TITLE OF THE INVENTIONModified natural killer cells having increased cytotoxicity and greater survival
FIELD OF THE INVENTIONThe present invention, in some embodiments thereof, relates to methods for improving the functionality of NK cell, such as their cytotoxic activity and survival, to be used in immunotherapy. In particular, these methods comprise a composition DNA fragments of
Fas-ligand (APTL; FASL; CD178; CD95L; ALPSIB; CD95-L; TNFSF6; TNLGIA;
APTILGI) and methods of producing modified NK cells with increased Fas-ligand — production. The invention encompasses also compositions of engineered NK cells and NK cell lines and uses thereof for treating or for preventing cancer and other immune related disorders.
BACKGROUND TO THE INVENTIONNK cells are the main cellular effectors of the innate immune system, destroying various targets, including infected or transformed cells, and in some cases senescent or stressed cells (Shimasaki et al., Nat Rev Drug Discov. 2020 Mar;19(3):200-2181; Giannoula et al.,
Biomed J. 2023 Feb 4:S2319-4170(23) 00005-7). NK cells do not reguire prior antigen exposure or MHC restriction (Schattner, Duggan. Am J Hematol. 1985 Apr;18(4):435-43; — Brix et al. US 10030065B2/2018). NK cells lack surface T cell receptors (TCRs) and do not induce graft-versus-host disease (GVHD). As such, they are considered as turnkey ("off-shelf") cell therapy product that can be prepared in advance, optimized, and administered to patients. These attributes endow NK cells with unique advantages for autologous as well as allogeneic therapeutic applications.
NK cell functions, including cytotoxicity, cytokine synthesis, and degranulation, are regulated by signals mediated by inhibitory receptors (particularly killer Ig-like receptors (KIRs) and heterodimeric C-type lectin receptor (NKG2A)), and activating receptors (particularly natural cytotoxicity (NCRs) NKp46, NKp30, NKp44 and lectin-like C-type activator immunoreceptor NKG2D7) that recognize ligands on target cells (Toledo et al.,
Sci Adv. 2021 Jun 11;7(24): eabc16405- 8; Rascle et al., Front Immunol. 2023 Jan 20; 14:1087155; Valton et al., JP2022101530A/2022; Andre, Kubler, ES2772307T3/2020).
NK cells can directly kill tumor cells by a) releasing cytoplasmic granules containing perforin and granzyme, and b) expressing TNF family proteins such as FasL or TRAIL, which induce tumor cell apoptosis by interacting with their respective receptors. Immature
NK cells likely use TRAIL-dependent cytotoxicity rather than FasL- or granule release- dependent cytotoxicity, while mature NK cells mainly use the latter two (Zamai et al., J
Exp Med. 1998 Dec 21;188(12):2375-80). In addition, antibody-dependent cellular cytotoxicity (ADCC) mediated by the CD16 Fc receptor can cause NK-mediated death of — target cells that have interacted with antibodies (Bunting et al., Sci Adv. 2022 Mar 18;8(11): eabk3327; Orange, Nat Rev Immunol. 2008 Sep;8(9):713-25.). Besides, IFN-y produced by activated NK cells also affects the tumor, since IFN-y induces remodeling of the tumor microenvironment, inhibits tumor angiogenesis, and has antimetastatic activity (von Locquenghien et al., J Clin Invest. 2021 Jan 4;131(1):e143296; Granzin et al., Front
Immunol. 2017 Apr 26;8:458; Chawla-Sarkar et al., Apoptosis. 2003 Jun;8(3):237-49;
JP7204643B2/2023).
In NK cells, FasL is stored in secretory lysosomes, which also contain granzymes and perforin. The co-localization of FasL, perforin, and granzymes in the same subcellular structures implies that the simultaneous delivery of these apoptosis-inducing molecules to — the immunological synapse between effector and target cells can lead to more efficient and faster killing of target cells. The intracellular accumulation of FasL is tightly regulated by its cytoplasmic tail and interacting molecules (Bossi et al., Nat Med. 1999 Jan;5(1):90-6;
Glukhova et al., Cell Death Dis. 2018 Jan 22;9(2):73.). When NK cells collide with target cells these granules transport to the site of intercellular contact, where they fuse with the — plasma membrane, thereby exposing FasL within the immunological synapse. The selective association with lipid rafts on the cell surface increases the death-promoting activity of FasL (Lettau et al., Curr Med Chem. 2008;15(17):1684-96; Kassahn et al., Cell
Death Differ, 2009 Jan;16(1):115-24).
It has been shown for T cells that after antigenic stimulation they undergo clonal expansion, rapidly increasing in number, after which their number decreases as a result of programmed cell death. This secondary phase is known as peripheral deletion and is due to the engagement of apoptosis pathways during signaling for clonal expansion. This phenomenon has been termed "activation-induced cell death" (AICD) and is mediated by the interaction of Fas and FasL on activated T cells. Upon antigen stimulation, T cells up- regulate FasL and increase their sensitivity to Fas-mediated apoptosis. The number of T cells declines as Fas-FasL interactions between T cells cause their cell death (Yamauchi et al., Blood. 1996 Jun 15;87(12):5127-35; Hennessy et al, J Leukoc Biol. 2019 —Jun;105(6):1341-1354). A similar phenomenon was also shown for NK cells. During the immune response, the expanded activated NK cells come to express Fas and eventually undergo AICD mediated by their own secretion of FasL (Masuda et al., Cancer Sci. 2020
Mar;111(3):807-816; Lopez-Verges et al., Blood. 2010 Nov 11;116(19):3865-74; Lee et al., Cytokine. 2012; 59 (3): 547).
Methods and compositions of modifying AICD of T cells and/or NK cells has been previously implicated for diabetes treatment and for anti-cancer therapy, they are summarized below:
US Patent US9624469B2/2017 (Regulatory immune cells with enhanced targeted cell death effect) discloses that targeted simulation of the process of activation-induced cell — death (AICD) at the site of inflammation ameliorate inflammatory insulitis. Inventors have generated regulatory T cells (Tregs) with enhanced cell death effect by chemically attaching to the surface of these cells a chimeric Fas-ligand (FasL) protein and use them for suppression of diabetogenic effector cells at the site of inflammation and for diabetes treatment. These results substantiate the value of modified Treg cells overexpressing a — death molecule, such as FasL, for treatment of immune related diseases.
Patent WO2019014684A 1/2019 provides methods of inhibiting AICD of T cells and/or
NK cells in a subject with chronic lymphocytic leukemia comprising administering to the subject an inhibitor of interleukin-2 inducible T cell kinase (ITK), ibrutinib. The data demonstrate that ibrutinib therapy is contemplated as an cellular immune modulating agent for CLL and potentially other types of hematologic and solid cancers.
Authors of Australian patent AU2019347873A1/2021 publish methods of obtaining genetically modified immunoresponsive cells (e.g., T cells or NK cells) comprising the antigen-recognizing receptor and the dominant negative Fas polypeptide. Such T or NK cells are endowed with augmented and selective cytolytic activity at the tumor site.
US Patent US20190038671A1/2019 discloses the pharmaceutical compositions based on the engineered mammalian cells containing a vector comprising the heterologous nucleic acid encoding the immunomodulator (an immune checkpoint inhibitors or immunoactivators) and a second heterologous nucleic acid encoding the CAR or the TCR.
Such T cells can be more resistant to activation-induced cell death and can be widely applicable in cancer immunotherapy.
US Pat. No. US20210246423A1/2021 proposes methods for improving in vitro expansion and activation of immune cells and preventing AICD. It is based on the discovery that activation of CAR expressed (e.g., transiently expressed) on the surface of immune effector cells provides an effective means to expand and/or activate a population of immune effector cells.
Canadian patent CA2706445C/2019 describes methods for protecting immune cells from cell death with IRX-2. IRX-2, also known as "citoplurikin", is a leukocyte-derived, natural primary cell derived biologic produced by mononuclear cells stimulated by phytohemagglutinin and ciprofloxacin. IRX-2 protects activated T cells from both extrinsic apoptosis and intrinsic metabolic apoptosis and enhance their anti-tumor activity.
An excess of Fas-ligand on the plasma membrane (in lipid rafts) can lead to the death of cells producing it. Preservation of the Fas-ligand inside the cell, in particular, in secretory lysosomes, is both a protective mechanism and one of the key factors in the cytotoxic activity of the NK cell (Krzewski et al., Front Immunol. 2012 Nov 9;3:335; Lee et al.,
Immun Inflamm Dis. 2018 Jun;6(2):312-321.). Known “trafficking domains”, primarily the LAMP lumenal domain, effectively target proteins containing them to lysosomal vesicles. Methods of modifying the protein for targeting of the protein to the — endosomal/lysosomal compartment are summarized below.
US patent US5633234A/1997 (Lysosomal targeting of immunogens. Expired) discloses a targeting signal that directs proteins to the endosomal/lysosomal compartment. Authors demonstrated that chimeric proteins containing a cytoplasmic endosomal/lysosomal targeting signal will effectively target antigens to that compartment.
US patent US20040157307A1/2004 (Chimeric vaccines) describes a chimeric protein comprising an antigen sequence and a domain for trafficking the protein to an endosomal compartment, irrespective of whether the antigen is derived from a membrane or non- membrane protein. In one preferred aspect of the invention, the trafficking domain comprises a lumenal domain of a LAMP polypeptide.
US patent US9993546B2/2018 (Lysosomal targeting of antigens employing nucleic acids encoding lysosomal membrane polypeptide/antigen chimeras) discloses lysosomal targeting of antigens employing nucleic acids encoding lysosomal membrane polypeptide/antigen chimeras. In one preferred aspect, the trafficking domain comprises a lumenal domain of a LAMP polypeptide. Alternatively, or additionally, the chimeric protein comprises a trafficking domain of an endocytic receptor (e.g., such as DEC-205 or gp200-MR6).
Australian patent AU2019250227B2/2021 (Nucleic acids for treatment of allergies) 5 provides DNA vaccines for the treatment of allergies. The vaccines comprise the coding seguence for one or more allergenic epitopes, and preferably the full protein seguence, of the allergenic protein from which the epitope(s) is derived, fused inframe with the lumenal domain of the lysosomal associated membrane protein (LAMP) and the targeting seguence of LAMP.
The ability to regulate AICD in NK cells has important implications to NK cell therapy for cancer. A common disadvantage to known AICD-modifying technigues is that a) it difficult to control persistent side effects caused by these treatments, and b) AICD inhibition results in greater NK cell survival but less cytotoxicity, and vice versa, increased
AICD causes poor survival and higher cytotoxic activity.
Thus, there is a need for alternative and preferably improved human NK cells, with a greater cytotoxicity and more pronounced survival.
PURPOSE OF THE INVENTIONCells of the invention, expressing FasL variants, offer a significant advantage in immunotherapy by redistributing FasL to secretory lysosomes but not to the cell membrane of NK cells. This reduces AICD during NK activation and, consequently, increases survival and enhances cytotoxic activity of the cells of the invention. In addition, the use of said compositions makes it possible to obtain a greater yield of NK cells with high cytotoxic activity during in vitro cultivation.
DESCRIPTION OF THE INVENTIONIt should be understood that the disclosure is not limited in its application to the details set forth in the following embodiments, the claims, the description, and the figures. The invention is capable of other embodiments and may be practiced or carried out in a variety of other ways.
To address this need for more efficient destruction of cancer cells, there are provided for herein nucleotide and amino acids sequences and vectors that encode genetic constructions that confer both greater survival and increased cytotoxicity on natural killer cells. A further object is to provide methods for producing modified NK cells with increased Fas-ligand — production, compositions containing the cells and uses of said compositions in the treatment of cancers.
According to an aspect of some embodiments of the NK cell is derived from umbilical cord blood, peripheral blood, bone marrow, CD34 + cells, iPSCs or ESC. In some respect the NK cell is human NK cell lines, e. g. NKL (CVCL_0466), YTS (CVCL_D324), NK 3.3 (CVCL 7994), NK-92 (CVCL 2142), KHYG-1 (CVCL 2976), haNK (CVCL_IM23), laNK (CVCL VN54) and others. According to some embodiments of the invention, NK cells are cells infiltrated into tissues.
According to some embodiments of the invention, amino acid substitutions for the modified forms of the Fas-ligand were chosen in accordance with the data of Bonifacino and Traub (Annu Rev Biochem. 2003; 72:395-447.). The selected amino acid substitutions are designed to redistribute Fas-ligand transport towards the intracellular depot, but not to the plasma membrane.
According to some embodiments of the invention, the YXX® site in the intracellular domain of the Fas-ligand is involved in interaction with proteins of the adapter protein — (AP) complex and is responsible for the internalization of the ligand and its transport into secretory lysosomes. In some embodiments, glycine residue preceding tyrosine promotes protein transport into the lysosomal compartment.
According to some embodiments of the invention, modified forms of the Fas-ligand comprise amino acid substitutions resulting in GYXXO repeat sites, where X is any amino — acid, Dis a hydrophobic amino acid (leucine, isoleucine or valine (L; I; V)), that promotes their transport to secretory lysosomes.
According to some embodiments of the invention, the modified form of the Fas-ligand,
FasLmodl, contains the following sites that facilitate their transportation to secretory lysosomes and increases cytotoxicity:
SGYGYL!, 3GYLQI'?, BYWVL!®, where G — glycine, Y — tyrosine, L — leucine, O — glutamine, I — isoleucine, W — tryptophan, V — valine.
According to some embodiments of the invention, the modified form of the Fas-ligand,
FasLmod2, contains the following sites that facilitate their transportation to secretory lysosomes and increases cytotoxicity:
SGYGYT!?, $SGYIOT'?, BSYWVI'*, where amino acid one letter code as previously.
According to some embodiments of the invention, the modified form of the Fas-ligand,
FasLmod3, contains the following sites that facilitate their transportation to secretory lysosomes and increases cytotoxicity:
SGYGYV', SGYVOI'?, BPYWVV'*, where amino acid one letter code as previously.
According to some embodiments of the invention, the modified form of the Fas-ligand,
FasLmod4, contains the following sites that facilitate their transportation to secretory lysosomes and increases cytotoxicity:
GYGYL!, SGYLOI!?, BYWVL!®, GYPPL’!, where amino acid one letter code as previously.
According to some embodiments of the invention, the modified form of the Fas-ligand,
FasLmodS, contains the following sites that facilitate their transportation to secretory lysosomes and increases cytotoxicity:
GYGYT!, SGYIOI?, BYWVI'®, YGYPPI”'!, where amino acid one letter code as previously.
According to some embodiments of the invention, the modified form of the Fas-ligand,
FasLmod6, contains the following sites that facilitate their transportation to secretory lysosomes and increases cytotoxicity:
SGYGYV'I, SGYVOI?, BYWVV!S GYPPV”! where amino acid one letter code as previously.
According to some embodiments of the invention, substituted amino acids (, leucine, isoleucine or valine (L; I; V) in the modified forms of the Fas-ligand, FasLmodl-6, are encoded by codons that is optimized for the use of human codon for expression in human cells. Unless otherwise specified, a particular nucleic acid seguence of the modified forms of the Fas-ligand implicitly encompasses its conservatively modified variants (eg, degenerate codon substitutions).
It is an object of the present invention to provide a method of transfection of NK cells, wherein cells with the modified forms of the Fas-ligand are individually characterized by higher cytotoxicity compared to unmodified NK cells and greater viability compared to cells producing unmodified FasL, and use of those cells for therapy of cancer and other diseases.
In some embodiments, truncated versions of the FASLG gene promoter may be included in an expression vector encoding modified forms of FasL. to enhance the Fas-ligand expression inside the cells. The truncated variant of the FASLG gene promoter (hfaslg) was chosen according with the data of Holtz-Heppelmann and others (Holtz-Heppelmann et al., J Biol Chem. 1998 Feb 20;273(8):4416-23; Rivera et al., J Biol Chem. 1998 Aug 28:273(35):22382-8; McClure et al., J Biol Chem. 1999 Mar 19;274(12):7756-62; Bodor et al., Eur J Immunol. 2002 Jan;32(1):203-12). The activity of the truncated promoters is — higher than that of the natural one, but lower than that of the widely used CMV promoter.
The use of such a promoter makes it possible to achieve a high level of Fas-ligand expression and has a less toxic effect on cells than CMV.
According to some embodiments of the invention, an additional positive effect in obtaining modified cells can be achieved by adding all-trans-retinoic acid (ATRA) when culturing modified NK cells in vitro. ATRA downregulates FasL. expression according to data of
Yang and others (Yang et al., J Exp Med. 1995 May 1;181(5):1673-82; Bissonnette et al.,
Mol Cell Biol. 1995 Oct;15(10):5576-85; Cui et al., Cell Immunol. 1996 Feb 1;167(2):276- 84; Lee et al., Eur J Biochem. 2002 Feb:269(4):1162-70).
According to some embodiments of the invention, an additional positive effect in obtaining — modified cells can be achieved by adding vitamin E and its derivatives when culturing modified NK cells in vitro. Vitamin E downregulates FasL expression according to data of
Li-Weber et al. (J Clin Invest. 2002 Sep;110(5):681-90; Lee et al., Nutrients. 2018 Nov 1;10(11):1614).
According to some embodiments of the invention, the compositions with the use of a truncated promoter and the addition of ATRA during cultivation leads to the suppression of FasL expression during cell production in vitro. As a result, the viability of the modified
NK cells and the yield of the cell product at the end of the cycle of cultivation are increased. After removal of ATRA at the final stage of cultivation, the expression level of
FasL and the cytotoxic activity of cells are restored. Thus, the use of a truncated promoter — for the expression of modified FasL. forms in combination with ATRA makes it possible to obtain a greater yield of NK cells with high cytotoxic activity during in vitro cultivation.
According to some embodiments of the invention, the compositions with the use of a truncated promoter and the addition of vitamin E or its derivatives during cultivation leads to the suppression of FasL expression during cell production in vitro. As a result, the viability of the modified NK cells and the yield of the cell product at the end of the cycle of cultivation are increased. After removal of vitamin E or its derivatives at the final stage of cultivation, the expression level of FasL and the cytotoxic activity of cells are restored.
Thus, the use of a truncated promoter for the expression of modified FasL forms in combination with vitamin E or its derivatives makes it possible to obtain a greater yield of
NK cells with high cytotoxic activity during in vitro cultivation.
According to some embodiments of the invention, an exemplary strategy for improving
NK cells for immunotherapy by redistributing FasL transport towards the intracellular — depot preferentially, enhancing anti-cancer cytotoxicity and improving NK cell survival is shown in Fig. 12.
Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understandable to a person skilled in the art, to which the invention pertains. The materials, methods, and examples described herein are illustrative only and are not intended to be necessarily limiting.
LIST OF THE FIGURESFIG. 1 depicts a map of the plasmid vector pFasLmod illustrating the point of insertion of certain constructs according to several embodiments into plasmids. Human faslg promoter —-233-685 bp, FasLmodl-6 - 686-1531 bp, BGH pA - 1575-1799 bp, {1 ori - 1845-2273 bp,
SV40 early promoter - 2278-2647 bp, Neo(R) - 2683-3477 bp, SV40 pA - 3651-3781 bp, pUC origin - 4164-4834 bp, Amp(R) - 4979-5839 bp (a complementary chain), bla promoter - 5840-5938 bp (a complementary chain).
FIG. 2 is a photomicrograph illustrating the cytotoxicity of NK cells modified with various constructs according to several embodiments against target cells HEK293 (cells of a human embryonal kidney). NK cells, NK-FasLmod1, NK-FasLmod2, NK-FasLmod3, NK-
FasLmod4, NK-FasLmod5, NK-FasLmod6, were incubated with HEK293 target cells at a ratio of 3:1 (effector:target) for 5 hours and photographed.
FIG. 3 is a photomicrograph illustrating the cytotoxicity of NK cells modified with various constructs according to several embodiments against target cells HeLa (human cervical adenocarcinoma cells). NK cells, NK-FasLmodl, NK-FasLmod2, NK-FasLmod3, NK-
FasLmod4, NK-FasLmod5, NK-FasLmod6, were incubated with Hela target cells at a ratio of 3:1 (effector:target) for 5 hours and photographed.
FIG. 4 is a photomicrograph illustrating the cytotoxicity of NK cells modified with various constructs according to several embodiments against target cells A172 (human glioblastoma cells). NK cells, NK-FasLmodl, NK-FasLmod2, NK-FasLmod3, NK-
FasLmod4, NK-FasLmod5, NK-FasLmod6, were incubated with A172 target cells at a ratio of 3:1 (effector:target) for 5 hours and photographed.
FIG. 5 depicts percentage of surviving HeLa target cells after incubation with NK92,
NKO92-FasLLmodl, NK92-FasLmod2, NK92-FasLmod3, NK92-FasLmod4, NK92- — FasLmod5, NK92-FasLmod6 cells for 5 hours at a ratio of 2:1 or 5:1 (effector:target).
FIG. 6 depicts percentage of surviving HEK293 target cells after incubation with NK92,
NKO92-FasLLmodl, NK92-FasLmod2, NK92-FasLmod3, NK92-FasLmod4, NK92-
FasLmod5, NK92-FasLmod6 cells for 5 hours at a ratio of 2:1 or 5:1 (effector:target).
FIG. 7 depicts percentage of surviving A172 target cells after incubation with NK92,
NK92-FasLmodl, NK92-FasLmod2, NK92-FasLmod3, NK92-FasLmodd, NK92-
FasLmod5, NK92-FasLmod6 cells for 5 hours at a ratio of 2:1 or 5:1 (effector:target).
FIG. 8 depicts comparative growth kinetics of NK92, NK92-FasLmod1, NK92-FasL mod2,
NK92-FasLmod3, NK92-FasLmod4, NK92-FasLmod5, NK92-FasLmod6 cell cultures.
FIG. 9A depicts Fas ligand expression in NK92 cells, transfected with a vector encoding — FasLmodl under transcriptional control of the natural faslg promoter (1), the truncated faslg promoter (2), or the cytomegalovirus promoter (3). FIG. 9B shows the growth rate of
NK92-FasLmod! cells with the natural faslg promoter (1), the truncated faslg promoter (2), or the cytomegalovirus promoter (3). M - marker.
FIG. 10A depicts Fas Ligand expression in NK92 cells (1), NK92-FasLmod!l cells with a truncated faslg promoter in the presence of ATRA (2), NK92-FasLmod! cells with a truncated faslg promoter after removal of ATRA (3). FIG. 10B shows growth rate of
NK92-FasLmod!1 cells with truncated faslg promoter with or without ATRA. NK92 cells were used as controls. FIG. 10C depicts a viability of HeLa target cells after incubation with NK92-FasLmod! cells with truncated faslg promoter after incubation with or without — ATRA for 5 hours in a ratio of 2:1 or 5:1 (effector:target). NK92 cells were used as controls.
FIG.11A depicts Fas Ligand expression in NK92 cells (1), NK92-FasLmod! cells with a truncated faslg promoter in the presence of vitamin E (2), NK92-FasLmod! cells with a truncated faslg promoter after removal of vitamin E (3). Fig. 10B shows proliferation rate of NK92-FasLLmod]1 cells with truncated faslg promoter with or without vitamin E. NK92 cells were used as controls. Fig. 10C depicts a viability of HeLa target cells after incubation with NK92-FasL mod] cells with truncated faslg promoter after incubation with — or without vitamin E, for 5 hours in a ratio of 2:1 or 5:1 (effector:target). NK92 cells were used as controls.
FIG. 12 is an illustration showing an exemplary strategy to improve NK cells for immunotherapy by redistributing FasL transport towards the intracellular depot preferentially, enhancing anti-cancer cytotoxicity and improving NK cell survival.
DETAILED DESCRIPTION OF THE INVENTIONIt should be understood that the disclosure is not limited in its application to the details set forth in the following embodiments, the claims, the description, and the figures. The invention is capable of other embodiments and may be practiced or carried out in a variety of other ways.
DEFINITIONSUnless otherwise noted, all technical and scientific terms used herein have a meaning commonly understood by a person skilled in the art. The following references provide the — skilled person with a general definition of many of the terms used in the subject matter of the invention disclosed herein: Dictionary of microbiology and molecular biology. (2nd
Edition, Singleton, P. & Sainsbury, D. 1988); Concise Dictionary of Biomedicine and
Molecular Biology (2nd Edition, 2001, Pei-Show Juo); Oxford Dictionary of Biochemistry and Molecular Biology (2nd Edition, Eds. Richard Cammack et al.,2006); The Dictionary of Cell and Molecular Biology (5th Edition, 2012, Ed. John Lackie).
All publications referred to herein are expressly incorporated by reference into this document for the disclosure and description of the methods and/or materials in connection with which they are cited.
The term "immunotherapy" refers to the treatment of a disease by a method, including the induction, enhancement, suppression or other change in the immune response. Examples of immunotherapy include, but are not limited to, NK cell therapy. It should be understood that the methods disclosed herein enhance the effectiveness of any NK cell therapy.
The term “NK cell” or “natural killer cell” refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell — receptor (CD3). In some embodiments the NK cell is derived from umbilical cord blood, peripheral blood, bone marrow, CD34 + cells, iPSCs, ESC or the NK cell is infiltrated into tissues. In some respect the NK cell is human NK cell lines, e. g. NKL (CVCL_0466),
YTS (CVCL_D324), NK 3.3 (CVCL_7994), NK-92 (CVCL_2142), KHYG-1 (CVCL_2976), haNK (CVCL IM23), laNK (CVCL VN54) and others.
The term "cytotoxicity" refers to the ability to kill living cells, and specifically describes the characteristics of NK cell activity that kills target cells. The extent of cell death can be expressed as the percentage of target cell death in excess of the background, with total target cell death taken as 100%.
The term "cell survival" refers to the span that encompasses the viability of a cell and its — ability to subsist and maintain the integrity of cellular processes. Survival mechanisms ensure that the cell will be able to carry on cellular activities such as metabolism, growth, reproduction, some form of responsiveness, and adaptability.
The term "secretory lysosomes" refers to the lysosome-related effector vesicles (LREVs), which serve as a common storage site for cytotoxic effector proteins and are released only — into the immunological synapse formed between the effector and the target cell. As used herein, the term "secretory lysosomes" includes all membrane-bound vesicles that are smaller in diameter than the cell from which they are derived. In the present invention, the term "secretory lysosomes" includes any selected from the group consisting of exosomes, ectosomes, microvesicles and apoptotic body, as well as any other vesicles.
The term "genetic modification" refers to a method of altering the genome of a cell, including, but not limited to, removing a coding or non-coding region or portion thereof, or inserting a coding region or portion thereof. The construct or sequence may include regulatory or control sequences such as start, stop, promoter, signal, secretion, or other sequences used by the cell's genetic machinery. In some embodiments, the cell to be — modified is an NK cell, which can be obtained from a patient or a donor. The cell may be modified to express an exogenous construct, such as FasL, which is inserted into the cell's genome.
Various aspects of the disclosure of the invention are described in more detail in the following subsections.
NK CELL IMMUNOTHERAPY
Immunotherapy, in particular CAR-modified cell therapy, has great potential due to its high cytotoxicity and specificity, and CAR-T-cell immunotherapy is an example of a major form of immunotherapy that is well studied and fairly widely used. However, there are numerous limitations to the use of CAR-modified T cells. The production of personalized
CAR-T products is time-consuming and expensive. A drawback of this approach is the necessity to use autologous cells to prevent the induction of a graft vs. host reaction in the patient. CAR-T cells can also cause marked toxic effects by cytokine release syndrome. In addition, the results of CAR-T cell therapy for solid tumors are not optimal. These disadvantages of CAR-T cells have led to great interest in NK-cell therapy. NK cells are cytotoxic lymphocytes of the innate immune system characterized by their ability to spontaneously detect and kill infected or malignant cells and also participate in the regulation of the adaptive immune response by producing a large number of cytokines and chemokines. NK cells use activating receptors to recognize germline-encoded ligands upregulated on cancer cells without requiring tumor neoantigen presentation by MHC molecules as T cells do. Activated NKs are capable of destroying tumor cells by a) releasing cytoplasmic granules containing perforin and granzyme; b) expressing TNF family proteins such as FasL. and TRAIL that induce tumor cell apoptosis, and c) antibody- dependent cellular cytotoxicity mediated by Fc-receptor CD16. Overexpression of Fas ligand on the plasma membrane (in lipid rafts) can lead to the death of the cells producing it (AICD). Retention of Fas ligand inside the cell, in particular in secretory lysosomes, is both a protective mechanism and one of the key factors of cytotoxic activity of NK cells.
The ability to regulate AICD in NK cells is essential for NK cell therapy of cancer.
Accordingly, in several embodiments of the present invention there is provided a method to — solve this problem by creating genetic constructions that confer both greater survival and increased cytotoxicity on natural killer cells in order to promote NK killing of target cells.
The genetic constructions are designed to redistribute Fas-ligand transport towards the intracellular depot, but not to the plasma membrane.
FAS LIGAND INTRACELLULAR DOMAIN
As mentioned above, NK cells can recognize and destroy tumor cells and infected cells by means of FasL that induces target cell apoptosis. Fas ligand (FasL or CD95L or CD178) is a type-II transmembrane protein that belongs to the tumor necrosis factor (TNF) family and induces apoptosis through the death receptor Fas/CD95, or by the reverse signalling pathway. FasL contains extracellular, transmembrane, and intracellular domains. The extracellular part is responsible for recognition of the corresponding receptors, Fas-antigen and DcR3, as well as for ligand self-association (Orlinick et al., J Biol Chem. 1997 Dec 19;272(51):32221-9). The transmembrane region of FasL is responsible for the "anchoring" and/or movement of this molecule in/on the plasma membrane. The intracellular part of FasL is required for sorting into secretory lysosomes, translocation of — the ligand into rafts, the "signaling platforms” of the plasma membrane, and for FasL- dependent "reverse signaling. The 45-65 a.a. polyproline region (PRD), is required for interaction with a number of enzymes and adaptor proteins, as well as directed ligand transport. The ubiquitinylation sites of lysine residues at positions 72 and 73 and the phosphorylation of tyrosine residues (7, 9, and 13 a.s.) play a role in the intracellular distribution of the ligand (Zuccato et al., J Cell Sci. 2007 Jan 1;120(Pt 1):191-9). The target signal directing proteins to the lysosomal compartment includes the amino acid sequence YXX®, where X is any amino acid, P is a hydrophobic amino acid (leucine, isoleucine or valine (L; I; V)). Chimeric proteins containing this amino acid motif are efficiently directed to this compartment (Wu et al., Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11671-5). As noted herein, the ability to reduce AICD in NK cells has important implications to NK cell therapy. The Fasl redistribution from cell surface to lysosomal compartment may reduce AICD of activated NK cells. Accordingly, in one embodiment, the intracellular domain of the Fas-ligand comprises the YXXO site (9YPOII2) responsible for its transport into secretory lysosomes. In some embodiments, glycine residue preceding tyrosine promotes protein transport into the lysosomal compartment.
The presence of several sequentially arranged “trafficking domains” (GYXX®) should enhance protein targeting into the lysosomal compartment. In some embodiments, modified forms of the Fas-ligand comprise amino acid substitutions resulting in several sequentially repeated GY XX® sites, that promotes their transport to secretory lysosomes.
In certain embodiments, the Fas-ligand comprises the amino acid sequence is set forth in
SEO ID NO: 1). SEQ ID NO: 1 is provided below.
MQQPFGYGYL QIYWVLSSAS SPWAPPGTVL PCPTSVPRRP GORRPPPPPP
PPPLPPPPPP PPLPPLPLPP LKKRGNBSTG LCLLVMFFMV LVALVGLGLG
MFOLFHLOKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT
GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGO
SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGOMWA RSSYLGAVEN
LTSADHLYVN VSELSLVNFE ESOTFFGLYK L*
An exemplary nucleic acid seguence encoding the amino acid seguence of SEO ID NO: 1 is set forth in SEO ID NO: 7. SEQ ID NO: 7 is provided below. atgcagcagce ccttcggeta cggctatctg cagatctact gggtgctgag cagtgecage tetceetggg cceetccagg cacagttctt cectgtccaa cetetgtgee cagaaggect ggtcaaagga ggecaccace accaccgeca cegeccaccac taccacetcec gecgecgeeg ccaccactge ctccactacec gectgccacecc ctgaagaaga gagggaacca cagcacagge ctgtgtctcc ttgtgatgtt tttcatggtt ctggttgcet tggtaggatt gggcetgggg atgtttcage tettecacet acagaaggag ctggcagaac tccgagagtc taccagccag atgcacacag catcatcttt ggagaagcaa — ataggccacc ccagtccacc ccctgaaaaa aaggagectga ggaaagtgge ccatttaaca ggcaagtcca actcaaggte catgcctctg gaatgggaag acacctatgg aattgtectg ctttctggag tgaagtataa gaagggtgge cttgtgatca atgaaactgg gctgtacttt gtatattcca aagtatactt ccggggtcaa tcttgcaaca acctgecect gagecacaag gtctacatga ggaactctaa gtatccccag gatctggtga tgatggaggg gaagatgatg agcetactgea ctactgggca gatgtgggcc cgeageagcet acctggggec agtgttcaat cttaccagtg ctgatcattt atatgtcaac gtatctgage — tctetetggt caattttgag gaatctcaga cgtttttcgg cttatataag ctctaa
In certain embodiments, the Fas-ligand comprises the amino acid seguence is set forth in
SEO ID NO: 2. SEO ID NO: 2 is provided below.
MOOPFGYGYI OIYWVISSAS SPWAPPGTVL PCPTSVPRRP GQRRPPPPPP
PPPLPPPPPP PPLPPLPLPP LKKRGNHSTG LCLLVMFFMV LVALVGLGLG
MFOLFHLOKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT
GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGO
SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGOMWA RSSYLGAVEN
LTSADHLYVN VSELSLVNFE ESOTFFGLYK L*
An exemplary nucleic acid sequence encoding amino acids of SEO ID NO: 2 is set forth in
SEO ID NO: 8, which is provided below. atgcagcagc cettcggcta cggctatatc cagatctact gggtgatcag cagtgecage tetceetggg cccetccagg cacagttctt cectgtccaa cetetgtgee cagaaggect ggtcaaagga ggecaccace accaccgeca cecgeeaccac taccacetcec gecgecgeeg ccaccactge ctccactacec gectgccacecc ctgaagaaga gagggaacca cagcacagge ctgtgtctcc ttgtgatgtt tttcatggtt ctggttgcet tggtaggatt gggcetgggg atgtttcage tettecacet acagaaggag ctggcagaac tccgagagtc taccagccag atgcacacag catcatcttt ggagaagcaa ataggccacc ccagtccace ccetgaaaaa aaggagctga ggaaagtgge ccatttaaca ggcaagtcca actcaaggtc catgcctctg gaatgggaag acacctatgg aattgtectg ctttctggag tgaagtataa gaagggtgge cttgtgatca atgaaactgg gctgtacttt gtatattcca aagtatactt ccggggtcaa tcttgcaaca acetgcecet gageccacaag gtctacatga ggaactctaa gtatccccag gatctggtga tgatggaggg gaagatgatg agcetactgea ctactgggca gatgtgggcc cgcagcaget acctggggec agtgttcaat cttaccagtg ctgatcattt atatgtcaac gtatctgage — tetetetggt caattttgag gaatctcaga cgtttttcgg cttatataag ctctaa
In certain embodiments, the Fas-ligand comprises the amino acid seguence is set forth in
SEO ID NO: 3, which is provided below.
MOOPFGYGYV OIYWVVSSAS SPWAPPGTVL PCPTSVPRRP GORRPPPPPP
PPPLPPPPPP PPLPPLPLPP LKKRGNHSTG LCLLVMFFMV LVALVGLGLG
MFOLFHLOKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT
GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGO
SCNNLPLSHK VYMRNSKYPO DLVMMEGKMM SYCTTGOMWA RSSYLGAVEN
LTSADHLYVN VSELSLVNFE ESOTFFGLYK L*
An exemplary nucleic acid sequence encoding amino acids of SEQ ID NO: 3 is set forth in
SEO ID NO: 9, which is provided below. atgcagcagec cettcggcta cggctatgtg cagatctact gggtggtgag cagtgecage tctceetggg cecctecagg cacagttctt cectgtccaa cetetgtgee cagaaggect ggtcaaagga ggecaccace accaccgeca cecgeeaccac taccacetcec gecgeecgeeg ccaccactge ctccactacec getgecacce ctgaagaaga gagggaacca — cagcacaggc ctgtgtctcc ttgtgatgtt tttcatggtt ctggttgcct tggtaggatt gggcetgggg atgtttcagc tettecacet acagaaggag ctggcagaac tcegagagtc taccagccag atgcacacag catcatcttt ggagaagcaa ataggccace ccagtccacec ccctgaaaaa aaggagectga ggaaagtgge ccatttaaca ggcaagtcca actcaaggtc catgcctctg gaatgggaag acacctatgg aattgtectg ctttctggag tgaagtataa gaagggtgge cttgtgatca atgaaactgg gctgtacttt gtatattcca aagtatactt ccggggtcaa tcttgcaaca acetgecect gagecacaag gtctacatga ggaactctaa gtatccccag gatctggtga tgatggaggg gaagatgatg agctactgca ctactgggca gatgtgggcc cgcagcaget acctggggec agtgttcaat cttaccagtg ctgatcattt atatgtcaac gtatctgage tctetetggt caattttgag gaatctcaga cgtttttcgg cttatataag ctctaa
In certain embodiments, the Fas-ligand comprises the amino acid sequence is set forth in
SEQ ID NO: 4, which is provided below.
MQQPFGYGYL QIYWVLSSAS SPWAPPGTVL PCPTSVPRRP GORRPPPPPP
PPPLPPPPPP PPLPPLGYPP LKKRGNHSTG LCLLVMFFMV LVALVGLGLG
MFOLFHLOKE LAELRESTSO MHTASSLEKO IGHPSPPPEK KELRKVAHLT
GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGO
SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGOMWA RSSYLGAVEN
LTSADHLYVN VSELSLVNFE ESOTFFGLYK L*
An exemplary nucleic acid sequence encoding amino acids of SEO ID NO: 4 is set forth in
SEQ ID NO: 10, which is provided below. atgcagcagce cecttcggcta cggctatctg cagatctact gggtgctgag cagtgecage tetecetggg cecetecagg cacagttctt cectgtccaa cetetgtgee cagaaggect ggtcaaagga ggecaccace accaccgeca cecgeeaccac taccacetcec gecgeecgeeg ccaccactge ctccactagg ctacccacecc ctgaagaaga gagggaacca cagcacagge ctgtgtctcc ttgtgatgtt tttcatggtt ctggttgcet tggtaggatt gggcetgggg atgtttcage — tettecacet acagaaggag ctggcagaac teegagagtc taccagccag atgcacacag catcatettt ggagaagcaa ataggccace ccagtccace ccctgaaaaa aaggagetga ggaaagtgge ccatttaaca ggcaagtcca actcaaggte catgcctctg gaatgggaag acacctatgg aattgtectg ctttctggag tgaagtataa gaagggtgge cttgtgatca atgaaactgg gctgtacttt gtatattcca aagtatactt ccggggtcaa tcttgcaaca acctgcccet gagecacaag gtctacatga ggaactctaa gtatccccag gatctggtga tgatggaggg gaagatgatg agctactgca ctactgggca — gatgtgggcc cgcagcagct acctgggggc agtgttcaat cttaccagtg ctgatcattt atatgtcaac gtatctgage tetetetggt caattttgag gaatctcaga cgtttttcgg cttatataag ctctaa
In certain embodiments, the Fas-ligand comprises the amino acid seguence is set forth in
SEO ID NO: 5. SEO ID NO: 5 is provided below.
MQQPFGYGYI QIYWVISSAS SPWAPPGTVL PCPTSVPRRP GQRRPPPPPP
PPPLPPPPPP PPLPPLGYPP IKKRGNBSTG LCLLVMFFMV LVALVGLGLG
MFOLFHLOKE LAELRESTSQ MHTASSLEKO IGHPSPPPEK KELRKVAHLT
GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGO
SCNNLPLSHK VYMRNSKYPO DLVMMEGKMM SYCTTGOMWA RSSYLGAVEN
LTSADHLYVN VSELSLVNFE ESOTFFGLYK L*
An exemplary nucleic acid seguence encoding amino acids of SEO ID NO: 5 is set forth in
SEO ID NO: 11, which is provided below. atgcagcagc cettcggcta cggctatatc cagatctact gggtgatcag cagtgecage tetceetggg cccetccagg cacagttctt cectgtccaa cetetgtgee cagaaggect ggtcaaagga ggecaccace accaccgeca cecgeeaccac taccacetcec gecgeegeeg ccaccactge ctccactagg ctacccaccec atcaagaaga gagggaacca cagcacagge ctgtgtctcc ttgtgatgtt tttcatggtt ctggttgcet tggtaggatt gggcetgggg atgtttcage tettecacet acagaaggag ctggcagaac tcegagagtc taccagccag atgcacacag catcatcttt ggagaagcaa ataggccacc ccagtccace ccetgaaaaa aaggagetga ggaaagtgge ccatttaaca ggcaagtcca actcaaggte catgcctctg gaatgggaag acacctatgg aattgtectg ctttctggag tgaagtataa gaagggtgge cttgtgatca atgaaactgg gctgtacttt gtatattcca aagtatactt ccggggtcaa tcttgcaaca acetgecect gagecacaag gtctacatga ggaactctaa gtatccccag gatctggtga tgatggaggg gaagatgatg agctactgca ctactgggca gatgtgggcc cgcagcaget acctggggec agtgttcaat cttaccagtg ctgatcattt atatgtcaac gtatctgage — tetetetggt caattttgag gaatctcaga cgtttttcgg cttatataag ctctaa
In certain embodiments, the Fas-ligand comprises the amino acid seguence is set forth in
SEO ID NO: 6, which is provided below.
MOOPFGYGYV OIYWVVSSAS SPWAPPGTVL PCPTSVPRRP GORRPPPPPP
PPPLPPPPPP PPLPPLGYPP VKKRGNHSTG LCLLVMFFMV LVALVGLGLG
MFOLFHLOKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT
GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGO
SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGOMWA RSSYLGAVEN
LTSADHLYVN VSELSLVNFE ESOTFFGLYK L*
An exemplary nucleic acid sequence encoding amino acids of SEO ID NO: 6 is set forth in
SEO ID NO: 12. SEO ID NO: 12 is provided below. atgcagcagec cettcggcta cggctatgtg cagatctact gggtggtgag cagtgecage tctceetggg cecctecagg cacagttctt cectgtccaa cetetgtgee cagaaggect ggtcaaagga ggecaccace accaccgeca cecgeeaccac taccacetce geecgecgeeg ccaccactgc ctccactagg ctacccacce gtgaagaaga gagggaacca — cagcacaggc ctgtgtctcc ttgtgatgtt tttcatggtt ctggttgcct tggtaggatt gggcetgggg atgtttcagc tettecacet acagaaggag ctggcagaac tcegagagtc taccagccag atgcacacag catcatcttt ggagaagcaa ataggccace ccagtccacec ccctgaaaaa aaggagetga ggaaagtgge ccatttaaca ggcaagtcca actcaaggtc catgcctctg gaatgggaag acacctatgg aattgtectg ctttctggag tgaagtataa gaagggtgge cttgtgatca atgaaactgg gctgtacttt gtatattcca aagtatactt ccggggtcaa tcttgcaaca acetgecect gagecacaag gtctacatga ggaactctaa gtatccccag gatctggtga tgatggaggg gaagatgatg agctactgca ctactgggca gatgtgggcc cgcagcaget acctggggec agtgttcaat cttaccagtg ctgatcattt atatgtcaac gtatctgage tctetetggt caattttgag gaatctcaga cgtttttcgg cttatataag ctctaa
Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons CTT, CTC, CTA, CTG,
TTA and TTG all encode the amino acid leucine. Thus, at every position where a leucine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. In some embodiments, substituted amino acids (leucine, isoleucine or valine (L; I; V) in the modified forms of the Fas-ligand, FasL. mod1- 6, are encoded by codons that is optimized for the use of human codon for expression in — human cells. Unless otherwise specified, a particular nucleic acid sequence of the modified forms of the Fas-ligand implicitly encompasses its conservatively modified variants (eg, degenerate codon substitutions).
Due to the fact that increased expression of FasL can cause death of the cells producing it, a shortened hfaslg promoter can be included in the expression vector encoding the modified form of FasL to increase the viability of the modified NK cells. The sequence of the shortened vector was chosen based on data from Holtz-Heppelmann et al. where it was shown on the Jurkat T-cell line that deletion of the -2365 -452 region of the FASLG gene promoter leads to a several-fold increase in gene. The activity of the shortened promoter is higher than that of the natural promoter, but lower than that of the commonly used viral
CMV promoter. The use of such a promoter makes it possible to achieve a high level of
Fas ligand expression. At the same time, the truncated promoter has less toxic effect on cells than CMV, which is illustrated in Fig. 9. Accordingly, in some embodiments,
truncated version of hfaslg promoter may be included in an expression vector encoding modified forms of FasL to enhance the Fas-ligand expression inside the cells.
The sequence of a truncated version of the FasL gene promoter (hfaslg promoter) SEQ ID
NO: 13 is provided below: ttatagccee actgaccatt ctcctgtagc tgggagcagt tcacactaac agggctatac ccccatgctg acctgctctg caggatccca ggaaggtgag catagectac taacctgttt gggtagcaca gegacageaa ctgaggeett gaaggetgtt atcagaaaat tgtggecgga aacttccagg ggtttgctct gagettcttg aggettctca gettcagetg caaagtgagt gggtgtttct ttgagaagca gaatcagaga gagagagata gagaaagaga aagacagagg tgtttecett agctatggaa actctataag agagatccag cttgcetcet cttgagcagt cagcaacagg gtcccgtcet tgacacetca gectctacag — gactgagaag aagtaaaacc gtttgctggg getggectga ctcaccaget gee
An additional positive effect on the viability of modified cells can be achieved by adding all-trans-retinoic acid (ATRA) during the cultivation of modified NK cells in vitro. ATRA is known to suppress FasL expression and, conseguently, death of activated thymocytes and T cells (Iwata et al., J Immunol. 1992 Nov 15;149(10):3302-8; Szondy et al., J Infect
Dis. 1998 Nov;178(5):1288-98). One of the mechanisms of ATRA action is based on inhibition of NFAT (nuclear factors of activated T-cells) protein activity (Lee et al., Eur J
Biochem. 2002 Feb;269(4):1162-70). NFAT is one of the main effectors that trigger FasL. transcription by interacting with the GGAAA seguence at position -276 relative to the transcription initiation point. Accordingly, in some embodiments of the invention, an additional positive effect in obtaining modified cells can be achieved by adding all-trans- retinoic acid (ATRA) when culturing modified NK cells in vitro. NK cells can be cultured in the presence of ATRA prior to addition to the target cells. In some embodiments of the invention, the compositions with the use of a truncated promoter and the addition of ATRA — during cultivation leads to the suppression of FasL. expression during cell production in vitro. As a result, the viability of the modified NK cells and the yield of the cell product at the end of the cycle of cultivation are increased. After removal of ATRA at the final stage of cultivation, the expression level of FasL. and the cytotoxic activity of cells are restored.
Thus, the use of a truncated promoter for the expression of modified FasL. forms in combination with ATRA makes it possible to obtain a greater yield of NK cells with high cytotoxic activity during in vitro cultivation.
The natural free radical scavenger vitamin E suppresses the activity of the transcription factors NF-kappa B and AP-1, thus blocking expression of CD95L and preventing AICD of immune cells. Administration of vitamin E suppresses CD95L mRNA expression and protects immune cells from CD95-mediated apoptosis. Accordingly, in some embodiments, an additional positive effect in obtaining modified cells can be achieved by adding vitamin E and its derivatives when culturing modified NK cells in vitro. In some embodiments of the invention, the compositions with the use of a truncated promoter and the addition of vitamin E or its derivatives during cultivation leads to the suppression of
FasL expression during cell production in vitro. As a result, the viability of the modified
NK cells and the yield of the cell product at the end of the cycle of cultivation are increased. After removal of vitamin E or its derivatives at the final stage of cultivation, the expression level of FasL and the cytotoxic activity of cells are restored. Thus, the use of a truncated promoter for the expression of modified FasL forms in combination with vitamin
E or its derivatives makes it possible to obtain a greater yield of NK cells with high cytotoxic activity during in vitro cultivation.
COMPOSITIONS AND USES
In some embodiments, a composition comprising NK cells disclosed herein comprises a pharmaceutically acceptable carrier, diluent, emulsifier, preservative and/or adjuvant. In some embodiments, the composition comprises an excipient. Suitable carrier and their formulation are described, for example, in Remington: The Science and Practice of
Pharmacy (23rd Edition, Adejare A., Ed., Academic Press, 2020). Compositions should not include agents that may inactivate or kill NK cells. In some embodiments, the pharmaceutical cell composition is a physiological solution, preferably a phosphate- buffered saline or sterile physiological solution or tissue culture medium.
The present invention provides methods of treating hematological malignancies in a — subject. In certain embodiments, the method comprises administering to the subject an effective amount of NK cells, wherein the NK cell comprises the modified Fas ligand variants, that increase their cytotoxicity and survival.
The present invention provides methods of treating a solid tumor in a subject. In certain embodiments of the invention, the method comprises administering to a subject an effective amount of NK cells, wherein the NK cells include modified variants of the Fas ligand, thereby increasing their cytotoxicity and survival rate.
The methods disclosed herein may be used to treat cancer in a subject, reduce tumor size, kill tumor cells, prevent tumor growth, prevent tumor recurrence, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In some embodiments, the methods elicit a complete response. In other embodiments, the methods cause a partial response.
The present invention provides methods of preventing and/or treating pathogenic infection ina subject. In certain embodiments of the invention, the method comprises administering to the subject an effective amount of NK cells, where the NK cells include modified variants of the Fas ligand, thereby increasing their cytotoxicity and survival rate. In some embodiments, the pathogen is selected from the group consisting of a virus, bacterium, fungus, parasite, and protozoa capable of causing disease.
A variety of additional therapeutic agents may be used in conjunction with the compositions described herein. For example, potentially useful additional therapeutic agents include including, but not limited to, PD1, PDL1, CTLA4, LAG-3 inhibitors such as nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), pidilizumab (CureTech), atezolizumab (Tecentrig&), avelumab (Bavencio®), cemiplimab (Libtayo®), dostarlimab (Jemperli), durvalumab (Imfinzi™), Ipilimumab (Yervoy&), Relatlimab (BMS).
The skilled person can readily determine the amount of cells and optional additives and/or carrier in the compositions and to be administered. For any composition to be administered to an animal or human, the following can be determined: toxicity by determining the lethal dose (LD) and LD50 in a suitable animal model; the dosage of the composition(s), the — concentration of components therein and the time of administration of the composition(s) that induce a suitable response.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the presently disclosed cells and compositions, and are not intended to limit the scope of what the inventors regard as their invention.
EXAMPLESExample 1 - Construction of expression vectors encoding modified forms of Fas ligand.
The expression vector with a length of 5975 bp, the physical map of which is shown in Fig. — 1, consists of: 233-685 bp. — hfaslg promoter; 686-1531 bp — FasLmod1-6; 1575-1799 bp —
BGH pA; 1845-2273 bp. — fl ori; 2278-2647 bp — SV40 early promoter; 2683-3477 bp —
Neo(R); 3651-3781 bp — SV40 pA; 4164-4834 bp — pUC origin; 4979-5839 bp (complementary chain) — Amp(R); 5840-5938 bp (complementary chain) — bla promoter.
A fragment representing a shortened FASLG gene promoter (hfaslg promoter) was synthesized by polymerase chain reaction with primers hfaslgprom f and hfaslgprom_r,
SEQ ID NO: 14 and SEQ ID NO: 15, respectively. The primer sequences for the polymerase chain reaction were designed with the OLIGO 4.0 software. hfaslgprom_f{ ttatagccccactgaccattctectgtagetg hfaslgprom r ctgcatggcagctggtgagtcaggc
Chromosomal DNA from peripheral blood mononuclear cells was used as a matrix. “Thermo Fisher Scientific” Pfu DNA polymerase was used for synthesis according to the manufacturer's instructions. PCR reactions were performed in an Eppendorf thermal cycler. 10 ng of DNA was used as a template in a reaction volume of 25 ul containing 20 mM
Tris-HCI (pH 8,8 at 25°C), 10 mM (NH4)2S04, 10 mM KCl, 0,1 mg/mL BSA, 0,1% (v/v)
Triton X-100, 2 mM MgS04, 0.2 mM dNTPs (Thermo Scientific, USA), 0.4 uM of the primers and 1,25 U of PFU DNA polymerase (Thermo Scientific, USA). The amplification — protocol was as follow: an initial denaturation at 95° C for 2 min, followed by 30 cycles at 95°C for 30 s, 59°C for 30 s, 72°C for 1 min, and a final extension at 72°C for 10 min. The size of each amplification product was resolved by electrophoresis in a 1,2% agarose gel (w / v) prepared in TAE buffer (40 mM Tris, pH 8.3, 20 mM acetic acid, I mM EDTA) with 0,4 ug / ml ethidium bromide. 6X DNA Loading Dye containing 10 mM Tris-HCI (pH 7.6) 0.03 % bromophenol blue, 0.03 % xylene cyanol FF, 60 % glycerol, 60 mM
EDTA was used for loading samples.
Fragments encoding sequences 1 - 258 bp of the modified Fas ligand were synthesized by polymerase chain reaction with overlapping oligonucleotides using the method described by Stemmer et al. (Gene. 1995 Oct 16;164(1):49-53).
Thefollowing oligonucleotide mixture was used to synthesize FasLmod!1:
F1GL, F2L, F3, F4, F5, F6, RI, R2, R3, R4, R5, R6GL.
The following oligonucleotide mixture was used to synthesize FasLmod2:
FI1GI, F2I, F3, F4, F5, F6, RI, R2, R3, R4, R5, R6GI.
The following oligonucleotide mixture was used to synthesize FasLmod3:
FIGV,F2V, F3, F4, F5, F6, RI, R2, R3, R4, R5, R6GV.
The following oligonucleotide mixture was used to synthesize FasLmod4:
F1GL, F2L, F3, F4, F5, F6GYL, RI, R2GYL, R3, R4, R5, R6GL.
The following oligonucleotide mixture was used to synthesize FasLmod5:
FIGI, F2I, F3, F4, F5, F6GYI, RI, R2GYI, R3, R4, R5, R6GI.
The following oligonucleotide mixture was used to synthesize FasLmod6:
FIGV, F2V, F3, F4, F5, FOGYV, RI, R2GYV, R3, R4, RS, R6GV.
The oligonucleotide sequences are shown below:
SEO ID NO: 16 F1GL atgcagcagcccttcggctacggctatctgcagatctact
SEO ID NO: 17 F1GI atgcagcagc ccttcggeta cggctatatc cagatctact
SEO ID NO: 18 FIGV atgcagcagc cettcggcta cggctatgtg cagatctact
SEO ID NO: 19 F2L gggtgctgagcagtgccagctctccctgggcccctccagg
SEOIDNO:20 F2I gggtgatcag cagtgecage tetceetggg cecctecagg
SEQ ID NO: 21 F2V gggtggtgag cagtgccage tetceetggg cccetccagg
SEO ID NO: 22 F3 cacagttcttccctgtccaacctctgtgcccagaaggcet
SEO ID NO: 23 F4 ggtcaaaggaggccaccaccaccaccgccaccgccaccac
SEO ID NO: 24 F5 taccacctccgcecgccgccgccaccactgcctccacta
SEOIDNO:25 F6 ccgectgccacccctgaagaagagagggaaccacagcacag
SEO ID NO: 26 F6GYL ggctacccacccctgaagaagagagggaaccacagcacag
SEO ID NO: 27 F6GYI ggctacccacccatcaagaagagagggaaccacagcacag
SEO ID NO: 28 F6GYV ggctacccacccgtgaagaagagagggaaccacagcacag
SEO ID NO: 29 RI ggagacacaggcctgtgctgtggttccctctcttctt
SEOIDNO:30 R2 caggggtggcagcggtagtggaggcagtggtggcggcggc
SEO ID NO: 31 R2GYL caggggtgggtagcctagtggaggcagtggtggcggcggc
SEO ID NO: 32 R2GYI gatgggtgggtagcctagtggaggcagtggtggcggcggc
SEO ID NO: 33 R2GYV cacgggtgggtagcctagtggaggcagtggtggcggcggc
SEO ID NO: 34 R3 ggeeggagetggtagtgetggeggtggcgetgetggtggtg
SEOIDNO:35 R4 gcctcetttgaccaggcettctgggcacagaggttggaca
SEO ID NO: 36 RS gggaagaactgtgcctggaggggceccagggagagctggca
SEO ID NO: 37 R6GL ctgctcagcacccagtagatctgcagatagccgtagccga
SEO ID NO: 38 R6GI ctgctgatcacccagtagatctggatatageegtagecga
SEQ ID NO: 39 R6GV ctgctcaccacccagtagatctgcacatagccgtagccga
PCR reactions were performed in an Eppendorf thermal cycler. Egual volumes of oligonucleotides were combined to a final concentration 100 uM mixed oligonucleotides.
0,5 ul of oligonucleotide mixture was used as a template in a reaction volume of 25 ul containing 20 mM Tris-HCI (pH 8,8 at 25°C), 10 mM (NH4)2S04, 10 mM KCI, 0,1 mg/mL BSA, 0,1% (v/v) Triton X-100, 2 mM MgSO4, 0.2 mM dNTPs (Thermo Scientific,
USA), 0.4 uM of the primers and 1,25 U of PFU DNA polymerase (Thermo Scientific,
USA) PCR reactions were performed as follow: an initial denaturation at 95° C for 2 min, followed by 55 cycles at 95°C for 30 s, 52°C for 30 s, 72°C for 30 min, and a final extension at 72°C for 5 min. “Thermo Fisher Scientific” Pfu DNA polymerase was used for synthesis according to the manufacturer's instructions. The size of each amplification product was resolved by electrophoresis in a 1,2% agarose gel (w / v) prepared in TAE buffer (40 mM Tris, pH 8.3, 20 mM acetic acid, I mM EDTA) with 0,4 ug / ml ethidium bromide. 6X DNA Loading Dye containing 10 mM Tris-HCl (pH 7.6) 0.03 % bromophenol blue, 0.03 % xylene cyanol FF, 60 % glycerol, 60 mM EDTA was used for loading samples.
At the next stage, the synthesized fragments were amplified using LNKFI and LNKRI primers, SEQ ID NO: 40 and SEO ID NO: 41, respectively. The oligonucleotide sequences are shown below:
SEO ID NO: 40 LNKFI tgccatgcagcagccettcgg
SEO ID NO: 41 LNKRI catcacaaggagacacaggcctgtgctg — 8 ul of synthesized fragments mix was used as a template in a reaction volume of 25 ul containing 20 mM Tris-HCl (pH 8,8 at 25°C), 10 mM (NH4):SO4, 10 mM KCI, 0,1 mg/mL BSA, 0,1% (v/v) Triton X-100, 2 mM MgSO4, 0.2 mM dNTPs (Thermo Scientific,
USA), 0.4 uM of the primers and 1,25 U of PFU DNA polymerase (Thermo Scientific,
USA). PCR reactions were performed as follow: an initial denaturation at 95° C for 2 min, followed by 25 cycles at 95°C for 30 s, 59°C for 30 s, 72°C for 30 min, and a final extension at 72°C for 5 min. “Thermo Fisher Scientific” Pfu DNA polymerase was used for synthesis according to the manufacturer's instructions. The size of each amplification product was resolved by electrophoresis in a 1,2% agarose gel (w / v) prepared in TAE buffer (40 mM Tris, pH 8.3, 20 mM acetic acid, I mM EDTA) with 0,4 ug / ml ethidium bromide. 6X DNA Loading Dye containing 10 mM Tris-HCl (pH 7.6) 0.03 % bromophenol blue, 0.03 % xylene cyanol FF, 60 % glycerol, 60 mM EDTA was used for loading samples.
The fragment encoding the sequence 231-846 bp of the Fas ligand was synthesized by polymerase chain reaction with primers LNKF3 and FaslR_Xho, SEQ ID NO: 42 and SEQ
ID NO: 43, respectively. The oligonucleotide sequences are shown below:
SEQ ID NO: 42 LNKF3 cagcacaggcctgtgtctcettgtgatg
SEOIDNO:43 FaslR Xho cacctcgagttagagcttatataagccgaaaaacgtc
The plasmid vector pcDNA4/TO-FasL previously described by Glukhova et al., 2018 (17), was used as a matrix. 5 ng of plasmid DNA was used as a template in a reaction volume of 25 pl containing 20 mM Tris-HCI (pH 8,8 at 25°C), 10 mM (NH4)2S04, 10 mM KCI, 0,1 mg/mL BSA, 0,1% (v/v) Triton X-100, 2 mM MgSO4, 0.2 mM dNTPs (Thermo Scientific,
USA), 0.4 uM of the primers and 1,25 U of PFU DNA polymerase (Thermo Scientific,
USA). PCR reactions were performed as follow: an initial denaturation at 95° C for 2 min, followed by 30 cycles at 95°C for 30 s, 59°C for 30 s, 72°C for 80 s, and a final extension at 72°C for 10 min. “Thermo Fisher Scientific” Pfu DNA polymerase was used for synthesis according to the manufacturer's instructions. The size of each amplification product was resolved by electrophoresis in a 1,2% agarose gel (w / v) prepared in TAE buffer (40 mM Tris, pH 8.3, 20 mM acetic acid, I mM EDTA) with 0,4 ug / ml ethidium bromide. 6X DNA Loading Dye containing 10 mM Tris-HCl (pH 7.6) 0.03 % bromophenol blue, 0.03 % xylene cyanol FF, 60 % glycerol, 60 mM EDTA was used for — loading samples.
The FaslP Mlu (SEO ID NO: 44) and FasIR Xho ( SEQ ID NO: 43) primers were used to synthesize DNA fragments encoding the FasLmod 1 (SEQ ID NO: 1) - FasLmod 6 (SEO
ID NO: 6) seguences and the truncated hfaslg promoter. The oligonucleotide seguences are shown below:
SEOIDNO:44 FaslP Mlu tttacgcgttatagccccactgaccattctc
A mixture of DNA fragments representing the truncated hfaslg promoter, a fragment encoding the sequence 1 - 258 bp of FasLmod!l - FasLmod6, and a fragment encoding the seguence 231 - 846 bp of Fas ligand were used as a matrix. 1 ng of a fragment encoding — the sequence 1 - 258 bp of FasLmodl - FasLmod6, and 2 ng of a fragment encoding the sequence 231 - 846 bp of Fas ligand was used as a template in a reaction volume of 25 ul containing 20 mM Tris-HCI (pH 8,8 at 25°C), 10 mM (NH4)2S04, 10 mM KCI, 0,1 mg/mL BSA, 0,1% (v/v) Triton X-100, 2 mM MgSO4, 0.2 mM dNTPs (Thermo Scientific,
USA), 0.4 uM of the primers and 1,25 U of PFU DNA polymerase (Thermo Scientific,
USA). PCR reactions were performed as follow: an initial denaturation at 95° C for 2 min, followed by 30 cycles at 95°C for 30 s, 59°C for 30 s, 72°C for 105 s, and a final extension at 72°C for 10 min. “Thermo Fisher Scientific” Pfu DNA polymerase was used for synthesis according to the manufacturer's instructions. The size of each amplification product was resolved by electrophoresis in a 1,2% agarose gel (w / v) prepared in TAE buffer (40 mM Tris, pH 8.3, 20 mM acetic acid, I mM EDTA) with 0,4 ug / ml ethidium bromide. 6X DNA Loading Dye containing 10 mM Tris-HCl (pH 7.6) 0.03 % bromophenol blue, 0.03 % xylene cyanol FF, 60 % glycerol, 60 mM EDTA was used for — loading samples.
The fragments encoding the seguences of the modified Fas ligand with the hfaslg promoter promoter were cloned into the pcDNA3.1 (+) vector (Invitrogen) at the Mlul and Xhol restriction sites. DNA seguences of the selected plasmid vectors pFasLmod 1, pFasLmod 2, pFasLmod3, pFasLmod4, pFasLmod5, pFasLmod6 were confirmed by DNA sequencing.
Modified NK cells were obtained by introducing plasmid vectors pFasLmod1, pFasLmod2, pFasLmod3, pFasLmod4, pFasLmod5, and pFasLmod6 into cells using Lipofectamine 2000 or Lipofectamine 3000 transfection (Thermo Fisher Scientific) or electroporation using a Gene Pulser Xcell device (Bio-Rad) according to the manufacturer's instructions.
Example 2 - Changes in target cell morphology during interaction with NK cells.
Human NK cells were isolated from peripheral blood mononuclear cell samples from a healthy donor using the NK Cell Isolation Kit (Miltenyi Biotec). NK cells were transfected with the pFasLmod1, pFasLmod2, pFasLmod3, pFasLmod4, pFasLmod5, and pFasLmod6 — vectors by electroporation using the Gene Pulser Xcell System (Bio-Rad) according to the manufacturer's instructions. Three days after electroporation, the cells were purified using the Dead Cell Removal Kit (Miltenyi Biotec) and then incubated in DMEM medium containing 500 iu/ml IL-2, 10% FBS, for 6 h with target cells HEK293 (transformed human embryonic kidney cells) (Fig. 2), HeLa (human cervical adenocarcinoma) (Fig. 3), or A172 (human glioblastoma) (Fig. 4) in a 1:3 ratio (target:effector). Cells were observed under phase-contrast optics with a 10x objective.
The data in Fig. 2-4 illustrate typical morphological changes induced in susceptible tumor target cells during an interaction with NK cells. All lines of target cells tested underwent morphological changes associated with cell death. Dying tumor target cells showed rounding-up, shrinkage, plasma membrane blebbing and the presence of apoptotic bodies (Ziegler, Groscurth. News Physiol Sci. 2004 Jun; 19:124-8). At the same time NK cells completely rounded up and form multi-cellular clusters which surrounded and covered — target cells. It is known that these homotypic NK-NK interactions that shape multicellular clusters are critical for optimal cytolytic activation of NK cells, IFN-y secretion and elimination of tumor cells in vivo (Lee et al., Blood. 2006 Apr 15;107(8):3181-8; Kim et al., Sci Rep. 2017 Jan 11; 7:40623).
All NK cells tested caused HEK293 cell death. However, all modified NK cells were more — devastating to target cells compared to control NK cells. Noteworthy is the greater number of live NK-FasLmod cells compared to mock NK cells. The most pronounced changes in the morphology of HEK293 cells were observed when target cells collided with NK-
FasLmod2 cells.
All tested NK cells resulted in death of HeLa cells (Fig. 3). However, all modified NK — cells were more destructive to target cells compared to control NK cells. Also noteworthy is the higher number of live NK-FasLmod cells compared with unmodified NK cells. The most pronounced changes in HeLa cell morphology were observed when target cells encountered with NK-FasLmod2 or NK-FasLmodd4 cells.
All NK cells tested resulted in A172 cell death (Fig. 4). However, all modified NK cells — were more detrimental to target cells compared to control NK cells. Also of note is the higher number of live NK-FasLmod cells compared with mock NK cells. The most pronounced changes in the morphology of A172 cells were observed when target cells interacted with NK-FasLmod2, NK-FasLmod3, or NK-FasLmod5 cells.
Taken together, these data demonstrate that, in accordance with several embodiments — disclosed herein, NK cells that express the FasL muteins are able to be activated and successfully generate enhanced cytotoxic effects against tumor target cells.
Example 3 - Assessment of cytotoxic activity of NK92 and NK92- FasLmodl, NK92-
FasLmod2, NK92- FasLmod3, NK92- FasLmod4, NK92- FasLmod5, NK92- FasLmod6 — cells against HeLa target cells (human cervical adenocarcinoma).
To assess the potency of the modified NK92 cells, cytotoxicity assays were performed using NK cell-sensitive cell lines, HEK293, Hela, and A172 cells. NK92 cells were transfected with the pFasLmodl, pFasLmod2, pFasLmod3, pFasLmod4, pFasLmod5, and pFasLmod6 vectors by electroporation using the Gene Pulser Xcell System (Bio-Rad) according to the manufacturer's instructions. After electroporation, cells were incubated in aMEM selection medium supplemented with 2 mM L-glutamine, sodium bicarbonate (1.5 g/L), 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 400 U/ml recombinant interleukin 2, 25% fetal calf serum, 50 pg/ml G418. Target cells (HEK293,
Hela, or A172 cells) were seeded/seeded/planted into the wells of a 96-well plate. The next day, control NK92 cells and modified (NK92- FasLmod1, NK92- FasLmod2, NK92-
FasLmod3, NK92- FasLmod4, NK92- FasLmod5, and NK92- FasLmod6) cells were added to the wells of the 96-well plate with the previously seeded target cells and incubated for 5 h at 370C and 5% CO2 at a ratio of 2:1 or 5:1 (effector:target). The wells were then washed with buffered saline and the number of surviving target cells was estimated by staining with neutral red dye (Wallach J Immunol. 1984 May; 132(5):2464-9).
Data summarizing the percent cytotoxicity of different NK92 cell lines against target cells at two E:T ratios are shown in Figs. 5-7 (all experiments were performed in triplicate). — As depicted in Fig. 5, NK92 cells expressing FasL muteins had a significantly higher cytotoxicity against HeLa cells as compared to mock NK92 cells. Even at a low E:T ratio of 2:1, 60%, 57%, 62%, 45%, 50% and 52% of HeLa cells were killed by NK92-
FasLmodl, NK92- FasLmod2, NK92- FasLmod3, NK92- FasLmod4, NK92- FasLmod5,
NK92- FasLmod6 cells, respectively. The mock NK-92 cytotoxicity was 35% at this ratio.
At the E:T ratio of 5:1 the cytotoxic effects of NK92-FasL muteins were even more pronounced. NK92- FasLmod1, NK92- FasLmod2, NK92- FasLmod3, NK92- FasLmodd4,
NK92- FasLmod5, NK92- FasLmod6 cells killed 85%, 83%, 82%, 78%, 80% and 75% of
Hel a cells, respectively. NK92- FasLmod1, NK92- FasLmod2 and NK92- FasLmod3 cells demonstrated the highest potency of killing HeLa cells comparing to other modified NK92 — cells.
Fig. 6 shows the death rates of HEK293 cells when co-cultured with NK92 cells expressing FasL muteins, in the ratios of 2:1 and 5:1, demonstrating a significant increase in killing compared to mock NK92 cells. At the E:T ratio of 2:1 92%, 93%, 94%, 81%, 87% and 86% of HEK293 cells were killed by NK92- FasLmodl, NK92- FasLmod2,
NK92- FasLmod3, NK92- FasLmod4, NK92- FasLmod5, NK92- FasLmod6 cells, respectively. At the E:T ratio of 5:1 NK92- FasLmodl, NK92- FasLmod2, NK92-
FasLmod3, NK92- FasLmod4, NK92- FasLmod5, NK92- FasLmod6 cells killed 97%, 94%, 97%, 88%, 91% and 89% of HEK293 cells, respectively. As with HeLa cells, NK92-
FasLmod1, NK92- FasL mod2, and NK92- FasLmod3 cells showed the highest cytotoxicity against HEK293 comparing to other modified NK92 cells.
Fig. 7 depicts the cytotoxicity of mock NK92, NK92- FasLmodl, NK92- FasLmod2,
NK92- FasLmod3, NK92- FasL mod4, NK92- FasLmod5, NK92- FasL mod6 cells against
A172 cells. As with other tumor cells, the modified NK92 cells expressing FasL. muteins, exhibited higher killing activity against A172 cells than mock NK92 cells. At the E:T ratio of 2:1, 39%, 40%, 39%, 35%, 31% and 35% of A172 cells were killed by NK92-
FasLmodl, NK92- FasLmod2, NK92- FasLmod3, NK92- FasLmod4, NK92- FasLmod5,
NK92- FasLmod6 cells, respectively. The mock NK-92 cytotoxicity against A172 cells was 18% at this ratio. At the E:T ratio of 5:1, 65%, 68%, 68%, 68%, 67% and 60% of
A172 cells were killed by NK92- FasLmodl, NK92- FasLmod2, NK92- FasLmod3,
NK92- FasLmod4, NK92- FasLmod5, NK92- FasLmod6 cells, respectively. The mock
NK-92 cytotoxicity against A172 cells was 48% at this ratio. Note that against A172 cells, the cytotoxic activity of the various modified TL92 cells was about the same and did not differ as much as against the other tumor target cells described above (HeLa and HEK293 cells).
Noteworthy, these data show that even at a modest E:T ratio (2:1), the modified NK cells exhibit high cytotoxic activity. This suggests that the desirable cytotoxic effects of modified NK cells expressing FasL muteins can be realized even when NK cells are — present in moderate numbers relative to the target cells, which is likely to be the case in clinical applications. Moreover, these data show that the modified NK cells disclosed herein have significantly increased cytotoxicity compared to unmodified NK cells.
Example 4 - Comparative assessment of the proliferation rate of NK92 and NK92-
FasLmodl, NK92- FasLmod2, NK92- FasLmod3, NK92- FasLmod4, NK92- FasLmod5,
NK92- FasLmodo cell cultures.
Cell proliferation rate was assessed by trypan blue staining and light microscopic quantification of live cells at different time points (63). NK92 cells, NK92- FasLmodi,
NK92- FasLmod2, NK92- FasLmod3, NK92- FasLmod4, NK92- FasLmod5 and NK92- — FasLmod6 were seeded (0.5x106cells/mL) in aMEM medium with 2 mM L-glutamine, sodium bicarbonate (1.5 g/L), 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 400 U/ml recombinant interleukin 2, 25% fetal calf serum and incubated for 14 days at 370C and 5% CO2. Samples for analysis were taken on days 7, 11 and 14. All experiments were performed in triplicate, and data are expressed as the mean of three samples with standard deviation. The results of proliferative activity assessment are shown in Fig. 8.
In all groups, cell proliferation steadily increased up to the 14th day. However, as early as day 11, it was noticeable that the fold change of NK92 cells transfected with wild-type
FasL was significantly lower than that of control NK92 cells and NK92 cells transfected with FasL muteins. And by day 14, the difference with control NK92, NK92- FasLmod!1,
NK92- FasLmod2, NK92- FasLmod3, NK92- FasLmod4, NK92- FasLmod5, NK92-
FasLmod6 cells was 2.5, 1.6, 1.4, 1.5, 1.9, 2.0, and 2.1 times, respectively. The fold change of NK92- FasLmodl, NK92- FasLmod2, NK92- FasLmod3 cells was slightly lower than that of NK92- FasLmod4, NK92- FasLmod5 or NK92- FasLmod6 cells. The difference between NK92- FasLmodl, NK92- FasLmod2, NK92- FasLmod3 cells and NK92-
FasLmod4, NK92- FasLmod5, NK92- FasLmod6 cells in the levels of survival and cytotoxicity is apparently determined by the presence of additional “trafficking domains” — in the intracellular part of NK92- FasLmod4, NK92- FasLmod5, NK92- FasLmod6 cells.
Thus, according to some of the embodiments disclosed here, these data indicate that, in contrast to wild-type FasL, overexpression of FasL mutains promotes the survival of transfected cells and enhances their cytotoxic activity.
Example 5 - Effect of a truncated promoter, ATRA, and vitamin E on the level of Fas ligand expression and survival of modified NK92 cells.
NK92 cells were transfected with a plasmid vector encoding the recombinant FasLmod!l gene with the natural faslg promoter or a truncated faslg promoter or cytomegalovirus promoter. After selection in the medium with G418, the expression level of the recombinant gene was assessed by polymerase chain reaction coupled with reverse transcription (RT-PCR) using the primers ExF (SEO ID NO: 45) and ExR (SEO ID NO: 46). The oligonucleotide seguences are shown below:
SEO ID NO: 45 ExF gagaagcaaataggccacccagtccac
SEO ID NO: 46 ExR gaccttatataagccgaaaaaacgtctgagattctc
Two-step RT-PCR reactions were performed using Revertäid M-MuLV reverse transcriptase (Thermo Scientific, USA) and Tag DNA polymerase (Thermo Scientific,
USA) in accordance with the manufacturer's recommendations. 5 ug of RNA was combined with 0,5 ng of oligo dT12-18 mer and incubated in water at 65°C for 5 min followed by incubation on ice. First strand synthesis was performed in buffer containing 50 mM Tris-HCI (pH 8.3 at 25 °C), 50 mM KCI, 4 mM MgClz, 10 mM DTT, 1 mM dNTPs, 10 U/ul RevertAid M-MuLV reverse transcriptase for 60 min at 42°C. The reaction was terminated by heating at 70 °C for 10 min. A fifth of the reaction was used as a template in a reaction volume of 25 ul containing 10 mM Tris-HCI (pH 8.8 at 25°C), 50 mM KCI, 0.08% (v/v) Nonidet P40, 2 mM MgClz, 0.2 mM dNTPs (Thermo Scientific, USA), 0.2 uM of the primers and 0,6 U of Taq DNA polymerase (Thermo Scientific, USA). The amplification protocol was as follow: an initial denaturation at 95° C for 10 min, followed by 30 cycles at 95°C for 30 s, 59°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 10 min. The size of each amplification product was resolved by electrophoresis in a 1,2% agarose gel (w / v) prepared in TAE buffer (40 mM Tris, pH 8.3, 20 mM acetic acid, 1 mM EDTA) with 0,4 ug / ml ethidium bromide. 6X DNA Loading Dye containing 10 mM Tris-HCI (pH 7.6) 0.03 % bromophenol blue, 0.03 % xylene cyanol FF, 60 % glycerol, 60 mM EDTA was used for loading DNA markers and samples. GeneRuler 100 bp DNA Ladder (Thermo Scientific, USA) was used as molecular weight standard. The results are shown in Fig. 9A. The activity of the truncated promoter is higher than that of the natural promoter, but lower than that of the commonly used CMV promoter. These data demonstrate that, in accordance with several embodiments disclosed herein, the use of this truncated promoter makes it possible to achieve a significantly higher level of Fas ligand expression compared to the natural one.
Cell proliferation rate was assessed by trypan blue staining and light microscopic quantification of live cells at different time points (Strober. Curr Protoc Immunol. 2015
Nov 2; 111: A3.B.1-A3.B.3). Transfected cells were seeded (0.5x10%ells/mL) in aMEM — medium with 2 mM L-glutamine, sodium bicarbonate (1.5 g/L), 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 400 U/ml recombinant interleukin 2, 25% fetal calf serum and incubated for 15 days at 37°C and 5% CO2. Samples for analysis were taken on days 7, 11 and 15. All experiments were performed in triplicate, and data are expressed as the mean of three samples with standard deviation. The results of proliferative activity assessment are shown in Fig. 9B. Expression of the Fas ligand under the control of the truncated hfaslg promoter ensures greater survival of NK92 cells than under the action of the CMV promoter. According to some embodiments disclosed herein, a truncated version of the hfaslg promoter can be incorporated into an expression vector encoding modified forms of FasL to enhance Fas ligand expression within cells and preserve cell viability.
The effects of ATRA on the expression level of Fas ligand and survival of modified NK92 cells were assessed as follows. NK92-FasLmodl with a truncated faslg promoter was incubated for 72 h in the presence of 1 uM ATRA or without it. RNA was isolated from the cells, and the expression level of FasL was assessed by polymerase chain reaction coupled with reverse transcription (RT-PCR) using the primers ExF (SEQ ID NO: 45) and
ExR (SEQ ID NO: 46) as described above. The results are shown in Fig.10A.
Cell proliferation rate was assessed by trypan blue staining and light microscopic quantification of live cells at different time points (63). Control NK92 cells and NK92-
FasLmod! cells with a truncated faslg promoter were seeded (0.5x10°cells/mL) in MEM medium with 2 mM L-glutamine, sodium bicarbonate (1.5 g/L), 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 400 U/ml recombinant interleukin 2, 25% fetal calf serum and incubated for 72 h in the presence of 1 uM, or 0.1 uM ATRA or without it. — After it cells were incubated for 13 days. Samples for analysis were taken on days 7, and 13. All experiments were performed in triplicate, and data are expressed as the mean of three samples with standard deviation. The assessment of a fold change in cell number is shown in Fig. 10B.
To evaluate the effect of ATRA on the cytotoxic activity of control NK92 cells and NK92-
FasLmodl cells with a truncated faslg promoter, a cytotoxicity assay was performed using
Hela cells as a target. Modified cells were incubated in medium with 1 uM ATRA for 13 days, then replaced with medium without ATRA and cultured for additional 48 h. Then cells were added to the wells of the 96-well plate with the previously seeded target HeLa cells and incubated for 5 h at 37°C and 5% CO» at a ratio of 2:1 or 5:1 (effector:target).
The wells were then washed with buffered saline and the number of surviving target cells was estimated by staining with neutral red dye. Data summarizing the percent cytotoxicity of NK92 cells and NK92-FasLmod! cells preincubated with ATRA are shown in Fig. 10C.
These data demonstrate that, in accordance with several embodiments of the invention disclosed here, the use of a truncated promoter and the addition of ATRA during cultivation leads to suppression of FasL expression during in vitro cell cultivation. As a result, the viability of the modified NK cells and the yield of the cell product at the end of the cultivation cycle are increased. After removal of ATRA at the final stage of cultivation, the expression level of FasL and the cytotoxic activity of cells are restored. Thus, the use of a truncated promoter for the expression of modified FasL. forms in combination with
ATRA makes it possible to obtain a greater yield of NK cells with increased survival and high cytotoxic activity.
The effects of vitamin E on the expression level of Fas ligand and survival of modified
NK92 cells were assessed as follows. NK92-FasLmod1 with a truncated faslg promoter was incubated for 4 h in the presence of 40 uM vitamin E or without it. RNA was isolated from the cells, and the expression level of FasL. was assessed by polymerase chain reaction coupled with reverse transcription (RT-PCR) using the primers ExF (SEQ ID NO: 45) and
ExR (SEQ ID NO: 46) as described above. The results are shown in Fig.11A.
Cell proliferation rate was assessed by trypan blue staining and light microscopic quantification of live cells at different time points. Control NK92 cells and NK92-
FasLmod! cells with a truncated faslg promoter were seeded (0.5x10°cells/mL) in aMEM medium with 2 mM L-glutamine, sodium bicarbonate (1.5 g/L), 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 400 U/ml recombinant interleukin 2, 25% fetal — calf serum and incubated for 13 days in the presence of 25 uM vitamin E or without it.
Samples for analysis were taken on days 7, and 13. All experiments were performed in triplicate, and data are expressed as the mean of three samples with standard deviation. The assessment of a fold change in cell number is shown in Fig. 11B.
To evaluate the effect of vitamin E on the cytotoxic activity of control NK92 cells and
NK92-FasLmodl cells with a truncated faslg promoter, a cytotoxicity assay was performed using HeLa cells as a target. Modified cells were incubated in medium with 40 uM vitamin
E for 4 h, then replaced with medium without vitamin E and cultured for additional 24 h.
Then cells were added to the wells of the 96-well plate with the previously seeded target
Hela cells and incubated for 5 h at 37°C and 5% CO2 at a ratio of 2:1 or 5:1 — (effector:target). The wells were then washed with buffered saline and the number of surviving target cells was estimated by staining with neutral red dye (62). Data summarizing the percent cytotoxicity of NK92 cells and NK92-FasLmodl cells preincubated with vitamin E are shown in Fig. 11C.
These data demonstrate that, in accordance with several embodiments of the invention disclosed here, the use of a truncated promoter and the addition of ATRA during cultivation leads to suppression of FasL. expression during in vitro cell cultivation. As a result, the viability of the modified NK cells and the yield of the cell product at the end of the cultivation cycle are increased. After removal of ATRA at the final stage of cultivation,
the expression level of FasL and the cytotoxic activity of cells are restored. Thus, the use of a truncated promoter for the expression of modified FasL. forms in combination with
ATRA makes it possible to obtain a greater yield of NK cells with increased survival and high cytotoxic activity.
In accordance with some embodiments, an additional positive effect in obtaining modified cells can be achieved by adding vitamin E and its derivatives when culturing modified NK cells in vitro. In some embodiments of the invention, the compositions with the use of a truncated promoter and the addition of vitamin E during cultivation leads to the suppression of FasL. expression during cell production in vitro. As a result, the viability of — the modified NK cells and the yield of the cell product at the end of the cycle of cultivation are increased. After removal of vitamin E at the final stage of cultivation, the expression level of FasL and the cytotoxic activity of cells are restored. Thus, the use of a truncated promoter for the expression of modified FasL forms in combination with vitamin E makes it possible to obtain a greater yield of NK cells with increased survival and high cytotoxic activity.
Thus, the invention provides NK cells and their production, for use in the therapy of immune related diseases.
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