanded CD16+ natural killer (NK) cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period. Step (b) generally includes differentiation of HPCs in the pre-differentiated HPC population and expansion of the resulting NK cells. Therefore, the present invention provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, wherein expansion of NK cells also occurs in the second culture period. The present invention also provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period; (c) expanding the cells in vitro to produce an expanded NK cell population. During at least part of the first culture period, the HPCs may also be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity. Therefore, the culture medium that does not induce HPC differentiation may contain a compound that inhibits REV-ERB activity. Therefore, during at least part of step (a) of the method of the present invention, the HPCs may be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity.

[0319] The present invention also provides a method for increasing the number of CD16+NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period. Step (b) generally includes differentiation of HPCs in the pre-differentiated HPC population and expansion of the resulting NK cells. Therefore, the present invention provides a method for increasing the number of CD16+NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, wherein the expansion of NK cells also occurs in the second culture period. The present invention also provides a method for increasing the number of CD16+ NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period; (c) expanding the cells in vitro to produce an expanded NK cell population. During at least part of the first culture period, the HPCs may also be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity. Therefore, the culture medium that does not induce HPC differentiation may contain a compound that inhibits REV-ERB activity. Therefore, during at least part of step (a) of the method of the present invention, the HPCs may be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity.

[0320] As described herein, the first culture period is between about 2 days and about 8 days. The HPCs can be contacted with a compound that inhibits REV-ERB activity during at least a portion of the first culture period, up to the entire first culture period. Thus, the HPCs can be contacted with a compound that inhibits REV-ERB activity during 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, or 8 days of the first culture period, or any time period between 2 days and 8 days, for example, between 3 days and 8 days or between 2.5 days and 8 days. Preferably, HPCs can be contacted with a compound that inhibits REV-ERB activity within about 2 days to about 6 days (e.g., 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, or 6 days, or any time between 2 days and 6 days, such as between 2.5 days and 6 days, between 3 days and 6 days, or between about 4 days and 6 days) of the first culture period. More preferably, HPCs can be contacted with a compound that inhibits REV-ERB activity within about 2 days to about 4 days (e.g., 2 days, 2.5 days, 3 days, 3.5 days, or 4 days, or any time between 2 days and 4 days, such as between about 2.5 days and 4 days, or between about 3 days and 4 days) of the first culture period.

[0321] Most preferably, the first culture period comprises a period of about 0 to about 2 days, particularly about 1 day, during which the compound that inhibits REV-ERB activity is absent, and the HPCs are then contacted with the compound that inhibits REV-ERB activity for the remainder of the first culture period. Thus, the compound that inhibits REV-ERB activity can be added to the culture medium that does not induce HPC differentiation after a period of about 0 to about 2 days, particularly after about 1 day.

[0322] If the culture medium that does not induce HPCs differentiation is replaced during the first culture period, the culture medium that does not induce HPCs differentiation after the replacement may or may not contain a compound that inhibits REV-ERB activity, depending on whether the presence of a compound that inhibits REV-ERB activity is required at that time point in the first culture period. As a non-limiting example, if the culture medium that does not induce HPCs differentiation is replaced on day 2, but HPCs are cultured with a compound that inhibits REV-ERB activity only from day 4 onwards, then the culture medium that does not induce HPCs differentiation replaced on day 2 will not include a compound that inhibits REV-ERB activity. As a further limiting example, or in addition, if the culture medium that does not induce HPCs differentiation is replaced on day 6, and HPCs are cultured with a compound that inhibits REV-ERB activity from day 4 onwards, then the culture medium that does not induce HPCs differentiation replaced on day 6 will include a compound that inhibits REV-ERB activity. The compound that inhibits REV-ERB activity can be added to the medium that does not induce HPCs differentiation before being used to replace the medium on HPCs, or the medium that does not induce HPCs differentiation can be replaced first and then the compound that inhibits REV-ERB activity can be added to HPCs.

[0323] Alternatively or additionally, in the methods described herein, the pre-differentiated HPC population can be cultured during at least a portion of the second culture period in the presence of a compound that inhibits REV-ERB activity. Thus, a compound that inhibits REV-ERB activity can be included in the culture medium that induces differentiation of HPCs.

[0324] Accordingly, the present invention provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period. Step (b) generally includes differentiation of HPCs in the pre-differentiated HPC population and expansion of the resulting NK cells. Therefore, the present invention provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, wherein expansion of NK cells also occurs in the second culture period. The present invention also provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period; and (c) expanding the cells in vitro to produce an expanded NK cell population. During at least a portion of the second culture period, the pre-differentiated HPCs may also be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity. Therefore, the culture medium that induces HPC differentiation may contain a compound that inhibits REV-ERB activity. Therefore, during at least a portion of step (b) of the method of the present invention, the pre-differentiated HPCs may be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity. This can serve as an alternative or supplement to the inclusion of a compound that inhibits REV-ERB activity in step (a) described herein.

[0325] The present invention also provides a method for increasing the number of CD16+NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period. Step (b) generally includes differentiation of HPCs in the pre-differentiated HPC population and expansion of the resulting NK cells. Therefore, the present invention provides a method for increasing the number of CD16+NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, wherein the expansion of NK cells also occurs in the second culture period. The present invention also provides a method for increasing the number of CD16+ NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period; and (c) expanding the cells in vitro to produce an expanded NK cell population. During at least a portion of the second culture period, the pre-differentiated HPCs may also be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity. Therefore, the culture medium that induces HPC differentiation may contain a compound that inhibits REV-ERB activity. Therefore, during at least a portion of step (b) of the method of the present invention, the pre-differentiated HPCs may be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity. This can serve as an alternative or supplement to the inclusion of a compound that inhibits REV-ERB activity in step (a) described herein.

[0326] As described herein, the second culture period can be between about 10 days and about 30 days. The pre-differentiated HPCs can be contacted with a compound that inhibits REV-ERB activity during at least a portion of the second culture period, up to the entire second culture period. Thus, the pre-differentiated HPCs can be contacted with a compound that inhibits REV-ERB activity on day 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the second culture period, or any time period between 10 and 30 days. Preferably, the pre-differentiated HPCs are contacted with a compound that inhibits REV-ERB activity within about 15 days to about 25 days (e.g., 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, or 25 days, or any time period between 15 days and 25 days) of the second culture period. More preferably, the pre-differentiated HPCs are contacted with a compound that inhibits REV-ERB activity within about 18 days to about 22 days (e.g., 18 days, 19 days, 20 days, 21 days, or 22 days, or any time period between 18 days and 22 days) of the second culture period. Most preferably, the pre-differentiated HPCs are contacted with a compound that inhibits REV-ERB activity within about 19 days to 21 days of the second culture period, particularly preferably about 20 days.

[0327] Preferably, the compound that inhibits REV-ERB activity is present only before inducing HPCs differentiation. In other words, the compound that inhibits REV-ERB activity is present only during at least part of the first culture period, and preferably is absent during at least part of the second culture period. Therefore, the compound that inhibits REV-ERB activity is preferably present in a culture medium that does not induce HPCs differentiation, and is not present in a culture medium that induces HPCs differentiation. Prior to step (b), the pre-differentiated HPC population obtained in step (a) of the method of the present invention can be transferred to a different culture container. This reduces the risk of contamination of the compound that inhibits REV-ERB activity in step (b) of the method.

[0328] The REV-ERB inhibitor compound can be added before, simultaneously with, or after the Notch ligand is added. In some embodiments, the compound is added at multiple time points, such as when the culture medium is changed. As a non-limiting example, a compound of the invention can be added on the first day of the first culture period and then added again on the fifth day of the first culture period.

[0329] The disclosures related to adding Notch ligands and / or compounds that inhibit REV-ERB activity apply independently to all methods of the invention, eg, to methods that also use compounds described herein that alter post-translational modifications of E4bp4.

[0330] As long as the compound that inhibits REV-ERB activity can inhibit the action of REV-ERB as described herein and is useful in expanding the NK cell population, any suitable concentration of the compound can be used. As a non-limiting example, in any aspect of the present invention, the final use concentration of the compound that inhibits REV-ERB activity can be about 2 micromol / L to about 20 micromol / L, about 2 micromol / L to about 15 micromol / L, about 5 micromol / L to about 15 micromol / L, about 5 micromol / L to about 14 micromol / L, about 4 micromol / L to about 13 micromol / L, about 5 micromol / L to about 12 micromol / L, about 5 micromol / L to about 11 micromol / L, or preferably about 2 micromol / L to about 10 micromol / L, for example, about 5 micromol / L to about 10 micromol / L.

[0331] As described above, HPCs can be cultured with or without stromal support cells or feeder cells or cell populations thereof. In some preferred embodiments using compounds that inhibit REV-ERB activity, the cells are cultured without stromal support cells or cell populations thereof, but may alternatively be cultured with extracellular matrix (ECM) or stromal cells as described herein.

[0332] The REV-ERB inhibitory compound can be added to the sample containing HPCs within one week, six days, five days, four days, three days, two days, or one day after the start of the method of the present invention. For example, the date of thawing and plating the HPCs, the date of isolating the HPCs from the sample, or the same day as the separation of NK cell precursors. The date of isolation of HPCs and / or NK cell precursors is usually the same day as the date on which the sample is obtained from the patient. Preferably, the REV-ERB inhibitory compound is added to the sample within five days after the start of the method of the present invention, more preferably within two days (for example, counting from the time the HPCs are thawed and plating or the time the HPCs are separated from the sample), such as on the day of initiation (day 0), or the first or second day after initiation (for example, counting from the time the HPCs are thawed and plating or the time the HPCs are separated). Most preferably, the REV-ERB inhibitory compound of the present invention is added to the sample one day after the start of the method of the present invention (for example, counting from the time the HPCs are thawed and plating or the time the HPCs are separated).

[0333] Thus, typically the REV-ERB inhibitory compound is present on and after the start of the process (e.g., counting from the time the HPCs are thawed and plated or the HPCs are isolated) until the pre-differentiated HPC population is cultured in a medium that induces the pre-differentiated HPCs to differentiate into NK cells (i.e., from the first day after the start of the process (e.g., counting from the time the HPCs are thawed and plated or isolated) until the end of step (a) / the start of step (b)).

[0334] Cells (e.g., HPCs in step (a)) can be cultured in the presence of a REV-ERB inhibitory compound for at least 6 hours, at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, or 8 days. Typically, the culture time is 1 to 7 days, preferably about 4 days.

[0335] Alternatively, these durations can be measured by the number of cell passages. For example, the cells have been passaged at least 1, 2, 3, 4, 5, 10, 20 or more times (in vivo, or under in vitro culture conditions). Typically, these durations can be up to 10 cell passages, i.e., any number of passages between 1 and 10 (1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0336] times), preferably 1, 2 or 3 times.

[0337] The methods of the present invention may include a recovery period, i.e., the hematopoietic progenitor cells (HPCs) are cultured for a recovery period of about 0 to about 5 days before contacting the hematopoietic progenitor cells (HPCs) with the REV-ERB inhibitory compound and undergoing a first culture period. As described herein, the first culture period is about 2 to about 8 days. The HPCs may be contacted with the REV-ERB inhibitory compound for at least a portion of the first culture period up to the entire culture period. Thus, during the first culture period, the HPCs may be contacted with the REV-ERB inhibitory compound for 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 64, 65, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92

[0338] 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 days, or any duration between 2 and 8 days. Preferably, during the first culture period, the HPCs are contacted with the REV-ERB inhibitory compound for a period of about 2 days to about 6 days (e.g., 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 or 6 days, or any duration between 2 and 6 days, such as about 4 to 6 days). More preferably, during the first culture period, the HPCs are contacted with the REV-ERB inhibitory compound for a period of about 2 days to about 4 days (e.g., 2, 2.5, 3, 3.5 or 4 days, or any duration between 2 and 4 days). A method may include a recovery period of about 0 to about 5 days (e.g., about 0 to about 2 days, particularly about 1 day), during which no REV-ERB inhibitory compound is added, and thereafter the HPCs are allowed to grow.

[0339] Contact with REV-ERB inhibitory compound and enter the first culture phase. Therefore, a REV-ERB inhibitory compound that does not induce HPCs differentiation can be added to the culture medium after a period of about 0 to about 5 days (e.g., about 0 to about 2 days), and in particular, the compound can be added to the culture medium after about 1 day.

[0340] ------------------------------------------------------------------

[0341] Methods for concurrently administering Notch ligands and compounds that inhibit REV-ERB activity

[0342] The methods of the present invention may include the simultaneous use of a Notch ligand (e.g., DLL4) and a compound that inhibits REV-ERB activity. Any disclosure herein related to methods using a Notch ligand (e.g., concentrations, timing, etc.) may be combined, without limitation, with any and all disclosures related to methods using a compound that inhibits REV-ERB activity (e.g., concentrations, timing, etc.).

[0343] Therefore, the present invention provides a method for producing an expanded CD16+ natural killer (NK) cell population, comprising the following steps: (a) culturing a sample containing hematopoietic progenitor cells (HPCs) obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period. Step (b) generally includes differentiation of HPCs in the pre-differentiated HPC population and expansion of the resulting NK cells. Therefore, the present invention provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, wherein expansion of NK cells also occurs in the second culture period. The present invention also provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period; (c) expanding the cells in vitro to produce an expanded NK cell population. During at least part of the first culture period, the HPCs may also be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4). Therefore, the culture medium that does not induce HPC differentiation may contain a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4). Therefore, during at least part of step (a) of the method of the present invention, the HPCs may be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4).

[0344] The present invention also provides a method for increasing the number of CD16+NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period. Step (b) generally includes differentiation of HPCs in the pre-differentiated HPC population and expansion of the resulting NK cells. Therefore, the present invention provides a method for increasing the number of CD16+NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, wherein the expansion of NK cells also occurs in the second culture period. The present invention also provides a method for increasing the number of CD16+ NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period; (c) expanding the cells in vitro to produce an expanded NK cell population. During at least part of the first culture period, the HPCs may also be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4). Therefore, the culture medium that does not induce HPC differentiation may contain a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4). Therefore, during at least part of step (a) of the method of the present invention, the HPCs may be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4).

[0345] As described herein, the first culture period is between about 2 days and about 8 days. HPCs can be contacted with a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4) during at least part of the first culture period, up to the entire first culture period. During the first culture period, the length of time that HPCs are in contact with the compound that inhibits REV-ERB activity and the Notch ligand (such as DLL4) can be different or the same. Thus, during a period of 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, or 8 days of the first culture period, or for any period between 2 days and 8 days, HPCs can be contacted with a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4), respectively, and the contact times can be independently selected. Preferably, HPCs can be contacted with a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4) during the first culture period of about 2 days to about 6 days (e.g., 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, or 6 days, or any time period between 2 days and 6 days, such as between about 4 days and 6 days), and the contact time can be independently selected. More preferably, HPCs can be contacted with a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4) during the first culture period of about 2 days to about 4 days (e.g., 2 days, 2.5 days, 3 days, 3.5 days, or 4 days, or any time period between 2 days and 4 days), and the contact time can be independently selected.

[0346] Most preferably, the first culture period comprises a period of about 0 days to about 2 days, particularly about 1 day, during which the compound that inhibits REV-ERB activity and the Notch ligand (such as DLL4) are absent, and then the HPCs are contacted with the compound that inhibits REV-ERB activity and the Notch ligand (such as DLL4) for the remainder of the first culture period. Thus, after a period of about 0 days to about 2 days, during which the compound that inhibits REV-ERB activity and the Notch ligand (such as DLL4) are each independently selected, the compound that inhibits REV-ERB activity and the Notch ligand (such as DLL4) can be added to a culture medium that does not induce differentiation of HPCs, particularly after about 1 day, the compound that inhibits REV-ERB activity and the Notch ligand (such as DLL4) can be added to a culture medium that does not induce differentiation of HPCs.

[0347] A method of the present invention may include a recovery period, such that the HPCs are cultured for a recovery period of about 0 to about 5 days before being contacted with a REV-ERB inhibitory compound and a Notch ligand (e.g., DLL4) and undergoing a first culture period. As described herein, the first culture period is between about 2 days and about 8 days. The HPCs can be contacted with a REV-ERB inhibitory compound and a Notch ligand (e.g., DLL4) for at least a portion of the first culture period, up to the entire first culture period. Thus, the HPCs can be contacted with a REV-ERB inhibitory compound and a Notch ligand (e.g., DLL4) for a period of 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, or 8 days of the first culture period, or for any period between 2 days and 8 days. Preferably, HPCs can be contacted with a REV-ERB inhibitory compound and a Notch ligand (such as DLL4) during the first culture period of about 2 days to about 6 days (e.g., 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, or 6 days, or any time period between 2 days and 6 days, such as between about 4 days and 6 days). More preferably, HPCs can be contacted with a REV-ERB inhibitory compound and a Notch ligand (such as DLL4) during the first culture period of about 2 days to about 4 days (e.g., 2 days, 2.5 days, 3 days, 3.5 days, or 4 days, or any time period between 2 days and 4 days). One method may include a recovery period of about 0 days to about 5 days, such as about 0 days to about 2 days, and particularly about 1 day, during which the REV-ERB inhibitory compound and Notch ligand (such as DLL4) are absent, and then contacting the HPCs with the REV-ERB inhibitory compound and Notch ligand (such as DLL4) during a first culture period. Thus, after a period of about 0 days to about 5 days, such as about 0 days to about 2 days, the REV-ERB inhibitory compound and Notch ligand (such as DLL4) may be added to a culture medium that does not induce differentiation of HPCs, and particularly after about 1 day, the REV-ERB inhibitory compound and Notch ligand (such as DLL4) may be added to a culture medium that does not induce differentiation of HPCs.

[0348] If the culture medium that does not induce HPCs differentiation is replaced during the first culture period, the culture medium that does not induce HPCs differentiation after the replacement may contain / not contain a compound that inhibits REV-ERB activity and / or a Notch ligand (such as DLL4), depending on whether the presence of the compound that inhibits REV-ERB activity and / or the Notch ligand (such as DLL4) is required at that time point during the first culture period. As a non-limiting example, if the culture medium that does not induce HPCs differentiation is replaced on day 2, but HPCs are cultured with a compound that inhibits REV-ERB activity and / or a Notch ligand (such as DLL4) only from day 4 onwards, then the culture medium that does not induce HPCs differentiation replaced on day 2 will not include a compound that inhibits REV-ERB activity and / or a Notch ligand (such as DLL4). As a further limiting example, or in addition, if the medium that does not induce HPCs differentiation is changed on day 6, and the HPCs have been cultured with a compound that inhibits REV-ERB activity and / or a Notch ligand (such as DLL4) since day 4, then the medium that does not induce HPCs differentiation changed on day 6 will include the compound that inhibits REV-ERB activity and the Notch ligand (such as DLL4). The compound that inhibits REV-ERB activity and / or the Notch ligand (such as DLL4) can be added to the medium that does not induce HPCs differentiation before being used to change the medium on the HPCs, or the medium that does not induce HPCs differentiation can be changed first, and then the compound that inhibits REV-ERB activity and / or the Notch ligand (such as DLL4) can be added to the HPCs.

[0349] Alternatively or additionally, in the methods described herein, the pre-differentiated HPC population can be cultured in the presence of a compound that inhibits REV-ERB activity and a Notch ligand (e.g., DLL4) during at least a portion of the second culture period. Thus, a compound that inhibits REV-ERB activity and a Notch ligand (e.g., DLL4) can be included in the culture medium that induces differentiation of HPCs.

[0350] Accordingly, the present invention provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period. Step (b) generally includes differentiation of HPCs in the pre-differentiated HPC population and expansion of the resulting NK cells. Therefore, the present invention provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, wherein expansion of NK cells also occurs in the second culture period. The present invention also provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period; (c) expanding the cells in vitro to produce an expanded NK cell population. During at least part of the second culture period, the pre-differentiated HPCs may also be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4). Therefore, the culture medium that induces HPC differentiation may contain a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4). Therefore, during at least part of step (b) of the method of the present invention, the pre-differentiated HPCs may be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4). This may be an alternative or additional approach to including a compound that inhibits REV-ERB activity and / or a Notch ligand (such as DLL4) in step (a) as described herein.

[0351] The present invention also provides a method for increasing the number of CD16+NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period. Step (b) generally includes differentiation of HPCs in the pre-differentiated HPC population and expansion of the resulting NK cells. Therefore, the present invention provides a method for increasing the number of CD16+NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period, wherein the expansion of NK cells also occurs in the second culture period. The present invention also provides a method for increasing the number of CD16+ NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period; (c) expanding the cells in vitro to produce an expanded NK cell population. During at least part of the second culture period, the pre-differentiated HPCs may also be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4). Therefore, the culture medium that induces HPC differentiation may contain a compound that inhibits REV-ERB activity. Therefore, during at least part of step (b) of the method of the present invention, the pre-differentiated HPCs may be contacted with (cultured in the presence of) a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4). This may be an alternative or additional approach to including a compound that inhibits REV-ERB activity and / or a Notch ligand (such as DLL4) in step (a) as described herein.

[0352] As described herein, the second culture period can be between about 10 days and about 30 days. During at least a portion of the second culture period, up to the entire second culture period, the pre-differentiated hematopoietic progenitor cells (HPCs) can be contacted with a compound that inhibits REV-ERB activity and a Notch ligand (e.g., DLL4). Thus, on day 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the second culture period, the pre-differentiated HPCs can be contacted with a compound that inhibits REV-ERB activity and a Notch ligand (e.g., DLL4), respectively, for a period of time independently selected from the aforementioned number of days, or for each of the compound that inhibits REV-ERB activity and the Notch ligand (e.g., DLL4), the contact time can be any length of time between 10 and 30 days. Preferably, the predifferentiated HPCs can be contacted with a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4) during about 15 days to about 25 days (e.g., 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, or 25 days, or any time period between 15 days and 25 days) of the second culture period, and the contact time can be independently selected from the above days. More preferably, the predifferentiated HPCs can be contacted with a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4) during about 18 days to about 22 days (e.g., 18 days, 19 days, 20 days, 21 days, or 22 days, or any time period between 18 days and 22 days) of the second culture period, and the contact time can be independently selected from the above days. Most preferably, during approximately 19 to 21 days of the second culture period, the pre-differentiated HPCs can be contacted with a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4), respectively, and the contact time can be independently selected from the above-mentioned days. For each of the compound that inhibits REV-ERB activity and the Notch ligand (such as DLL4), a contact time of approximately 20 days is particularly preferred.

[0353] Preferably, the compound that inhibits REV-ERB activity and the Notch ligand (such as DLL4) are present only before inducing HPCs differentiation. In other words, the compound that inhibits REV-ERB activity and the Notch ligand (such as DLL4) are present only during at least part of the first culture period, and preferably are not present during at least part of the second culture period. Therefore, the compound that inhibits REV-ERB activity and the Notch ligand (such as DLL4) are preferably present in a culture medium that does not induce HPCs differentiation, and are not present in a culture medium that induces HPCs differentiation. Prior to step (b) of the method of the present invention, the pre-differentiated HPC population obtained in step (a) can be transferred to a different culture container. This reduces the risk of contamination of the compound that inhibits REV-ERB activity and the Notch ligand (such as DLL4) in step (b) of the method.

[0354] When using a compound that inhibits REV-ERB activity and a Notch ligand (such as DLL4), they can be added simultaneously or in any order. Therefore, the cells can be first contacted with the REV-ERB inhibitory compound and then cultured in the presence of the Notch ligand. Alternatively, the cells can be cultured first in the presence of the Notch ligand and then in the presence of the REV-ERB inhibitory compound. In addition, the cells can be cultured in the presence of both the REV-ERB inhibitory compound and the Notch ligand. Preferably, the cells are first cultured in the presence of the REV-ERB inhibitory compound and then in the presence of the Notch ligand.

[0355] In some embodiments, the REV-ERB inhibitory compound of the present invention is added in step (a) and the Notch ligand is added in step (b). In other embodiments, the Notch ligand is added in step (a) and the REV-EB inhibitory compound is added in step (b). In yet other embodiments, both the REV-ERB inhibitory compound and the Notch ligand are added in step (a). In further embodiments, both the REV-ERB inhibitory compound and the Notch ligand are added in step (b). If the REV-ERB inhibitory compound and the Notch ligand are added in the same stage (step (a) or step (b)), then the stage can be further subdivided so that: (i) the REV-ERB inhibitory compound is added before the Notch ligand; or (ii) the Notch ligand is added before the REV-ERB inhibitory compound. Alternatively, the Notch ligand and the REV-ERB inhibitory compound can be added simultaneously in the same stage. Preferably, both the REV-ERB inhibitor and the Notch ligand are added in step (a), with the REV-ERB inhibitor added first (eg, on day 0 or day 2) and the Notch ligand added second (eg, on day 2 or day 4, respectively).

[0356] Preferred embodiments of the present invention include: (i) adding a REV-ERB inhibitory compound and a Notch ligand to the sample on the first day after the start of the method (e.g., calculated from the time the HPCs are thawed and plated or the HPCs are separated); (ii) adding a REV-ERB inhibitory compound to the sample on the day the method is started (e.g., calculated from the time the HPCs are thawed and plated or the HPCs are separated), and adding a Notch ligand to the sample on the first day after the start of the method (e.g., calculated from the time the HPCs are thawed and plated or the HPCs are separated); or (iii) adding a REV-ERB inhibitory compound to the sample on the second day after the start of the method (e.g., calculated from the time the HPCs are thawed and plated or the HPCs are separated), and adding a Notch ligand to the sample on the fourth day after the start of the method (e.g., calculated from the time the HPCs are thawed and plated or the HPCs are separated); among which scheme (ii) is particularly preferred. As demonstrated by the inventors, these specific conditions maximize the synergistic effect between REV-ERB inhibition and Notch ligands, thereby maximizing NK cell expansion.

[0357] The present invention provides a method for generating an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to generate a pre-differentiated HPC population, wherein (i) a REV-ERB inhibitory compound is added on day 1 after the start of the method, and (ii) after about one day of contact and culture with the REV-ERB inhibitory compound, the HPCs are transferred to a culture vessel coated with a Notch ligand (such as DLL4) and cultured for another about four days; and (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period to differentiate the HPCs in the pre-differentiated HPC population and expand the resulting NK cells. The culture medium that induces HPC differentiation into NK cells contains neither a REV-ERB inhibitory compound nor a Notch ligand.

[0358] The present invention also provides a method for increasing the number of CD16+ NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population, wherein (i) a REV-ERB inhibitory compound is added on day 1 after the start of the method, and (ii) after contact with the REV-ERB inhibitory compound for about 1 day, the HPCs are transferred to a culture vessel coated with a Notch ligand (such as DLL4) and cultured for another about 4 days; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period to differentiate the HPCs in the pre-differentiated HPC population and expand the resulting NK cells. The culture medium that induces HPC differentiation into NK cells contains neither a REV-ERB inhibitory compound nor a Notch ligand.

[0359] The present invention provides a method for producing an expanded CD16+ NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population, wherein (i) a Notch ligand (such as DLL4) is added on day 1 after the start of the method, or the HPCs are transferred to a culture vessel coated with the Notch ligand (such as DLL4) on day 1 after the start of the method, and (ii) after culturing with the Notch ligand (such as DLL4) for 4 days, a REV-ERB inhibitory compound is added and cultured for an additional approximately 24 hours; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period to differentiate the HPCs in the pre-differentiated HPC population and expand the resulting NK cells. The culture medium that induces HPC differentiation into NK cells contains neither a REV-ERB inhibitory compound nor a Notch ligand.

[0360] The present invention also provides a method for increasing the number of CD16+ NK cells in an expanded NK cell population, comprising the following steps: (a) culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPC differentiation for a first culture period to produce a pre-differentiated HPC population, wherein (i) a Notch ligand (such as DLL4) is added on the first day after the start of the method, or the HPCs are transferred to a culture vessel coated with the Notch ligand (such as DLL4) on the first day after the start of the method, and (ii) after culturing with the Notch ligand (such as DLL4) for 4 days, a REV-ERB inhibitory compound is added and cultured for another approximately 24 hours; (b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells for a second culture period to differentiate the HPCs in the pre-differentiated HPC population and expand the resulting NK cells. The culture medium that induces HPC differentiation into NK cells contains neither a REV-ERB inhibitory compound nor a Notch ligand.

[0361] Additional external stimulation

[0362] Any of the methods described herein, whether using one, both, or neither of Notch ligands, compounds that inhibit REV-ERB activity, or both, may include the use of one or more additional external stimuli. Any disclosure herein related to methods using Notch ligands (e.g., concentrations, timing, etc.) and / or compounds that inhibit REV-ERB activity (e.g., concentrations, timing, etc.) may be combined with any and all disclosures related to methods using any external stimulus (e.g., concentrations, timing, etc.) without limitation.

[0363] Additional external stimulation, such as growth factors and / or cytokines, can be used to further promote the generation of (CD16+) NK cells. The non-limiting example of suitable external stimulation includes interleukin-7 (IL-7), interleukin-15 (IL-15), Fms-like tyrosine kinase 3 ligand (Flt3L), stem cell factor (SCF), thrombopoietin (TPO), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and / or interleukin-6 (IL-6), or any combination thereof. Any suitable concentration of these factors can be used. The non-limiting example of the suitable concentration of these factors is described herein.

[0364] The inventors surprisingly found that omitting IL-3 from the culture medium for inducing HPCs to differentiate into NK cells, that is, in the culture medium used in the culturing step (step (b) of the method of the present invention) of subjecting the pre-differentiated HPC population to a second culture period in the culture medium for inducing HPCs to differentiate into NK cells, increases the number of CD16+ NK cells produced. As exemplified herein, when the pre-differentiated HPC population is contacted with a Notch ligand (such as DLL4) in step (b), and / or when the HPCs in step (a) are contacted with a Notch ligand (such as DLL4) in step (a), it is particularly important to omit IL-3 from the culture medium for inducing HPCs to differentiate into NK cells. Therefore, preferably, in the case where a Notch ligand (such as DLL4) is used in the method of the present invention (such as steps (a) and / or (b) described herein), IL-3 is omitted in step (b) of the method. Or in addition, in the case where the method of the present invention (such as steps (a) and / or (b) described herein) involves the use of stromal cells, IL-3 is omitted in step (b) of the method.

[0365] Therefore, a method of the present invention can omit IL-3 from the following situations: (i) a culture medium that induces HPCs to differentiate into NK cells, i.e., the culture medium used in the step of culturing a pre-differentiated HPC population in a culture medium that induces HPCs to differentiate into NK cells for a second culture period (step (b) of the method of the present invention); and / or (ii) a culture medium that does not induce HPCs to differentiate in the first culture period to produce a pre-differentiated HPC population, i.e., the culture medium used in the step of culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs to differentiate for a first culture period to produce a pre-differentiated HPC population (step (a) of the method of the present invention). Preferably, IL-3 is omitted from the culture medium for inducing HPCs to differentiate into NK cells, that is, the culture medium used in the step of culturing the pre-differentiated HPC population in a culture medium for inducing HPCs to differentiate into NK cells for a second culture period (step (b) of the method of the present invention); but IL-3 is not omitted from the culture medium for not inducing HPCs differentiation in the first culture period to produce a pre-differentiated HPC population, that is, the culture medium used in the step of culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiated HPC population (step (a) of the method of the present invention).

[0366] In the step of culturing a sample containing HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiation HPC population, the culture medium may include one or more of GM-CSF, IL-3, IL-6, Flt3, TPO and / or SCF, or any combination thereof. Typically, the exogenous cytokine added in step (a) of the present invention may include one or more of GM-CSF, IL-3, IL-6, Flt3, TPO and / or SCF, or consist of them, or be any combination thereof. The exogenous cytokine added in step (a) of the present invention may include GM-CSF, preferably in combination with one or more of IL-3, IL-6, Flt3, TPO and / or SCF, more preferably in combination with one or more of IL-6, Flt3, TPO and / or SCF. The exogenous cytokines added in step (a) of the method of the present invention may be composed of one or more combinations of GM-CSF and IL-3, IL-6, Flt3, TPO and / or SCF, more preferably one or more combinations of IL-6, Flt3, TPO and / or SCF. Preferably, the exogenous cytokines added in step (a) of the method of the present invention may comprise GM-CSF, IL-3, IL-6, Flt3, TPO and SCF, more preferably GM-CSF, IL-6, Flt3, TPO and SCF. More preferably, the exogenous cytokines added in step (a) of the method of the present invention may be composed of GM-CSF, IL-3, IL-6, Flt3, TPO and SCF, more preferably GM-CSF, IL-6, Flt3, TPO and SCF.

[0367] Therefore, step (a) of the method of the present invention may include culturing a sample comprising HPCs obtained from an individual in a culture medium that does not induce HPCs differentiation for a first culture period to produce a pre-differentiation HPC population, wherein the culture medium comprises one or more of GM-CSF, IL-3, IL-6, Flt3, TPO and / or SCF, or any combination thereof. Typically, the culture medium that does not induce HPCs differentiation comprises at least GM-CSF, preferably in combination with one or more of IL-3, IL-6, Flt3, TPO and / or SCF, more preferably in combination with one or more of IL-6, Flt3, TPO and / or SCF. Preferably, the culture medium that does not induce HPCs differentiation comprises GM-CSF, IL-3, IL-6, Flt3, TPO and SCF. Alternatively, the culture medium that does not induce HPCs differentiation may comprise GM-CSF, IL-6, Flt3, TPO and SCF. Exemplary concentrations of these external stimuli are described herein. Typically, HPCs are cultured in the absence of exogenous IL-15 in step (a), and exogenous IL-15 is added only in step (b).

[0368] Typically, the exogenous cytokine added in step (b) of the inventive method may comprise one or more of IL-7, IL-15, Flt3 and / or SCF, or any combination thereof. The exogenous cytokine added in step (b) of the inventive method may consist of one or more of IL-7, IL-15, Flt3 and / or SCF, or any combination thereof. Preferably, the exogenous cytokine added in step (b) of the inventive method may comprise IL-7, IL-15, Flt3 and SCF. More preferably, the exogenous cytokine added in step (b) of the inventive method may consist of IL-7, IL-15, Flt3 and SCF.

[0369] Therefore, in the culture step of carrying out the second culture period of the pre-differentiated HPC population in the culture medium for inducing HPCs to NK cell differentiation, the culture medium may include one or more of IL-7, IL-15, Flt3 and / or SCF, or any combination thereof. Therefore, step (b) of the inventive method may include carrying out the culture of the second culture period of the pre-differentiated HPC population in the culture medium for inducing HPCs to NK cell differentiation, wherein the culture medium includes one or more of IL-7, IL-15, Flt3 and / or SCF, or any combination thereof. Preferably, the culture medium for inducing HPCs differentiation includes IL-7, IL-15, Flt3 and SCF. The exemplary concentrations of these external stimuli are described herein. Typically, the culture medium for inducing HPCs to NK cell differentiation does not include IL-3.

[0370] Although IL-2 is traditionally used to generate NK cells, the present invention is generally independent of IL-2. Typically, the culture medium used in steps (a) and / or (b) does not contain IL-2. Preferably, the culture medium used in steps (a) and (b) does not contain IL-2, so that the method of the present invention does not use IL-2 and can be described as IL-2-free.

[0371] As non-limiting examples, IL-7 can be used at a concentration of about 1 ng / ml to about 100 ng / ml, about 1 ng / ml to about 50 ng / ml, about 1 ng / ml to about 25 ng / ml, about 1 ng / ml to about 10 ng / ml, or less. IL-7 can be used at a concentration of about 50 ng / ml, about 25 ng / ml, about 20 ng / ml, about 15 ng / ml, about 10 ng / ml, or about 5 ng / ml, preferably about 10 ng / ml, and more preferably about 20 ng / ml.

[0372] As non-limiting examples, Flt3L can be used at a concentration of about 1 ng / ml to about 100 ng / ml, about 10 ng / ml to about 100 ng / ml, about 1 ng / ml to about 50 ng / ml, about 1 ng / ml to about 25 ng / ml, about 1 ng / ml to about 10 ng / ml, or lower. Flt3L can be used at a concentration of about 100 ng / ml in a medium that does not induce differentiation, and preferably at about 10 ng / ml in a differentiation medium.

[0373] As non-limiting examples, SCF can be used at a concentration of about 1 ng / ml to about 200 ng / ml, about 1 ng / ml to about 150 ng / ml, about 1 ng / ml to about 100 ng / ml, about 20 ng / ml to about 100 ng / ml, about 1 ng / ml to about 50 ng / ml or less, preferably about 20 ng / ml to about 100 ng / ml. SCF can be used at a concentration of about 150 ng / ml, about 125 ng / ml, about 120 ng / ml, about 110 ng / ml, about 100 ng / ml, about 90 ng / ml, about 80 ng / ml, or about 75 ng / ml, preferably about 100 ng / ml.

[0374] As non-limiting examples, IL-15 can be used at a concentration of about 1 ng / ml to about 100 ng / ml, about 10 ng / ml to about 50 ng / ml, about 1 ng / ml to about 50 ng / ml, about 1 ng / ml to about 40 ng / ml, about 1 ng / ml to about 30 ng / ml, about 1 ng / ml to about 20 ng / ml, about 1 ng / ml to about 10 ng / ml or less, preferably about 10 ng / ml to about 50 ng / ml. IL-15 can be used at a concentration of about 50 ng / ml, about 40 ng / ml, about 35 ng / ml, about 30 ng / ml, about 25 ng / ml, about 20 ng / ml, or about 10 ng / ml, preferably about 30 ng / ml, and more preferably about 10 ng / ml.

[0375] As non-limiting examples, TPO can be used at a concentration of about 1 ng / ml to about 100 ng / ml, about 1 ng / ml to about 50 ng / ml, about 1 ng / ml to about 40 ng / ml, about 1 ng / ml to about 30 ng / ml, about 1 ng / ml to about 20 ng / ml, about 1 ng / ml to about 10 ng / ml, or less. TPO can be used at a concentration of about 50 ng / ml, about 40 ng / ml, about 35 ng / ml, about 30 ng / ml, about 25 ng / ml, about 20 ng / ml, or about 10 ng / ml, preferably about 30 ng / ml, and more preferably about 100 ng / ml.

[0376] As non-limiting examples, GM-CSF can be used at a concentration of about 1 ng / ml to about 100 ng / ml, about 1 ng / ml to about 50 ng / ml, about 1 ng / ml to about 40 ng / ml, about 1 ng / ml to about 30 ng / ml, about 1 ng / ml to about 20 ng / ml, about 1 ng / ml to about 10 ng / ml, or less. IL-15 can be used at a concentration of about 50 ng / ml, about 40 ng / ml, about 35 ng / ml, about 30 ng / ml, about 25 ng / ml, about 20 ng / ml, or about 10 ng / ml, preferably about 10 ng / ml or 30 ng / ml.

[0377] As non-limiting examples, IL-3 can be used at a concentration of about 1 ng / ml to about 100 ng / ml, about 1 ng / ml to about 50 ng / ml, about 1 ng / ml to about 40 ng / ml, about 1 ng / ml to about 30 ng / ml, about 1 ng / ml to about 20 ng / ml, about 1 ng / ml to about 10 ng / ml, or less. IL-15 can be used at a concentration of about 50 ng / ml, about 40 ng / ml, about 35 ng / ml, about 30 ng / ml, about 25 ng / ml, about 20 ng / ml, or about 10 ng / ml, preferably about 30 ng / ml, and more preferably about 10 ng / ml.

[0378] As non-limiting examples, IL-6 can be used at a concentration of about 1 ng / ml to about 100 ng / ml, about 10 ng / ml to 50 ng / ml, about 1 ng / ml to about 50 ng / ml, about 1 ng / ml to about 40 ng / ml, about 1 ng / ml to about 30 ng / ml, about 1 ng / ml to about 20 ng / ml, about 1 ng / ml to about 10 ng / ml or less, preferably about 10 ng / ml to 50 ng / ml. IL-6 can be used at a concentration of about 50 ng / ml, about 40 ng / ml, about 35 ng / ml, about 30 ng / ml, about 25 ng / ml, about 20 ng / ml, or about 10 ng / ml, preferably about 10 ng / ml or about 30 ng / ml.

[0379] The culture medium that does not induce HPCs differentiation (i.e., the culture medium used in step (a)) may contain: (i) GM-CSF at a concentration of 1 ng / ml to about 100 ng / ml, preferably about 10 ng / ml; (ii) IL-3 at a concentration of about 1 ng / ml to about 100 ng / ml, preferably about 10 ng / ml; (iii) IL-6 at a concentration of about 10 ng / ml to 50 ng / ml, preferably about 10 ng / ml; (iv) Flt3 at a concentration of about 10 ng / ml to about 100 ng / ml, preferably about 100 ng / ml; (v) TPO at a concentration of about 1 ng / ml to about 100 ng / ml, preferably about 100 ng / ml; and (vi) SCF at a concentration of about 20 ng / ml to about 100 ng / ml, preferably about 100 ng / ml.

[0380] The culture medium that does not induce HPCs differentiation (i.e., the culture medium used in step (a)) may contain: (i) about 10 ng / ml GM-CSF; (ii) about 10 ng / ml IL-3; (iii) about 10 ng / ml IL-6; (iv) about 100 ng / ml Flt3; (v) about 100 ng / ml TPO; and (vi) about 100 ng / ml SCF.

[0381] The culture medium that does not induce HPCs differentiation (i.e., the culture medium used in step (a)) may contain: (i) GM-CSF at a concentration of 1 ng / ml to about 100 ng / ml, preferably about 10 ng / ml; (ii) IL-6 at a concentration of about 10 ng / ml to 50 ng / ml, preferably about 10 ng / ml; (iii) Flt3 at a concentration of about 10 ng / ml to about 100 ng / ml, preferably about 100 ng / ml; (iv) TPO at a concentration of about 1 ng / ml to about 100 ng / ml, preferably about 100 ng / ml; and (v) SCF at a concentration of about 20 ng / ml to about 100 ng / ml, preferably about 100 ng / ml.

[0382] The culture medium that does not induce differentiation of hematopoietic progenitor cells (HPCs) (i.e., the culture medium used in step (a)) may contain: (i) granulocyte-macrophage colony-stimulating factor (GM-CSF) at a concentration of about 10 ng / ml, (ii) interleukin-6 (IL-6) at a concentration of about 10 ng / ml, (iii) Fms-like tyrosine kinase 3 ligand (Flt3) at a concentration of about 100 ng / ml, (iv) thrombopoietin (TPO) at a concentration of about 100 ng / ml, and (v) stem cell factor (SCF) at a concentration of about 100 ng / ml.

[0383] The culture medium for inducing HPCs differentiation may contain: (i) interleukin-7 (IL-7) at a concentration of about 1 ng / ml to about 100 ng / ml, preferably about 20 ng / ml, (ii) interleukin-15 (IL-15) at a concentration of about 10 ng / ml to about 50 ng / ml, preferably about 10 ng / ml, (iii) Flt3 at a concentration of about 10 ng / ml to about 100 ng / ml, preferably about 10 ng / ml, and (iv) SCF at a concentration of about 20 ng / ml to about 100 ng / ml, preferably about 20 ng / ml.

[0384] The culture medium for inducing HPCs differentiation may contain: (i) IL-7 at a concentration of about 20 ng / ml, (ii) IL-15 at a concentration of about 10 ng / ml, (iii) Flt3 at a concentration of about 10 ng / ml, and (iv) SCF at a concentration of about 20 ng / ml.

[0385] HPCs and / or predifferentiation of HPC colonies can be cultivated on suitable support / stromal cells or cell layers, or co-cultivated with them. Any suitable stromal cells can be used, including but not limited to OP9 stromal cells and / or ELO8 cells (for example EL08-ID2 stromal cells), wherein ELO8 cells are more preferred. In other words, the step (a) and / or step (b) of the inventive method can be carried out in the presence of matrix / supporting cells as described herein or matrix / supporting cell layer (particularly ELO8 cells). The step (a) of the inventive method can be carried out in the presence of matrix / supporting cells as described herein or matrix / supporting cell layer (particularly ELO8 cells). The step (b) of the inventive method can be carried out in the presence of matrix / supporting cells as described herein or matrix / supporting cell layer (particularly ELO8 cells). The steps (a) and (b) of the inventive method can be carried out in the presence of matrix / supporting cells as described herein or matrix / supporting cell layer (particularly ELO8 cells). Alternatively, HPCs and / or predifferentiation of HPC colonies can be cultivated without support / stromal cells or cell layers. In other words, steps (a) and / or step (b) of the method of the present invention can be performed in the absence of a matrix / supporting cells or a matrix / supporting cell layer. When support / matrix cells are not used, HPCs and / or pre-differentiated HPC populations can be cultured on an extracellular matrix (ECM) or ECM proteins. ECM proteins can be of natural origin, purified from human or animal tissues. Alternatively, ECM proteins can be genetically engineered recombinant proteins or synthetic proteins. ECM proteins can be complete proteins or in the form of natural or modified peptide fragments. Examples of defined and / or xenobiotic ECM proteins that may be useful in cell culture matrices include laminin, type I collagen, type IV collagen, fibronectin, and vitronectin. ECM compositions can include synthetic fibronectin peptide fragments or recombinant fibronectin, or at least a mixture of fibronectin and vitronectin.

[0386] One or more steps of the method of the present invention, or the entire process of the method can be carried out under "xeno-free (XF)", "animal-free (ACF)" or "animal-derived component-free" conditions. When the terms "XF" and "ACF" are used to describe culture medium, extracellular matrix or culture conditions, it refers to culture medium, ECM or culture conditions that are substantially free of xeno-animal derived components. For the cultivation of human cells, any protein from a non-human animal (such as a mouse) is a xeno-component. In some aspects, a xeno-free matrix can be substantially free of any components of non-human animal origin. The culture medium used in the method of the present invention (in steps (a) and / or (b)) can be "serum-free", which means that the culture medium does not contain untreated or unpurified serum, and therefore can include culture medium containing purified blood-derived components or animal tissue-derived components (such as growth factors). The culture medium according to the present invention may or may not contain any serum substitutes.

[0387] One or more steps of the method of the present invention, or the entire process of the method, can be carried out under "defined" conditions. When the term "defined" is used to describe a culture medium, extracellular matrix, or culture conditions, it refers to a culture medium, extracellular matrix, or culture conditions in which the properties and amounts of substantially all of the components are known. A "chemically defined medium" refers to a culture medium in which the chemical properties and amounts of substantially all of the components are known. These media are also referred to as synthetic media.

[0388] Any of the methods of the present invention, whether using one, both, or neither of a Notch ligand, a compound that inhibits REV-ERB activity, or both, can include administering one or more compounds that cause an alteration in the post-translational modification of E4bp4, thereby increasing E4bp4 activity as described herein. Alternatively, as described herein, the alteration in the post-translational modification of E4bp4 is a decrease in the level of SUMOylation and / or phosphorylation of E4bp4. In some preferred embodiments, the compound that causes an alteration in the post-translational modification of E4bp4: decreases the level of SUMOylation of one or more residues among K10, K116, K219, K337, and / or K394 (or residues corresponding thereto, or any combination thereof) of E4bp4; and / or decreases the level of phosphorylation of one or more residues among S286, S301, and / or S454 (or residues corresponding thereto, or any combination thereof) of E4bp4.

[0389] Any compound that can cause a change in the post-translational modification of E4bp4, as long as it can increase the activity of E4bp4 as described herein and is useful for expanding the (CD16+) NK cell population, can be used at a suitable concentration. As a non-limiting example, in any aspect of the present invention, the final use concentration of the compound that can cause a change in the post-translational modification of E4bp4 can be about 0.1 micromol / L to about 20 micromol / L, about 0.1 micromol / L to about 15 micromol / L, about 0.5 micromol / L to about 15 micromol / L, about 0.5 micromol / L to about 14 micromol / L, about 0.5 micromol / L to about 12 micromol / L, about 0.5 micromol / L to about 11 micromol / L, or about 0.5 micromol / L to about 10 micromol / L, for example, about 5 micromol / L to about 10 micromol / L. In some preferred embodiments, the final use concentration of the compound that can cause changes in the post-translational modification of E4bp4 can be about 0.5 μmol / L to about 5 μmol / L, more preferably about 0.5 μmol / L to about 2 μmol / L, and even more preferably about 0.5 μmol / L to about 1 μmol / L.

[0390] The REV-ERB inhibitor compound, Notch ligand, compound that alters the post-translational modification of E4bp4, and / or other external stimuli can be used simultaneously, separately, or sequentially as described herein.

[0391] Each of the REV-ERB inhibitor compounds, Notch ligand compounds that alter the post-translational modification of E4bp4, and / or other external stimuli can be independently applied as a single treatment or application, or multiple treatments or applications (all applicable in the in vitro, ex vivo, or in vivo methods described herein). For multiple applications, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or more applications can be used. Multiple applications can be performed at any suitable time point according to the methods or treatments of the present invention. As non-limiting examples, the REV-ERB inhibitory compounds, Notch ligands, compounds that alter the post-translational modification of E4bp4, and / or other external stimuli of the present invention can each be independently applied twice a day, once a day, once every other day, once every three days, or once a week. Typically, the REV-ERB inhibitory compounds, Notch ligands, compounds that alter the post-translational modification of E4bp4, and / or other external stimuli of the present invention can be applied independently as needed when changing the culture medium.

[0392] The methods of the present invention may further comprise modulating (increasing or decreasing) the expression and / or activity of one or more additional genes and / or proteins in HPCs to promote the expansion of (CD16+) NK cells. Such modulation may be initiated by a compound of the present invention, including the same compound of the present invention used to inhibit REV-ERB activity. Alternatively, one or more additional compounds may be used to modulate the expression and / or activity of one or more additional genes and / or proteins. The modulation may occur directly or indirectly. Indirect modulation includes downstream effects caused by the inhibition of REV-ERB activity by a compound of the present invention.

[0393] Treatment indications

[0394] As described herein, the present invention provides methods for preparing CD16+ natural killer (NK) cells, and expanded NK cell populations having a greater number of CD16+ NK cells. Also as described herein, CD16+ NK cells are generally more active than CD16- or CD16 low-expressing (CD16lo) cells. In particular, the CD16+ NK cells and expanded NK cell populations prepared by the methods of the present invention generally exhibit at least 50% higher antibody-dependent cell-mediated cytotoxicity (ADCC), preferably at least 70% higher ADCC, compared to the control NK cells described herein.

[0395] Therefore, the present invention also relates to the therapeutic use of CD16+ NK cells and expanded (CD16+) NK cell populations, and the therapeutic use of compositions comprising CD16+ NK cells and expanded (CD16+) NK cell populations.

[0396] Accordingly, the present invention provides CD16+ NK cells, expanded (CD16+) NK cell populations, and / or compositions comprising the same for use in therapeutic methods.

[0397] The present invention further provides a method of treatment comprising administering to a patient in need thereof a therapeutically effective amount of CD16+ NK cells, expanded (CD16+) NK cell populations, and / or compositions comprising the same.

[0398] The present invention also provides use of CD16+ NK cells, expanded (CD16+) NK cell populations and / or the composition of the present invention in preparing a medicament.

[0399] Typically, the method of treatment comprises administering a product (described herein) to a patient or subject.

[0400] Administration of the CD16+ NK cells, amplified (CD16+) NK cell populations and / or compositions of the present invention to a patient generally increases the number of NK cells in the patient. As used herein, the term "increase NK cell number" can be understood as meaning that the product of the present invention can significantly increase the number of NK cells in a patient. This increase in NK cell number can be measured relative to a control (as described herein).

[0401] The increase in the number of NK cells can be quantified as a fold increase relative to a control. Typically, the therapeutic applications of the present invention can increase the number of NK cells relative to a control by at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 3-fold or more.

[0402] Alternatively, reference to increasing the number of NK cells can be understood to mean an increase in the number of NK cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 300%, or more, compared to a control. Typically, the number of NK cells is increased by at least 50%, preferably by at least 70%, more preferably by at least 80%, and even more preferably by at least 90% or more, compared to a control.

[0403] In some embodiments, the increase in the number of NK cells can be defined based on the absolute number of NK cells in a sample or patient, such as the percentage of NK cells, such as the percentage of NK cells in a circulating lymphocyte population. For example, the therapeutic methods of the present invention may result in an increase in the number of NK cells such that the percentage of NK cells reaches at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or more.

[0404] The number of NK cells can be determined by quantitative and / or qualitative analysis, and can be measured directly or indirectly. Any suitable technology can be used to determine relative to the NK cell number of control. Suitable standard techniques, such as flow cytometry, fluorescence activated cell sorting (FACS) and magnetic cell sorting (MACS), are known in the art.

[0405] The number of NK cells can be increased for at least 6 hours, at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or longer compared to a control. Typically, this is assessed relative to the last administration of treatment.

[0406] The number of NK cells can be quantified based on the total number of NK cells in a sample from an individual / patient or in a cultured sample (from the ex vivo method of the present invention).

[0407] In the context of the therapeutic uses and methods of the present invention, "subject" or "patient" (these terms are used interchangeably herein) refers to any animal patient that would benefit from increasing the number of NK cells. Typical animal patients are mammals, such as primates. Preferably, the patient is a human.

[0408] The therapeutic uses or methods of the present invention may comprise administering to a subject or individual a therapeutically effective amount of CD16+ NK cells, expanded (CD16+) NK cell populations and / or compositions (as defined herein), either alone or in combination with other therapeutic agents.

[0409] As used herein, the terms "treat" or "treatment" include therapeutic or prophylactic / preventative measures.

[0410] The compounds or products of the present invention can also be used as preventive treatments. As used herein, the term "prevent" includes preventing the onset of symptoms associated with a disease or condition, which can be treated by increasing the number of NK cells and / or reducing the severity or intensity of the symptoms. The term "prevent" includes inducing or providing protective immunity against such diseases or conditions, particularly infectious diseases as described herein. Immunity can be quantified using any suitable technique, examples of which are known in the art.

[0411] The CD16+NK cells, amplified (CD16+) NK cell populations and / or compositions of the present invention can be administered to patients who already have a disease or condition that can be treated by increasing the number of NK cells. For example, a patient may be suspected of having an infectious disease or cancer as described herein and may be showing or not yet showing symptoms of the disease or condition. When administered to such patients, the compounds or products of the present invention can cure, delay, alleviate the severity of one or more symptoms, improve one or more symptoms, and / or prolong the survival of the subject beyond the survival expected in the absence of such treatment.

[0412] Alternatively, the CD16+ NK cells, expanded (CD16+) NK cell populations and / or compositions of the invention can be administered to a patient who may eventually become infected with a particular infectious disease, or develop a disease or condition described herein, in order to cure, delay, reduce the severity of one or more symptoms, ameliorate one or more symptoms, and / or prolong the subject's survival beyond what would be expected in the absence of such treatment, or, in the case of infectious diseases, to help prevent the patient from transmitting the disease.

[0413] The therapeutic and prophylactic treatments of the present invention are applicable to a variety of subjects of different ages. In humans, these treatment methods are applicable to children (e.g., infants, children under 5 years old, older children or adolescents) and adults. In other animal subjects (e.g., mammals, such as primates), these treatment methods are applicable to immature subjects and mature / adult subjects.

[0414] The present invention relates to treating any disease or condition that can be beneficially treated by increasing the number of NK cells in the patient's body. Such diseases and conditions include cancer, infectious diseases (acute and chronic), autoimmune diseases, and diseases or conditions associated with female infertility or pregnancy. Infectious diseases that can be treated according to the present invention include viral infections, and infections caused by other pathogens, including bacteria, protozoa, fungi, or helminthic pathogens. Typically, the pathogen is an intracellular pathogen that has at least one intracellular stage in its life cycle. Infections of particular concern include viral infections, and zoonotic infectious diseases that are particularly important from a public health perspective. Cancers that can be treated according to the present invention include bladder cancer, blood cancer, leukemia, bone cancer, intestinal cancer, brain tumors, breast cancer, kidney cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, and uterine cancer. Autoimmune diseases that can be treated according to the present invention include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and obesity-induced insulin resistance. As used herein, the term "diseases or conditions associated with female infertility or pregnancy" include, but are not limited to, fetal growth restriction, premature birth, uterine vascular remodeling defects, and preeclampsia.

[0415] The CD16+NK cells of the present invention, the (CD16+) NK cell colonies and / or compositions can be used in combination with one or more additional therapeutic agents or treatment methods, which can generally be selected from conventional treatment methods for the disease to be treated or the condition. As a non-limiting example, if the CD16+NK cells of the present invention, the (CD16+) NK cell colonies and / or compositions are used to treat cancer, such as lung cancer, then the CD16+NK cells, the (CD16+) NK cell colonies and / or compositions can be used in combination with conventional treatment methods for lung cancer, such as radiotherapy, chemotherapy or surgery. When used in combination with one or more additional therapeutic agents or treatment methods, the CD16+NK cells of the present invention, the (CD16+) NK cell colonies and / or compositions can be applied before, simultaneously or after applying one or more additional therapeutic agents or treatment methods.

[0416] In some preferred embodiments, the CD16+NK cells of the present invention, the (CD16+) NK cell colonies and / or compositions of the invention are used in combination with antibody-mediated immunotherapy. Antibody-mediated immunotherapy involves administering antibodies to the patient to target disease-specific antigens. Such antibodies can be used to increase the specificity and killing activity of NK cells, and NK cells express receptors for the FC region of IgG antibodies. Activation of these FC receptors can lead to NK cell activation, thereby causing the activated NK cells to secrete cytokines and release cytotoxic granules, resulting in cell lysis expressing disease antigens. This combination therapy is particularly preferred for treating cancer (using antibodies against tumor-specific antigens). Any antibody used in immunotherapy can be used in combination with the CD16+NK cells of the present invention, the (CD16+) NK cell colonies and / or compositions of the invention. Non-limiting examples of such antibodies include anti-CD20 monoclonal antibodies (for non-Hodgkin's lymphoma, chronic lymphocytic lymphoma), anti-ganglioside D2 (anti-GD2) monoclonal antibodies (for neuroblastoma, melanoma), anti-human epidermal growth factor (anti-HER2) monoclonal antibodies (for breast cancer and gastric cancer), and anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies (for colorectal cancer and head and neck cancer).

[0417] Pharmaceutical compositions and formulations

[0418] The terms "compound" or "product" are used interchangeably herein with the terms "therapeutic / prophylactic composition," "formulation," or "medicament."

[0419] The compounds of the present invention, products or amplified (CD16+) NK cell colonies (as defined above) can be combined with a pharmaceutically acceptable carrier, diluent and / or excipient or administered together. As an alternative or supplement, the compounds of the present invention, products or amplified (CD16+) NK cell colonies can also be combined with one or more of salt, excipient, diluent, adjuvant, immunomodulator and / or antimicrobial compound.

[0420] Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or salts formed with organic acids, such as acetic acid, oxalic acid, tartaric acid, maleic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0421] Immunogenic compositions, therapeutic formulations, drugs and prophylactic formulations are typically administered by conventional routes, such as intravenous, subcutaneous, intraperitoneal or mucosal routes. Administration can be by parenteral injection, such as subcutaneous, intradermal or intramuscular injection. For example, a formulation comprising an antibody of the present invention or amplified NK cell population may be particularly suitable for intravenous, intramuscular, intradermal or subcutaneous administration. Administration of small molecule REV-ERB inhibitors can be by injection, such as intravenous, intramuscular, intradermal or subcutaneous injection, or by oral administration (small molecules with a molecular weight of less than 500 daltons typically have oral bioavailability).

[0422] Thus, the immunogenic compositions, therapeutic formulations, medicaments, and prophylactic formulations of the present invention can be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for dissolution or suspension in liquids prior to injection can also be prepared. The formulations can also be emulsified, or the peptides can be encapsulated in liposomes or microcapsules.

[0423] The active immunogenic component (such as the compound, product or expanded (CD16+) NK cell population of the present invention) is usually mixed with a pharmaceutically acceptable excipient that is compatible with the active ingredient. Suitable excipients include water, saline, glucose, glycerol, ethanol, and the like, and combinations thereof. In addition, if necessary, the vaccine may contain a small amount of auxiliary substances, such as wetting agents or emulsifiers, pH buffers, and / or adjuvants that enhance the efficacy of the composition.

[0424] In general, the carrier is a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, physiological saline, and phosphate buffered saline. However, in some embodiments, if the composition comprises a compound or product of the invention, it may be in lyophilized form, in which case it may contain a stabilizer, such as bovine serum albumin (BSA). In some embodiments, it may be necessary to formulate the composition with a preservative (such as thimerosal or sodium azide) to facilitate long-term storage.

[0425] Examples of other adjuvants that may be effective include, but are not limited to, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), saponins, purified extract fractions of saponins (e.g., QuilA), saponin derivatives (e.g., QS-21), saponin-based lipid particles (e.g., ISCOM / ISCOMATRIX), Escherichia coli heat-labile toxin (LT) mutants (e.g., LTK63 and / or LTK72), aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (threonine-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, known as MTP-PE), as well as RIBI (which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and a cell wall skeleton (MPL+TDM+CWS), dispersed in a 2% squalene / Tween 80 emulsion), the MF59 formulation developed by Novartis, and the AS02, AS01, AS03, and AS04 adjuvant formulations developed by GlaxoSmithKline Biologicals (Rixensart, Belgium).

[0426] Examples of buffers include, but are not limited to, sodium succinate (pH 6.5) and phosphate buffered saline (PBS; pH 6.5 and 7.5).

[0427] Additional formulations suitable for other modes of administration include suppositories and, in some cases, oral formulations or formulations suitable for administration as aerosols. For suppositories, traditional binders and carriers may include, for example, polyethylene glycol or triglycerides; such suppositories may be made from mixtures containing 0.5% to 10% active ingredient, preferably 1% to 2%.

[0428] Oral formulations include commonly used excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. These compositions can be in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations or powders.

[0429] The dosage range for administering the compounds or products of the present invention is the range that can produce the desired therapeutic effect. It should be understood that the desired dosage range depends on the exact nature of the compound or product, the route of administration, the nature of the formulation, the age of the patient, the nature, degree or severity of the patient's condition, whether there are any contraindications, and the judgment of the attending physician. Standard empirical optimization procedures can be used to adjust the changes in these dosage levels. Similarly, the dosage of the compounds or products of the present invention used in the methods of the present invention (particularly in vitro methods) can be easily determined by those skilled in the art and is any dosage that can produce a desired increase in the number of NK cells and / or induce the desired NK cell expansion to produce an expanded NK cell population. As a non-limiting example, the dosage of SR8278 according to the present invention may produce a final concentration of about 2 to about 20 micromol / L, about 2 to about 15 micromol / L, about 5 to about 15 micromol / L, about 5 to about 14 micromol / L, about 4 to about 13 micromol / L, about 5 to about 12 micromol / L, about 5 to about 11 micromol / L, or preferably about 5 to about 10 micromol / L.

[0430] The present invention also provides the use of the expanded (CD16+) NK cell population (as described herein) in a pharmaceutical formulation. Any and all disclosures herein related to the formulation of the compounds of the invention can be equally and independently applied to the therapeutic application of the expanded (CD16+) NK cell population of the present invention.

[0431] Sequence information

[0432] Description of Sequence Identifier (SEQ ID NO)

[0433] Sequence identifier NO:1—E4bp4 messenger RNA (mRNA) sequence (X64318.1) Sequence identifier NO:2—E4bp4 amino acid sequence (X64318.1)

[0434] Sequence identifier NO: 3—REV-ERBα messenger RNA (mRNA) sequence (NM_021724.4)

[0435] Sequence identifier NO: 4—REV-ERBα amino acid sequence (NM_021724.4)

[0436] Sequence identifier NO: 5 - REV-ERBβ messenger RNA (mRNA) sequence (AB307693.1)

[0437] Sequence identifier NO:6——REV-ERBβ amino acid sequence (AB307693.1)

[0438] Sequence identifier NO:7—Delta-like ligand 4 messenger RNA (mRNA) sequence (AF253468.1)

[0439] Sequence identifier NO:8—Delta-like ligand 4 amino acid sequence (AF253468.1)

[0440] Sequence identifier NO:9——Human Notch1 complementary deoxyribonucleic acid (cDNA) sequence (CR457221.1)

[0441] Sequence identifier NO:10——Human Notch1 protein sequence (CR457221.1)

[0442] Sequence Description

[0443] Sequence identifier NO: 1——E4bp4 gene sequence (X64318.1)

[0444]

[0445] Sequence identifier number: 2—E4bp4 amino acid sequence (accession number: X64318.1)

[0446] MQLRKMQTVKKEQASLDASSNVDKMMVLNSALTEVSEDSTTGEDVLLSEGSVGKNKSSACRRKREFIPDEK

[0447] KDAMYWEKRRKNNEAAKRSREKRRLNDLVLENKLIALGEENATLKAELLSLKLKFGLISSTAYAQEIQKLS

[0448] NSTAVYFQDYQTSKSNVSSFVDEHEPSMVSSSCISVIKHSPQSSLSDVSEVSSVEHTQESSVQGSCRSPEN

[0449] KFQIIKQEPMELESYTREPRDDRGSYTASIYQNYMGNSFSGYSHSPPLLQVNRSSSNSPRTSETDDGVVGK

[0450] SSDGEDEQQVPKGPIHSPVELKHVHATVVKVPEVNSSALPHKLRIKAKAMQIKVEAFDNEFEATQKLSSPI

[0451] DMTSKRHFELEKHSAPSMVHSSLTPFSVQVTNIQDWSLKSEHWHQKELSGKTQNSFKTGVVEMKDSGYKVSDPENLYLKQGIANLSAEVVSLKRL IATQPISASDSG

[0452] Sequence identifier number 3 - REV-ERBα messenger ribonucleic acid sequence (Accession number: NM_021724.4)

[0453]

[0454]

[0455]

[0456] Sequence identifier number: 4 - REV-ERBα amino acid sequence (Accession number: NM_021724.4)

[0457] 8]MTTLDSNNNTGGVITYIGSSGSSPSRTSPESLYSDNSNGSFQSLTQGCPTYFPPSPTGSLTQDPARSFGSIPPSLSDDGSPSSSSSSSSSSSSFYNGSPPGSLQVAMEDSSRVSPSKSTSNITKLNGMVLLCKVCGDVASGFHYGVHACEGCKGFFRRSIQQNIQYKRCLKNENCSIVRINRNRCQQCRFKKCLSVGMSRDAVRFGRIPKREKQRMLAEMQSAMNLANNQLSSQCPLETSPTQHPTPGPMGPSPPPAPVPSPLVGFSQFPQQLTPPRSPSPEPTVEDVISQVARAHREIFTYAHDKLGSSPGNFNANHASGSPPATTPHRWENQGCPPAPNDNNTLAAQRHNEALNGLRQAPSSYPPTWPPGPAHHSCHQSNSNGHRLCPTHVYAAPEGKAPANSPRQGNSKNVLLACPMNMYPHGRSGRTVQEIWEDFSMSFTPAVREVVEFAKHIPGFRDLSQHDQVTLLKAGTFEVLMVRFASLFNVKDQTVMFLSRTTYSLQELGAMGMGDLLSAMFDFSEKLNSLALTEEELGLFTAVVLVSADRSGMENSASVEQLQETLLRALRALVLKNRPLETSRFTKLLLKLPDLRTLNNMHSEKLLSFRVDAQ

[0458] Sequence number 5-REV-ERBβ messenger RNA sequence (AB307693.1)

[0459]

[0460]

[0461] Sequence identifier number: 6 — REV-ERBβ amino acid sequence (accession number: AB307693.1)

[0462] MEVNAGGVIAYISSSSSASSPASCHSEGSENSFQSSSSSVPSSPNSSNSDTNGNPKNGDLANIEGILKNDRIDCSMKTSKSSAPGMTKNHSGVTKFSGMVLLCKVCGDVASGFHYGVHACEGCKGFFRRSIQQNIQYKKCLKNE NCSIMRMNRNRCQQCRFKKCLSVGMSRDAVRFGRIPKREKQRMLIEMQSAMKTMMNSQFSGHLQNDTLVEHHEQTALPAQEQLRPKPQLEQENIKSSSPPSSDFAKEEVIGMVTRAHKDTFMYNQEQQENSAESMQPQRGERIPK NMEQYNLNHDHCGNGLSSHFPCSESQQHLNGQFKGRNIMHYPXGHAICIANGHCMNFSNAYTQRVCDRVPIDGFSQNENKNSYLCNTGGRMHLVCPMSKSPYVDPHKSGHEIWEEFSMSFTPAVKEVVEFAKRIPGFRDLSQHDQ VNLLKAGTFEVLMVRFASLFDAKERTVTFLSGKKYSVDDLHSMGAGDLLNSMFEFSEKLNALQLSDEEMSLFTAVVLVSADRSGIENVNSVEALQETLIRALRTLIMKNHPNEASIFTKLLLKLPDLRSLNNMHSEELLAFKVHP

[0463] Sequence identifier number: 7 - Delta-like ligand 4 messenger RNA (mRNA) sequence (accession number: AF253468.1)

[0464] 1atggcggcag cgtcccggag cgcctctggc tgggcgctac tgctgctggt ggcactttgg

[0465] 61cagcagcgcg cggccggctc cggcgtcttc cagctgcagc tgcaggagtt catcaacgag

[0466] 121cgcggcgtac tggccagtgg gcggccttgc gagcccggct gccggacttt cttccgcgtc

[0467] 181tgccttaagc acttccaggc ggtcgtctcg cccggaccct gcaccttcgg gaccgtctcc

[0468] 241acgccggtat tgggcaccaa ctccttcgct gtccgggacg acagtagcgg cggggggcgc

[0469] 301aaccctctcc aactgccctt caatttcacc tggccgggta ccttctcgct catcatcgaa

[0470] 361gcttggcacg cgccaggaga cgacctgcgg ccagaggcct tgccaccaga tgcactcatc

[0471] 421agcaagatcg ccatccaggg ctccctagct gtgggtcaga actggttatt ggatgagcaa

[0472] 481accagcaccc tcacaaggct gcgctactct taccgggtca tctgcagtga caactactat

[0473] 541ggagacaact gctcccgcct gtgcaagaag cgcaatgacc acttcggcca ctatgtgtgc

[0474] 601cagccagatg gcaacttgtc ctgcctgccc ggttggactg gggaatattg ccaacagcct

[0475] 661atctgtcttt cgggctgtca tgaacagaat ggctactgca gcaagccagc agagtgcctc

[0476] 721tgccgcccag gctggcaggg ccggctgtgt aacgaatgca tcccccacaa tggctgtcgc

[0477] 781cacggcacct gcagcactcc ctggcaatgt acttgtgatg agggctgggg aggcc...

Claims

1. A method for preparing an expanded CD16+ natural killer (NK) cell population in vitro, comprising the following steps: a) culturing a sample containing hematopoietic progenitor cells (HPCs) obtained from an individual for about 2 to about 8 days in a medium that does not induce HPC differentiation to produce a pre-differentiated HPC population; and b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells.

2. An in vitro method for increasing the number of CD16+ NK cells in an expanded NK cell population, comprising the following steps: a) culturing a sample containing hematopoietic progenitor cells (HPCs) obtained from an individual for about 2 to about 8 days in a medium that does not induce HPC differentiation to produce a pre-differentiated HPC population; and b) culturing the pre-differentiated HPC population in a culture medium that induces HPC differentiation into NK cells.

3. The method of claim 1 or 2, wherein in step (a), the HPCs are cultured for about 2 days to about 6 days, optionally for about 4 days to about 6 days.

4. A method according to any preceding claim, wherein the HPCs are cultured in the presence of a Notch ligand during at least part of step (a).

5. The method according to claim 4, wherein: (i) during at least part of step (a), the HPCs are cultured in a vessel coated with a Notch ligand; and / or (ii) the Notch ligand is Delta-like ligand 4 (DLL4), or a fragment thereof that retains DLL4 function; wherein preferably, in step (a), the HPCs are cultured in the absence of the Notch ligand for about 1 day and then cultured in the presence of the Notch ligand for the remainder of step (a).

6. A method according to any preceding claim, wherein: (i) in step (a), the HPCs are cultured in the presence of a compound that inhibits the action of REV-ERB; and / or (ii) In step (b), the pre-differentiated HPC population is cultured in the presence of a compound that inhibits the action of REV-ERB.

7. The method of claim 6, wherein the compound: (i) Increased E4bp4 expression by reducing REV-ERB activity; (ii) reducing the activity of REV-ERB-α and / or REV-ERB-β, preferably reducing the activity of REV-ERB-β; (iii) reducing the activity of REV-ERB-α and REV-ERB-β; (iv) is a REV-ERB antagonist, preferably an antagonist of REV-ERB-α and REV-ERB-β; (v) selected from small molecules, proteolysis targeting chimeras (PROTAC) agents, double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA, antisense RNA, aptamer, antibody, ribozyme, peptide or peptidomimetic, preferably a small molecule; and / or (VI) is SR8278, or GSK1362.

8. The method according to any of the preceding claims, wherein the culture medium that does not induce differentiation of hematopoietic progenitor cells (HPCs) in step (a) and / or the culture medium that induces differentiation of HPCs into NK cells in step (b) does not contain interleukin-3 (IL-3), preferably, the culture medium that induces differentiation of HPCs into NK cells in step (b) does not contain IL-3.

9. A method according to any preceding claim, wherein: (i) the culture medium in step (a) comprises at least one of Fms-like tyrosine kinase 3 ligand (Flt3L), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, interleukin-6 (IL-6), thrombopoietin (TPO) and / or stem cell factor (SCF), preferably comprises Flt3L, GM-CSF, IL-3, IL-6, TPO and SCF; and / or (ii) The culture medium in step (b) contains interleukin-7 (IL-7), Flt3L, interleukin-15 (IL-15) and / or SCF, preferably comprising IL-7, Flt3L, IL-15 and SCF.

10. The method according to any one of the preceding claims, wherein step (a) and / or step (b) is performed in the absence of stromal support cells, preferably both step (a) and step (b) are performed in the absence of stromal support cells.

11. The method according to any one of the preceding claims, wherein the HPC sample is obtained from bone marrow, umbilical cord blood and / or peripheral blood.

12. A method according to any preceding claim, wherein the proportion of CD16+ NK cells is increased compared to the proportion of CD16+ NK cells produced by a corresponding method omitting step (a).

13. The method according to any of the preceding claims, wherein the expanded NK cell population comprises at least 10% CD16+ NK cells, preferably at least 15% CD16+ NK cells, more preferably at least 20% CD16+ NK cells, even more preferably at least 30% CD16+ NK cells.

14. A method according to any preceding claim, wherein the expanded NK cell population exhibits at least 30% higher antibody-dependent cell-mediated cytotoxicity (ADCC), preferably at least 50% higher ADCC, compared to NK cells produced by a corresponding method omitting step (a).

15. The method according to any one of the preceding claims, which does not include the further step of introducing exogenous nucleic acid into HPCs and / or NK cells.

16. An expanded CD16+ NK cell population, wherein at least 10% of the NK cells are CD16+ NK cells, preferably at least 15% of NK cells are CD16+ NK cells, more preferably at least 20% of NK cells are CD16+ NK cells, even more preferably at least 30% of NK cells are CD16+ NK cells.

17. An expanded CD16+ NK cell population obtained by the method of any one of claims 1 to 15, wherein at least 10% of the NK cells are CD16+ NK cells, preferably at least 15% of the NK cells are CD16+ NK cells, more preferably at least 20% of the NK cells are CD16+ NK cells, and even more preferably at least 30% of the NK cells are CD16+ NK cells.

18. The expanded CD16+ NK cell population according to claim 16 or 17, wherein the CD16+ NK cells do not contain exogenous nucleic acid.

19. The expanded CD16+ NK cell population according to any one of claims 16 to 18, wherein the expanded NK cell population exhibits at least 30% higher ADCC, preferably at least 50% higher ADCC, more preferably at least 70% higher ADCC, compared to NK cells produced by a corresponding method omitting step (a).

20. A composition comprising the expanded NK cell population as defined in any one of claims 16 to 19 and a pharmaceutically acceptable carrier, diluent and / or excipient.

21. An expanded CD16+ NK cell population as defined in any one of claims 16 to 19 or a composition as defined in claim 20, for use in a method of treatment.

22. The expanded CD16+ NK cell population or composition for use in therapy according to claim 21, wherein the therapeutic method is a method of treating a disease or condition selected from cancer, an infectious disease (acute or chronic), an autoimmune disease, or one associated with female infertility or pregnancy.

23. The expanded CD16+ NK cell population or composition for use in treatment according to claim 21 or 22, wherein the treatment method is a method for treating viral infection, bacterial infection, protozoan infection, fungal infection and / or helminth infection.

24. The expanded CD16+ NK cell population or composition for use in therapy according to any one of claims 21 to 23, used in combination with antibody-mediated immunotherapy.

25. The expanded CD16+ NK cell population or composition for use in treatment according to claim 24, wherein the expanded CD16+ NK cell population or composition is administered before, simultaneously with, or after administration of antibody-mediated immunotherapy.

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