NK cells having reduced CD38 expression and method for producing

Abstract

Provided herein are populations of Natural Killer (NK) cells having reduced expression of a gene of interest, methods of producing the NK cells, methods of using the NK cells, and pharmaceutical compositions comprising the NK cells.

Description

Attorney Docket No. :0171.0107-PCTNK CELLS HAVING REDUCED CD38 EXPRESSION AND METHOD FOR PRODUCINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and relies on the filing date of, U.S. provisional patent application number 63 / 689,467, filed 30 August 2024, the entire contents of which are incorporated by reference herein.BACKGROUND

[0002] Natural killer (NK) cells inherently possess therapeutic potential due to their functional capacity for targeting and eliminating tumor cells. Addition of genetic alteration(s) of NK cells can further enhance the functional capacity7of the cells and enable targeted treatments and potential for combination therapies. However, one significant barrier to use of NK cell therapy has been limited ex vivo expansion of genetically edited primary human NK cells to clinically relevant cell numbers.

[0003] The cell surface molecule, CD38, is expressed on multiple myeloma cells in most patients, and anti-CD38 monoclonal antibodies can be used to eliminate multiple myeloma cells. The killing activity of anti-CD38 monoclonal antibodies relies, in part, on antibody-dependent cellular toxicity (ADCC) mediated by endogenous NK cells. However, due to CD38 expression on endogenous NK cells, patients treated with anti-CD38 monoclonal antibodies experience a decrease in endogenous NK cell numbers during treatment. Administering anti-CD38 monoclonal antibody following prior anti-CD38 antibody treatment has proven to be less efficient than first use despite continued presence of CD38 on multiple myeloma cells. This may be the result of endogenous NK cell depletion caused by fratricide of NK cells. Therefore, providing exogenous NK cells with a genetic knockout of CD38, combined with anti-CD38 monoclonal antibody could restore the efficacy of treatment in addition to maintaining NK cell anti -tumor cytotoxicity.

[0004] The ability to generate clinical-scale batches of genetically modified primary NK cells remains a significant challenge for this form of therapy (Kang, et al. Curr Oncol, 2021. 28(2): p. 1077-1093, and Wu, et al., Mol Ther. Oncolytics, 2022. 27: p. 224-238. Therefore, ex vivo expansion of larger numbers of human primary NK cells, particularly genetically altered NK cells, and methods to produce them are needed.Attorney Docket No. :0171.0107-PCTSUMMARY

[0005] Provided herein are populations of NK cells having reduced expression of a gene of interest (CD38), methods of producing and expanding genetically modified NK cells, methods of using the genetically modified NK cells, and pharmaceutical compositions comprising the genetically modified NK cells.

[0006] In a first aspect, populations of NK cells are provided wherein the NK cells have reduced ability to express a gene of interest (such as CD38) compared to a population of NK cells isolated from human peripheral blood mononuclear cells (PBMCs) from healthy donors. In some embodiments, the population of NK cells is obtained from human peripheral blood mononuclear cells (PBMCs) and subjected to gene editing as described herein. In some embodiments, at least 90 % of the NK cells of the population have reduced expression of CD38 compared to NK cells that have been produced as described herein but that have not been subjected to gene editing. In some embodiments, the CD38 gene in at least 90% of NK cells of the populations has a disruption in exon 1 CD38.

[0007] In a second aspect, methods of producing a population of NK cells that have reduced expression of a gene of interest (such as CD38) are provided. In some embodiments, the method comprises obtaining a sample of human PBMCs. The sample of human PBMCs can be thawed from cryopreserved PBMCs. In some embodiments, of CD3 positive (CD3+) cells are then removed (depleted) from the PBMCs. In some embodiments, the PBMCs have already been depleted of CD3+ cells. The CD3+ depleted cells are subjected to gene editing by electroporation with mRNA encoding CasX and a guide RNA (gRNA) directed to a gene of interest (such as CD38). such that the gene of interest in the NK cells is disrupted. After electroporation, the cells are expanded, thereby producing a population of NK cells that have reduced expression of the gene of interest as described herein.

[0008] In a third aspect, populations of genetically modified NK cells produced by the methods described herein are provided.

[0009] In a fourth aspect, methods for treating a hematological cancer are provided. The methods comprise administering a population of NK cells or a pharmaceutical composition comprising a population of NK cells, where the NK cells have reduced expression of a gene of interest (such as CD38) to a patient in need thereof. The population of NK cells are produced from PBMCs that have been subjected to gene editing as described herein.

[0010] In a fifth aspect, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise a population of NK cells that have reduced expressionAttorney Docket No. :0171.0107-PCT of a gene of interest (such as CD38), Plasma-Lyte A. and Human Serum Albumin (HSA). In some embodiments, the population of NK cells was obtained from human PBMCs and subjected to gene editing. The population of NK cells are produced from PBMCs that have been subjected to gene editing as described herein.

[0011] In a sixth aspect, methods for producing pharmaceutical compositions comprising a population of genetically modified NK cells produced by methods described herein are provided.

[0012] Other objects, features and advantages will become apparent from the following detailed description. The detailed description and specific examples are given for illustration only since vanous changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 shows a schematic representation of CasX mRNA construct.

[0014] FIG 2 shows schematic overview of the production of CD38KO NK cells.

[0015] FIG. 3A-B show a bar graph of average fold expansion post-electroporation (FIG. 3A) and average percent CD38+ / CD56+ / CD3- cells (FIG. 3B).

[0016] FIG. 4 is a bar graph showing fold expansion of cells after electroporation.

[0017] FIG. 5 shows percent CD38 positive NK cells over time.

[0018] FIG. 6 shows the percent of CD38 positive cells obtained from manufacturing scale process.

[0019] FIG. 7A-B show expression levels of NK cell markers CD 16, NKG2D, and NKp46 (FIG. 7A) and percent cytotoxic abi 1 i ty of cells obtained from the manufacturing scale process (FIG 7B).

[0020] FIG. 8A-B show for control and CD38KO cells in the presence or absence of TGFP: average fold expansion post electroporation of cells (FIG. 8A) and average percentage of CD38+ cells (FIG. 8B).

[0021] FIG. 9A-C show for manufacturing scale production of NK cells: fold expansion (FIG. 9A), population doubling time as a function of time (FIG. 9B) and percentage of CD38 positive cells (FIG. 9C).DETAILED DESCRIPTION

[0022] To address the need for genetically modified human NK cells in quantities suitable forAttomey Docket No. :0171.0107-PCT medicinal use, including NK cells that have been genetically altered, processes for clinical-scale generation and expansion of genetically modified NK cells and NK cells that have been genetically modified are provided herein. As described herein for the first time, gene-editing materials, comprising CRISPR endonuclease CasX mRNA and gene-targeting gRNA is efficiently delivered (transfected) to primary human NK cells via electroporation. In addition, clinical-scale expansion of the transfected NK cells can be achieved using the methods provided herein. Using the methods provided herein, CD38-knockout (CD38KO) NK cells at clinically relevant scale have been produced for the first time. Additionally, the functionality of the CD38KO NK cells can be further enhanced by conditioning cells through exposure to TGF-(31.

[0023] The starting material for the process is peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. The PBMCs can be collected using leukapheresis in a qualified blood collection center such as Charles River Laboratories or Oklahoma Blood Institute.

[0024] In some embodiments, PBMCs can be isolated from healthy individuals meeting Universal donor criteria, as described in published PCT application W02021 / 051042, which is hereby incorporated by reference in its entirety. Universal donors exhibit a KIR / HLA expression profile that allows NK cell drug product to be administered to potential recipients with reduced risk for rejection or graft-versus-host disease (GVHD), thereby enabling an off- the-shelf therapy. Donors can be screened for CMV positivity in an infectious disease panel, for example. PBMCs can be shipped at 4°C overnight for next day processing or cryopreserved at the collection site and then shipped and stored in liquid nitrogen (LN2) for later use.

[0025] Provided herein are allogenic, CD38 knock-out natural killer (CD38KO NK) cell population, an off-the-shelf donor-derived NK cell investigational immunotherapy in which the CD38 locus has been genetically disrupted. The disruption in the CD38 gene sequence is generated using clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing technology. Using CRISPR-associated protein X (CasX) messenger RNA (mRNA) and CD38-specific guide RNA (gRNA) the CD38 locus of donor NK cells was edited ex vivo. The CD38KO NK cells are expanded ex vivo using a cell-free expansion regimen.

[0026] Herein, a method is described in which genetically modified NK cells, that have a disruption which causes the expression of a given protein to be reduced or knocked-out, can be manufactured in a continuous process, as shown in FIG 2. In some embodiments, the genetic disruption is in the CD38 gene.

[0027] The starting material (e.g., PBMCs) can be depleted of CD3+ cells. The resulting CD3+- depleted mononuclear fraction can be stimulated with membrane bound interleukin-21 (mlL-Attorney Docket No. :0171.0107-PCT21) and 4-1BB ligand (4-1BBL) and one or more of cytokines IL-2. IL-12, IL-15, and IL-18 to induce NK cell expansion ex vivo. PM21 particles can be used as the source of membrane mlL- 21 and 4-1 BBL. PM21 particles are generated from the plasma membranes of a K562 cell line transduced to constitutively express mbIL-21 and 4-1BBL as described in published PCT application WO2014 / 005072, which is hereby incorporated by reference in its entirety.

[0028] Following an initial expansion of the NK cells, the cells are transfected via electroporation with mRNA encoding CasX endonuclease and a gRNA specific to the gene being disrupted. Surprisingly, applicants found that electroporation of the NK cells with CasX mRNA together with the gRNA. following the methods provided herein, caused disruption of the gene of interest with high efficiency.

[0029] Following transfection, the NK cells can be stimulated again with mIL-21 and 4-1BBL to increase expansion of the NK cells until harvest. It was found that stimulating the cells 1 or 2 days after electroporation gave the highest yield of NK cells at harvest. In some embodiments, expanded NK cells have increased in number by at least 3000-fold compared to the number of NK cells that were transfected. In some embodiments, expanded NK cells have increased in number by at least 1000, 2000, 3000, 4000, 5000, or 6000-fold, including all values and subranges therebetween, compared to the number of NK cells that were transfected.

[0030] In one embodiment, the gRNA is specific for the CD38 gene. The resulting CD38KO NK cell product presents a phenotypic and functional profile characterized by potent inflammatory cytokine production and cytotoxicity against transformed cells, with reduced fratricide caused by anti-CD38 monoclonal antibody (e.g., isatuximab).

[0031] The harvested, genetically modified NK cells produced by the methods described herein can be formulated and cryopreserved for future use.

[0032] In some embodiments populations of NK cells are provided wherein at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99%, including all values and subranges therebetween, of the NK cells have a gene of interest disrupted, causing a reduction or elimination of gene expression compared to cells that have not been transfected / edited. In one embodiment, the CD38 gene is disrupted in human primary NK cells, reducing or eliminating the expression of CD38 on the NK cell surface. In all embodiments, the cells used to generate the population of NK cells having a gene of interest disrupted are obtained from human PBMCs from healthy volunteers.

[0033] In some embodiments, the population of cells is comprised of at least 90, 91, 92. 93. 94, 95, 96, 97. 98. or 99% NK cells (CD56+ / CD3- cells), including all values and subranges therebetween. In some embodiments, at least 90, 91, 92, 93, 94, 95, 96, 97, or 98%, includingAtorney Docket No. :0171.0107-PCT all values and subranges therebetween, of the NK cells have reduced expression of the gene of interest. In some embodiments, at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99%, including all values and subranges therebetween, of the NK cells are viable. In some embodiments, the population of NK cells provided herein were obtained from fresh or cryopreserved human PBMCs that were isolated from healthy donor leukapheresis material.

[0034] In one embodiment, at least 90, 91, 92, 93, 94, 95, 96, 97, or 98%, including all values and subranges therebetween, of the NK cells of the population of NK cells provided herein have reduced expression of CD38. In another embodiment the disruption of the CD38 gene is within exon 1 the human genomic sequence of human CD38.

[0035] Methods of producing the population of NK cells that have reduced expression of a gene of interest are provided. The population of NK cells produced according to the methods provided herein can have one or more of the attributes described herein.

[0036] In some embodiments, the methods provided herein comprise depleting human PBMCs of CD3 positive (CD3+) cells. In some embodiments, the PBMCs have previously been depleted of CD3+ cells. In some embodiments, the PBMCs are not subjected to density gradient separation prior to electroporation. In some embodiments, the PBMCs are subjected to density gradient separation. In some embodiments, the CD3+ depleted cells are transfected via electroporation with mRNA encoding CasX endonuclease and a gRNA directed to gene of interest, such that the gene of interest of the cells is disrupted. In some embodiments, the cells are washed with buffer (suitable buffers include HBSS buffer and Maxcyte Electroporation Buffer) at least once before electroporation. In some embodiments, the CD3 -depleted PBMCs are stimulated with mIL-21 and 4-1 BBL (e.g., PM21 particles) on day 0 after thawing the sample.

[0037] In some embodiments, the CasX mRNA has the nucleotide sequence of SEQ ID No. 54. In some embodiments, the gene of interest is CD38 and the gRNA has the sequence of SEQ ID Nos. 4-6, 12, 20, or 40. In some embodiments, the NK cell concentration during electroporation is 30-50xl06NK cells / mL. In some embodiments, the CasX mRNA is at a concentration of 0. 1 - 5.0 pg per IxlO6NK cells during electroporation. In some embodiments, the CD38-specific gRNA is at a concentration of 0.15-4.00 pg per IxlO6NK cells during electroporation.

[0038] After electroporation, the cells are expanded, thereby producing a population of NK cells that have reduced expression of a gene of interest. In some embodiments, the cells are stimulated with mIL-21 and 4-1IBBL (e.g., PM21 particles) two days after electroporation (dayAttorney Docket No. :0171.0107-PCT7 after thawing).

[0039] In a third aspect, populations of NK cells produced by the methods described herein are provided. The population of NK cells produced according to the methods provided herein can have one or more of the attributes described herein.

[0040] In a fourth aspect, methods for treating a hematological cancer are provided. The methods comprise administering a population of NK cells or a pharmaceutical composition comprising a population of NK cells wherein the NK cells have reduced expression of a gene of interest to a patient in need thereof. The population of NK cells are as described herein and can be produced as described herein. In some embodiments, the NK cells have reduced expression of CD38. In some embodiments, the population of NK cells was obtained from human PMBCs and subjected to gene editing such that the CD38 gene in the NK cells has a disruption in exon 1 compared to parental NK cells prior to gene editing.

[0041] In a fifth aspect, pharmaceutical compositions are provided. The pharmaceutical compositions comprise a population of NK cells that have reduced expression of a gene of interest and a liquid formulation suitable for maintaining viability of the population of NK cells. In some embodiments, the formulation comprises Plasma-Lyte A, and Human Serum Albumin (HSA). In some embodiments, the population of NK cells was obtained from human PBMCs and subjected to gene editing, such that the CD38 gene in at least 90, 91, 92, 93, 94, 95. 96. 97, of 98%. including all values and subranges therebetween, of the NK cells have a disruption in exon 1 compared to parental NK cells prior to exposure to the gene editing.

[0042] In a sixth aspect, methods for producing pharmaceutical compositions comprising a population of NK produced by methods described herein are provided.EXAMPLES

[0043] Examples have been set forth below for the purpose of illustration and to describe certain specific embodiments of the invention. However, the scope of the claims is not to be in any way limited by the examples set forth herein.Example 1: Preparing Genetically Altered Natural Killer Cells

[0044] CD3 depleted peripheral blood mononuclear cells (CD3- PBMCs) were obtained as described below (Pharmaceutical Product). NK cell base media (NK MACs®, Miltenyi Biotec) was prepared with 5. 10 or 20 ng / mL IL-15 and warmed to 37°C. Frozen CD3‘ PBMCs were thawed using a ThawStar Controlled-Rate Cell Thawer. Slowly and dropwise, 1 mL ofNK cellAttorney Docket No. :0171.0107-PCT base media was added to cells and carefully agitated with a ImL pipette. Total volume of container was transferred to a 15 mL conical tube. The final volume was adjusted to 10 mL using complete media (NK MACs®, Miltenyi Biotec, 10% human AB serum) supplemented with IL- 15 and cells were counted. Cells were centrifuged at 1000 rpm for 5 min then resuspended in 3 mL NK cell base media.

[0045] One milliliter of NC cell base media was transferred to each of 3 wells of a G-REX® 6 well plate (Wilson Wolf), then 1 mL of the newly thawed cell suspension was added to each well. The media in each well was adjusted up to 35 mL using complete NK cell media with IL- 15. Cells were placed in an incubator at 37°C and 5% CO2.

[0046] Seven days after thaw, cells were carefully resuspended and counted. Each well was split 1 : 1 and leveled to 35 mL with fresh NK cell media supplemented with IL-15. Cells were placed into an incubator at 37°C and 5% CO2 for seven days. Fourteen days following thaw, cells were again counted and then harvested. Cells were centrifuged at 1000 rpm for 10 minutes and supernatant was removed. Cells were resuspended in lOmL of lx HBSS buffer and centrifuged at 1000 rpm for 10 minutes and supernatant was removed. Cells were then resuspended to 50 mL with lx HBSS, counted, and held at 37°C in a bath until time of electroporation.

[0047] CasX mRNA was prepared as described in W02022082001, which is hereby incorporated by reference in its entirety. FIG. 1 shows a schematic representation of CasX mRNA. Table 1 shows the sequences of the gRNA.

[0048] TABLE 1 gRNA sequencesAttorney Docket No. :0171.0107-PCTAttorney Docket No. :0171.0107-PCTAtorney Docket No. :0171.0107-PCT

[0049] Five pL of CasX mRNA were added to each well of a V-bottom plate. Subsequently, 1.08 pL of 100 uM unique CasX gRNA were added to each well of a V-bottom plate. After material plate was prepared, cells were centrifuged at 1000 rpm for 10 minutes and supernatant was removed. Cells were then resuspended to 25 xl 06- 50 xl 06 / mL in Lonza P3 buffer (Lonza). Twenty pL of cells were then added to each well of the material plate, and total well contents were next moved to a Lonza electroporation plate (Lonza). Cells were electroporated using pulse code CM137 and immediately thereafter removed from the electroporation well and placed in 200 pL of warm NK base media supplemented with 5. 10 or 20 ng / mL IL-15 in a 96- well plate. This process was repeated for a technical duplicate for each unique NK cell donor. Plates were placed in an incubator at 37°C and 5% CO2 for 48 h. At 48 h, plates were removed from incubator and cells were prepared for K562 feeder cell restimulation. Briefly, K562 feeder cells expressing membrane bound IL-21 and 4-1BBL were cultured for 21 days, then treated with mitomycin C to arrest cell division. Cells were then aliquoted and frozen until use. At the time of restimulation, feeder cells were thawed and adjusted to add at a 1 : 1 Feeder: NK cell ratio in 20 pL NK cell base media. Cells were placed in the incubator at 37°C and 5% CO2 and split 1 : 1 as needed until Day 7.Attorney Docket No. :0171.0107-PCTAttorney Docket No. :0171.0107-PCT

[0050] At Day 7 post electroporation, cells were harvested for analysis by flow cytometry. Briefly, cells were pelleted by centrifugation at 1000 rpm for 5 min. A fluorescent antibody staining cocktail was prepared using fluorescently labeled anti-CD38 and anti-CD56 antibodies, as well as a viability dye and added to the pelleted NK cells for 20 min at room temperature. After 20 min, cells were washed with Phosphate Buffered Saline (PBS), centrifuged at 1000 rpm for 5 min, supernatant discarded and cells were resuspended in 50 pL of PBS for analysis by flow cytometry. Data were analyzed in NovoExpress® or FlowJo™ and quantified in Tableau®.Attorney Docket No. :0171.0107-PCTResults

[0051] Samples were analyzed by flow cytometry. Cells were gated to include live, CD56 positive, singlet lymphocytes. As shown in Table 2, about 46 % of the 52 gRNA tested resulted in at least 50 % knock out of CD38. CD57+ cells per w ell were counted, and as shown by Table 2. gRNAs did not differently impact CD57+ cell number 7 days post electroporation. CD57+ viable cells were also counted, and as shown by Table 2, gRNAs did not differently impact average cell viability seven days post electroporation. Observed variability' was determined to be donor driven. In silico off-target analyses were also performed, and the sequences of SEQ ID NOS: 4-6, 12, 20, and 40 demonstrated the combination of high knock out of CD38 and low predicted off-target effects.TABLE 2Example 2: Titration of Payload ConcentrationsAtorney Docket No. :0171.0107-PCT

[0052] To maximize post-electroporation NK cell expansion and gene editing efficiency, the payload concentrations of CasX mRNA and gRNA were titrated in combination.

[0053] Cells were prepared following the Research Scale Example. For the electroporation step three concentrations of CasX (5, 1 , and 0. 1 pg of CasX mRNA per 1 x 106cells) and four concentrations of gRNA (3.79, 1.89, 0.379, and 0.189 pg of gRNA per 1 x 106cells) were tested in combination.

[0054] As shown in Table 3, gRNA concentration was inversely correlated with grow th when the cells treated with the highest dose of CasX mRNA. Conversely, lower doses of CasX mRNA. either 1 pg or 0.1 pg per l* 106NK cells, resulted in improved NK cell expansion regardless of gRNA dosage.

[0055] To determine gene editing efficiency, expression of CD38 via flow cytometry was measured. Whereas lower doses of CasX mRNA generally improved post-editing expansion, higher doses of CasX resulted in more efficient gene editing as demonstrated by lower proportions of CD38+ NK cells after electroporation. While the editing was successful across a range of payload concentrations, it w as determined that the optimal dosage to be 1 pg of CasX and 0.379 pg of gRNA per 1 * 106NK cells.

[0056] TABLE 3Example 3: Electroporation and Testing Conditions

[0057] It was determined that 48 hours of rest following electroporation resulted in the highest level of NK cell survival. Despite lower NK cell purity earlier in the process, it w as also found that electroporation as early as day 5 with PM21 stimulation on day 7 resulted in the highest fold expansion post-electroporation (FIG. 3A). Fold expansion was highest overall in theAttorney Docket No. :0171.0107-PCT corresponding day 5 control sample, suggesting the NK cells possess a greater capacity to respond to PM21 stimulation on day 7 rather than day 8 or 9. Regardless of when electroporation was performed, CD38 knockout efficiency was comparable, underscoring the robustness of this gene editing approach (FIG. 3B).

[0058] Despite successful gene editing using the MaxCyte system, fold expansion of under CD38 knock out conditions was only as high as 17.59X following electroporation whereas the non-electroporated control expanded up to 60.08X. Since the MaxCyte system restricts users to pre-set electroporation programs without the ability7to fine tune parameters, the Thermo Fisher Neon™Nxt system, which allows users to manipulate voltage settings and pulse durations was tested. In addition to testing different electroporation parameters, three different electroporation buffers were tested as shown in Table 4.

[0059] TABLE 4

[0060] Electroporation via Thermo 1 -6 resulted in post-editing fold expansion equivalent to or greater than the MaxCyte control, electroporation via Thermo 7, 8, or 9, which were characterized by higher voltages and the use of T buffer, resulted in severely diminished cell growth (FIG. 4). This result could be explained by greater levels of cell death immediately following electroporation. Of the remaining Thermo Conditions using lower voltages, the highest fold expansion resulted from the use of GE buffer (Thermo 4, 5, and 6, FIG. 4). Gene editing efficiency was comparable between Thermo 1-6 and the MaxCyte control, but lower in Thermo 7-9.Attorney Docket No. :0171.0107-PCT

[0061] Next, to confirm the applicability of the top performing Conditions (Thermo 4 and 6) at a larger scale, the MaxCyte system was compared to the CTS Xenon system using the SingleShot electroporation chambers which can accommodate up to lOOxlO6cells. Simultaneously, the impact of post-electroporation seeding concentration on expansion was tested. Thermo 6 outperformed both the MaxCyte control and Thermo 4 in terms of expansion, which was consistent with the results observed in the Neon™Nxt. However, Thermo 6 performed the worst in terms of gene editing efficiency with 11.6% residual CD38+ NK cells, followed by Thermo 4 with 4.6% and MaxCyte with 0.86% residual CD38+ NK cells (FIG. 5 average values). Across all conditions, seeding cells at 0.5 x lO6rather than I xlO6NK cells resulted in improved post-electroporation expansion without impacting gene editing efficiency. Taken together, these results demonstrate scalability of the MaxCyte and Thermo Fisher electroporation systems and the importance of electroporation parameters for successful gene- edited NK cell therapy platforms.

[0062] To ensure the feasibility of the NK knockout manufacturing process, we tested the optimized MaxCyte electroporation protocol at clinical scale. Compared to a control sample which comprised 99.43% CD38+ NK cells by the end of the process, the large-scale electroporation with CasX mRNA and CD38 specific gRNA followed by expansion resulted in only 2.17% CD38+ NK cells (FIG. 6). Cells were harvested on day 14 when the population doubling time (PDT) reached 48.38 hours. Importantly, the process yielded 162 billion NK cells, a 3000-fold increase.

[0063] Following harvest and cryopreservation, the NK cell population obtained exhibited expression of classic NK cell functional markers including CD 16, NKG2D, and NKp46 (FIG. 7A). Furthermore, the NK cell population also exhibited cytotoxicity against K562 cells (FIG. 7B). Taken together, these results demonstrated the success of a clinical-scale platform capable of producing a functional, genetically modified NK cell therapy.Example 4: Modifications to cell therapy are modular and can be combined

[0064] Having established GMP-compliant ex vivo primary NK cell expansion platforms amenable to both cytokine conditioning and gene editing, the feasibility of combining modifications was tested. Customization of genetic and non-genetic modifications will allow the production of NK cell therapies for a broader array of indications across oncology and autoimmunitv.Attorney Docket No. :0171.0107-PCT

[0065] Primary NK cells (prepared as described in the Research Scale example, were treated with TGF|3 throughout the expansion process and underwent gene editing on day 5 of the process. Interestingly, the addition of TGF|3 improved expansion of the control, non-edited cells and the cells subjected to CD38 knockout conditions (CD38KO NK) (FIG. 8 A). TGF(3 treatment was associated with lower CD38 expression in the non-edited control (FIG. 8B). Furthermore, gene editing efficiency was performed successfully with or without TGF(3 treatment (FIG. 8B).

[0066] Finally, TGF(3 conditioning and gene editing modifications were combined at manufacturing scale, resulting in the production of 558 billion NK cells (FIG. 9A), a 5580-fold increase. The PDT remained below 20 hours for the first three days of wave bioreactor expansion, and slowly increased to 41.95 hours on the seventh day of bioreactor expansion (FIG. 9B). By the end of the process, 3.8% of TGF|3-conditioned, gene-edited NK cells expressed CD38, compared to 64.6% in the TGFP -conditioned, non-edited control NK cells (FIG. 9C). Expression of surface receptors including CD 16, NKG2D, and NKp46 varied between the non-electroporated control and the manufacturing-scale CD38 knockout NK cells, but cytotoxic functionality of the TGFP -conditioned, gene-edited NK cells performed as expected. This experiment demonstrates the adaptability of the manufacturing platform provided herein and the feasibility of creating customizable production methods using a modular approach to manufacturing.Example 5: Research Scale

[0067] On day 0 of the process CD3 depleted PBMCs were obtained from the Prodigy7(see Manufacturing-scale CD38KO NK expansion methods, PBMC processing and CD3+ cell depletion) or from cryopreserved stocks and counted by flow cytometry (see Analytical testing methods, MTH0038). Cells were cultured in T-flasks at 0.3X106NK cells / mL in cSCGM supplemented with 100 lU / rnL of IL-2 (Cytiva Europe, 29062790), lO ng / mL of MACS® GMP Recombinant Human IL-12 (Miltenyi, 170-076-173). 100 ng / mL of MACS® GMP Recombinant Human IL-15 (Miltenyi. 170-076-114), and 50 ng / mL of MACS® GMP Recombinant Human IL-18 (Miltenyi, 170-076-183). At the time of seeding, cells were stimulated with 0.833 mg of PM21 per 1X1O6NK cells.

[0068] On day 4 of the process, an 80% volumetric medium exchange was performed using cSCGM supplemented with 100 lU / mL IL-2.

[0069] On day 5. cells were counted by flow cytometry. In preparation for electroporation, cells were washed with 10 mL HBSS. Cells were electroporated at 30 - 50X106NK cells / mL in finalAttorney Docket No. :0171.0107-PCT volume of 100 pL with 1 pg of CasX mRNA and 0.379 jj.g of CD38 gRNA per 1X106NK cells. Electroporation was performed using the GTxTM NK2 program (Maxcyte) or the ThermoFisher Neon (various programs tested). Following electroporation, cells were diluted to a concentration of 0.5X106NK cells / mL in cSCGM supplemented with IL-2.

[0070] On day 6, cells were counted by flow cytometry, then a 50% volumetric medium exchange was performed using cSCGM supplemented with 100 lU / mL IL-2.

[0071] On day 7, cells were counted by flow cytometry, then re-stimulated with PM21 according to the following parameters: 3X106NK cells / mL in cSCGM supplemented with 100 lU / mL IL-2, with 0.4 mg / mL PM21 for 6 hours at 37°C without motion. Following stimulation, cells were diluted to 0.42X106NK cells / mL in RPMI-1640 (ThermoFisher, 11875-101) supplemented with 10% Heat-inactivated Fetal Bovine Serum, 1% GlutaMAX™, (ThermoFisher, 35050-061), 1% Glucose (ThermoFisher, A2494001), 0.8% KnockOut™ Serum (ThermoFisher, 10828-010) (cRPMI) and 100 lU / mL IL-2. Cells were incubated at 37°C on a rocker. Rock rate and angle was set according to culture volume: 8 rpm / 8° for 3-15 mL, 10 rpm / 10ofor 15.1-25 mL, and 12 rpm / 12° for 25.1-40 mL.

[0072] On days 8-11 or 8-12, cells were counted by flow cytometry and diluted to 0.42X106using cRPMI supplemented with 100 lU / mL IL-2 until a maximum volume of 40 mL was reached. At maximum volume. 100% volumetric medium exchanges were performed.

[0073] On day 12 or 13, cells were counted by flow cytometry, and cytotoxicity, surface marker, and characterization flow cytometry panels were completed. Remaining cells were cryopreserved at 50X106NK cells / mL in 1: 1 solution of CryoStor®10 (BioLife Solutions, 210102) and Plasma-Lyte A (NDC, 0338-0221) supplemented with 1% Human Serum Albumin (HSA, 0.5% final).

[0074] In some experiments, cells were also treated with 10 ng / mL TGFP starting from day 0 and throughout the process concurrently with all IL-2 treatments.Example 6: Pharmaceutical Product I Manufacturing MethodPharmaceutical Preparation: PBMC processing and CD3+ cell depletion

[0075] PBMCs can be obtained as cryopreserved peripheral leukapheresis donations from healthy individuals from a suitable blood banking facility such as Oklahoma Blood Institute. The CliniMACS® Prodigy Automated Cell Processing System (Miltenyi, 200-075-301) was used for clinical-scale expansion of NK cells.Attomey Docket No. :0171.0107-PCT

[0076] On day 0. cryopreserved leukapheresis packs were thawed using the blood protocol on a Plasmatherm (Barkey, 00000448). For every 3X109nucleated cells, thawed material was diluted with 70 mL of 37°C CliniMACS® PBS / EDTA (Miltenyi Biotec, 130-070-525) supplemented with Human Serum Albumin (HSA) to 1% (Octapharma). Diluted starting material was passed through a Pall Blood Transfusion Filter (Fisher, NC9567012) and loaded into the Prodigy Centricult Unit (CCU) of a TS520 tubing set (Miltenyi Biotec, 170-076-600). PBMCs were then further washed with PBS / EDTA buffer supplemented with HSA to 0.5% and centrifuged to remove platelet content. When using cryopreserved leukapheresis samples, density gradient separation (DGS) can be omitted. If DGS is omitted, cells are counted after passing the material through the Pall Blood Transfusion Filter, otherwise were counted following the DGS procedure. Total cell counts were determined by flow cytometry.

[0077] Cells in the CCU were blocked by addition of Intravenous Immunoglobulin (IVIG) (Octapharma AG, 5600967). Cells were then incubated with anti-CD3 iron-conjugated microbeads (Miltenyi, 130-050-101) for 30 minutes. Post-incubation, cellular material was sequentially passed over the magnetic column 600X106CD3+ cells per round. The total number of column passes was determined by the in-process analysis described below (see section titled “Analytical testing methods’'). During CD3 depletion, PBMCs were transferred to an external cell bag on the TS520 tubing set. The column and CCU were washed with sterile water (S. A.L.F. S.P.A, ACQPVF10AIC030649305) followed by PBS / EDTA / HSA buffer to deplete any remaining T cells. The CD3+ cell depleted PBMCs were passed over the column a second time to further deplete CD3+ cells.Culturing of NK cells and CD38 gene editing

[0078] CD3-depleted PBMCs were exchanged out of buffer and into completed GMP SCGM Media (Cellgenix GMBH, 20806-0500) in the Prodigy CCU. Completed SCGM (cSCGM) is made by supplementing heat-inactivated, gamma-irradiated Fetal Bovine Serum (FBS) (Life Technologies Italia, 01190015 M) to 10%, GlutaMAX™ (Fisher. 35050-061) to 1% and adding IL-2 (Cytiva Europe, 29062790) to 100 lU / mL. The cells were then eluted from the Prodigy and a sample was taken for flow cytometry7analysis described previously. Between 60x106and 150xl06NK cells were loaded into a TS620 tubing set (Miltenyi, 170-076-614). NK cells were stimulated by addition of 0.8 - 1.0 mg PM21 particles (produced internally, as described in W02014 / 005072) per IxlO6NK cells for 15 minutes in 100 mL. After the PM21 stimulation, the culture was supplemented with 10 ng / mL TGF|3 (Miltenyi Biotec, 170-076-166, 170-076-Atorney Docket No. :0171.0107-PCT167, 170-076-168). 10 ng / mL IL- 12 (Miltenyi. 170-076-173), 100 ng / mL IL- 15 (Miltenyi, 170- 076-1 14) and 50 ng / mL IL-18 (Miltenyi, 170-076-183) according to the final culture volume, which was calculated to reach a cell concentration of 0.3xl06NK cells / mL in cSCGM. The cell culture was maintained at 37°C and 5% CO2.

[0079] On day 4 after thaw, an 80% media exchange was performed using cSCGM supplemented with 100 lU / mL IL-2 and 10 ng / mL TGF(3. Culture conditions were maintained at 37 °C and 5% CO2 with agitation: 75 rpm spinning for 5 seconds every 5 minutes.

[0080] On day 5 after thaw, cells were eluted from the Prodigy and counted via flow cytometry7. Cells were transferred to a 250 mL conical centrifuge tube (Coming) and pelleted at 300 x g for 10 minutes. Supernatant was removed and cells were washed with 50-100 mL of HBSS (Gibco. ThermoFisher, 14025092), then pelleted again. Supernatant was removed and cells were resuspended at 50xl06NK cells / mL in electroporation buffer (MaxCyte, EPB-1) with 1 pg of CRISPR-CasX endonuclease mRNA and 0.379 pg of CD38-specific gRNA per 1x106NK cells. Cells were then transferred to a CL- 1.1 RUO processing assembly (MaxCyte) and electroporated using the GTx NK2 program (Maxcyte). Following payload delivery7, cells were diluted in 50 mL of cSCGM, transferred into a new TS520 or TS620 tubing set and cultured at a final concentration of 0.5xl06NK cells / mL in cSCGM supplemented with 100 lU / mL IL-2 and 10 ng / mL TGFfl in the CliniMACS® Prodigy. Culture conditions were maintained at 37 °C and 5% CO2 with agitation: 75 rpm spinning for 5 seconds every 5 minutes.

[0081] On day 6, a sample was obtained for flow cytometry, then 50% media exchange was performed with cSCGM supplemented with 100 lU / mL IL-2 and 10 ng / mL TGFff Culture conditions were maintained at 37 °C and 5% CO2 with agitation: 75 rpm spinning for 5 seconds every 5 minutes. Next, a 10 L Flexsafe® RM bag (Sartorius Stedim Biotech. DFC050L — 05SP) was installed on a Sartorius Biostat wave bioreactor. IL of RPMI-1640 (Life Technologies Italia, 11875176) supplemented with FBS to 10%, GlutaMAX™ (Fisher, 35050-061) to 1%, Glucose (Fisher, A2494001) to 1%, and KnockOut™ Serum (ThermoFisher, 10828010) to 0.8% was equilibrated overnight to 50% dissolved O2, and 37°C, rocking at 8 rpm and a 4° angle.

[0082] On day 7, a sample was collected from the Prodigy and analyzed via flow cytometry to determine cell count. The cells were then stimulated by addition of 40 mg PM21 particles for every 30xl06NK cells seeded on day 0 in 100 mL of cSCGM supplemented with 100 lU / mL IL-2 and 10 ng / mL TGF(3. After 15 minutes elapsed, the culture volume was raised to maximumAttorney Docket No. :0171.0107-PCT volume of 500 mL with cSCGM. Cells were further incubated with PM21 for 6-7 hours while being maintained at 37°C, 5% CO2, with agitation.Seeding and culturing of NK cells (post-electroporation) in Wave bioreactor

[0083] Following stimulation on day 7, cells were eluted from the Prodigy CCU and counted via flow cytometry. The entire elution volume was seeded into the Sartorius wave reactor that was previously installed and equilibrated on day 6. A post-seed sample was taken to determine NK cell count. The wave culture volume was adjusted with additional cRPMI to reach an NK cell concentration of 4.2xl05NK cells / mL. The final culture was supplemented with 100 lU / mL IL-2 and 10 ng / mL TGF0.

[0084] On day 8, a sample was collected and analyzed by flow cytometry to determine NK cell count. If necessary, cRPMI was added to maintain the cell concentration at 4.2x105NK cells / mL. The culture as supplemented with 100 lU / mL IL-2 and 10 ng / mL TGF0 according to the final culture volume. Wave reactor parameters such as rock rate, and angle change according to cell density and culture volume and are outlined in Table 5 below. Once a maximum working volume of 5L was reached, perfusion was initiated at one vessel volume per day (VVD). Once perfusion was initiated, both the reactor culture and the perfusion medium were supplemented with 100 lU / mL IL-2 and 10 ng / mL TGF0.TABLE 5: 10L Wave Bioreactor Fed-batch and Perfusion Scheme

[0085] On day 8. a 50 L Flexsafe® RM bag (Sartorius Stedim Biotech, DFC050L — 05SP) was installed on a Sartorius wave bioreactor. 5L of RPMI-1 40 (Life Technologies Italia, 11875176) supplemented with 10% gamma-irradiated heat-inactivated FBS, 1% GlutaMAX™ (Fisher, 35050-061), 1% Glucose (Fisher, A2494001), and 0.8% KnockOut™ Serum (ThermoFisher, 10828010) was equilibrated overnight to 50% O2, and 37°C, rocking at 8 rpm and a 4° angle.Attorney Docket No. :0171.0107-PCT

[0086] On day 9. a sample was collected and analyzed by flow cytometry to determine NK cell count. The 50L wave bag volume was raised to 11 L with additional cRPMI, and then the entire 5L wave reactor culture was transferred to the 50L bag. After transfer, the culture volume was increased to 18L with additional cRPMI, and the final culture was supplemented with 100 lU / mL IL-2 and 10 ng / mL TGF0. Perfusion was initiated as described in Table 6 below using cRPMI supplemented with 100 lU / mL IL-2 and 10 ng / mL TGFp.Table 6: 50L Wave Bioreactor Perfusion scheme

[0087] On subsequent days, a sample was collected and analyzed by flow cytometry to determine NK cell count. Perfusion was increased stepwise by 0.5 VVD each day up to a maximum perfusion rate of 2.5 VVD until harvest as triggered when population doubling time (PDT) reached greater than 40 hours.NK Cell Harvesting and Drug Product Fill & Finish

[0088] To harvest, cells were washed and concentrated with a LOVO Cell Processing Kit (Fresenius Kabi, X6R4909A) and a LOVO Cell Processing system (Fresenius Kabi, 6R4900). During concentration, NK cells were exchanged into Plasma-Lyte A (NDC, 0338-0221) supplemented with 1% HSA (Octapharma) and chilled to 4 °C. The final cell population was sampled and analyzed by flow cytometry' before dilution to lOOxlO6NK cells / mL with Plasma- Lyte / HSA buffer. Drug product was finalized by then combining 1 : 1 with CryoStor®10 (BioLife Solutions, 210102) to achieve a final concentration of 50x106NK cells / mL. Drug product (formulated NK cell population) was filled into CryoStore bags (OriGen Biomedical Inc, CS50S and CS50N) and placed into a Planer Kryosave controlled rate freezer (Integra) for cryopreservation. Cryopreserved drug products can be stored in liquid nitrogen indefinitely.Analytical testing methodsIn-process Monitoring of NK Cell Growth (ATM2629)Attorney Docket No. :0171.0107-PCT

[0089] Samples were blocked with Human TruStain FcX™ (Biolegend. 422302) and then immunostained with antibodies for CD56 (Biolegend, 362508, Clone 5.1H1 1) and CD3 (Biolegend, 344812, Clone SK7). Samples were then stained with DRAQ7 (Abeam, abl09202) and acquired in Cell staining buffer (Biolegend, 420201) on a BD FACSLyric™ flow cytometer (BD Biosciences, 3 laser 12 color) in TruCount™ tubes (BD Biosciences, 340334). Isotype control samples were also prepared using the following antibodies: APC Mouse IgGl, K Isotype (Biolegend, 400120) and PE Mouse IgGl, K Isotype (Biolegend, 400112).In-process Monitoring of CD38 Gene-edited NK Cell Growth and Viability (MTH0038)

[0090] The same process was followed as described in "‘In-process monitoring of NK cell growth” with one modification: CD38 (Biolegend, 303504, Clone HIT2) was added to the stained sample and IgGl, K FITC (Biolegend, 400108) was added to the isotype control.

[0091] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the present disclosure and may be practiced within the scope of the appended claims. For example, all constructs, methods, and / or component features, steps, elements, or other aspects thereof can be used in various combinations.

[0092] While there have been shown and described what are at present considered the preferred embodiments of the invention, those skilled in the art may make various changes and modifications which remain within the scope of the appended claims.

[0093] Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would beAttorney Docket No. :0171.0107-PCT evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the present disclosure can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.

[0094] Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0095] All patents, patent applications, websites, other publications or documents, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.

Claims

Attorney Docket No. :0171.0107-PCTWE CLAIM:

1. A population of Natural Killer (NK) cells wherein at least 90% of the NK cells have reduced CD38 expression compared to NK cells isolated from human Peripheral Blood Mononuclear cells (PBMCs) from healthy volunteers, wherein the population of cells was obtained from human PBMCs, and wherein the CD38 gene in the at least 95% of NK cells has a disruption in exon 1.

2. The population of NK cells of claim 1, wherein the population comprises at least 90% NK cells.

3. The population of NK cells of claim 1 or 2, wherein least 90 % of the NK cells have reduced CD38 expression.

4. The population of NK cells of any one of claims 1-3, wherein 95% of the NK cells are viable.

5. The population of NK cells of any one of claims 1-4, wherein the cells were obtained from previously frozen human PBMCs.

6. The population of cells of any one of claims 1-5, wherein the disruption of the CD38 gene is between within exon 1 of human genomic sequence of human CD38.

7. A method of producing a population of Natural Killer (NK) cells that have reduced CD38 expression comprising: culturing a population of CD3+ depleted human Peripheral Blood Mononuclear Cells (PBMCs), a transfecting the population of CD3+ depleted human Peripheral Blood Mononuclear Cells (PBMCs) with mRNA encoding CasX and a gRNA directed to CD38, wherein the transfection is by electroporation, such that the CD38 gene in the NK cells is disrupted, and expanding the transfected CD3+ depleted PBMCs. thereby producing a population of NK cells that have reduced CD38 expression.Attorney Docket No. :0171.0107-PCT8. The method of claim 7, wherein the population of NK cells has one or more of the following attributes: the population of NK cells comprises at least 90% NK cells, or at least 90 % of the cells in the population of NK cells have reduced CD38 expression, or95% of the cells in the population of NK cells are viable, or the disruption of the CD38 gene is within exon 1 of genomic sequence of human CD38.

9. The method of claim 7 or 8, wherein the PBMCs were not subjected density gradient separation prior to electroporation.

10. The method of any one of claims 7-9, wherein the cells are stimulated with PM21 particles on day 0 of culturing the sample.

11. The method of any one of claims 7-10, wherein prior to electroporation, the cells are washed.

12. The method of any one of claims 7-11, wherein the CasX mRNA comprises a nucleotide sequence of SEQ ID NO: 54.

13. The method of any one of claims 7-12. wherein the gRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos: 4-6, 12, 20, and 40.

14. The method of any one of claims 7-13, wherein during electroporation, the mRNA is at a concentration of 0. 1-5.0 pg CasX mRNA per IxlO6cells.

15. The method of any one of claims 7-14, wherein during electroporation, the gRNA is at a concentration of 0.15-4.00 pg gRNA per 1x106cells.

16. The method of any one of claims 7-15. wherein during electroporation, the NK cells are a concentration of 30-50 xlO6viable cells / mL.Atorney Docket No. :0171.0107-PCT17. The method of any one of claims 7-16, wherein the cells are stimulated with PM21 particles 2 days after electroporation.

18. The method of any one of claims 7-17. wherein the cells are expanded to 3000-6000 fold more cells compared to the number of electroporated cells.

19. A population of Natural Killer (NK) cells that have reduced CD38 expression, said cells produced by a method comprising: culturing a sample of human Peripheral Blood Mononuclear Cells (PBMCs), depleting the thawed PBMCs of CD3 positive (CD3+) cells, transfecting the CD3 depleted PBMCs with mRNA encoding CasX and a gRNA directed to CD38, wherein the transfection is by electroporation, such that the CD38 gene in the NK cells is disrupted, and expanding the transfected CD3+ depleted PBMCs, thereby producing a population of NK cells that have reduced CD38 expression.

20. The population of NK cells of claim 19, having one or more of the following attributes: the population comprises at least 90% NK cells, or at least 90 % of the NK cells have reduced CD38 expression, or95% of the NK cells are viable, or the disruption of the CD38 gene is in exon 1 of genomic sequence of human CD38.

21. The population of NK cells of claim 19, wherein the PBMCs were not subjected density gradient separation prior to electroporation, and / or the cells are stimulated with PM21 particles on day 0 after culturing the sample, and / or prior to electroporation, the cells are washed, and / or the CasX mRNA comprises a nucleotide sequence of SEQ ID NO: 54, and / or the gRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos: 4-6, 12, 20, and 40, and / or during electroporation, the mRNA is at a concentration of 0. 1 - 5.0 pg CasX mRNA per 1x106cells, and / orAttorney Docket No. :0171.0107-PCT electroporation, the gRNA is at a concentration of 0. 15-4.00 pg gRNA per IxlO6cells, and / or during electroporation, the NK cells are a concentration of 30-50x106viable cells / mL, and / or the cells are stimulated with PM21 particles on day 7 after culturing the sample.

22. A method for treating a hematological cancer comprising: administering to a patient in need thereof a population of Natural Killer (NK) cells that have reduced CD38 expression, wherein the population of NK cells was obtained from human Peripheral Blood Mononuclear Cells (PBMCs) and subjected to gene editing, and the CD38 gene in the NK cells has a disruption in exon 1 compared to parental NK cells prior to gene editing.

23. The method of claim 22, wherein the population of NK cells comprises at least 90% NK cells, and / or at least 90 % of the NK cells have reduced CD38 expression, and / or95% of the NK cells are viable, and / or the disruption of the CD38 gene is exon 1 of genomic sequence of human CD38, and / or the PBMCs were not subjected density gradient separation prior to electroporation.

24. A pharmaceutical composition comprising: a population of Natural Killer (NK) cells that have reduced CD38 expression. Plasma-Lyte A, and Human Serum Albumin (HSA), wherein the population of NK cells was obtained from human Peripheral Blood Mononuclear Cells (PBMCs), and subjected to gene editing, the CD38 gene in at least 90% of the NK cells have a disruption in exon 1 compared to parental NK cells prior to exposure to the gene editing.

25. The pharmaceutical composition of claim 24 wherein the population of NK cells has one or more of the following attributes: the population of cells comprises at least 90% NK cells, and / or at least 90% of the NK cells have reduced CD38 expression, and / orAttorney Docket No. :0171.0107-PCT95% of the NK cells are viable, and / or the cells were obtained from previously frozen human PBMCs, and / or the disruption of the CD38 gene is in exon 1 of genomic sequence of human CD38.

26. A method of producing a pharmaceutical composition comprising a population of Natural Killer (NK) cells that have reduced CD38 expression, comprising: culturing a sample of human Peripheral Blood Mononuclear Cells (PBMCs), depleting the thawed PBMCs of CD3 positive (CD3+) cells, transfecting the CD3+ depleted PBMCs with mRNA encoding CasX and a gRNA directed to CD38, wherein the transfection is by electroporation, such that the CD38 gene in the NK cells is disrupted, expanding the transfected CD3+ depleted cells, and formulating the transfected population of cells by transferring the cells to Plasma-Lyte A containing Human Serum Albumin (HSA), thereby producing a pharmaceutical composition comprising a population of NK cells having reduced CD38 expression.

27. The method of claim 26, wherein the population of cells comprises at least 90% NK cells, and / or at least 90 % of the NK cells have reduced CD38 expression, and / or95% of the NK cells are viable, and / or the disruption of the CD38 gene is in exon 1 of genomic sequence of human CD38.

28. The method of claim 26 or 27, wherein: the PBMCs were not subjected density gradient separation prior to electroporation, and / or the cells are stimulated with PM21 particles on day 0 after culturing the sample, and / or prior to electroporation, the cells are washed, and / or the CasX mRNA comprises a nucleotide sequence selected of SEQ ID NO: 54, and / or the gRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos: 4-6, 12, 20, and 40, and / or during electroporation, the mRNA is at a concentration of 0.1 -5.0 pg CasX mRNA per IxlO6cells, and / or electroporation, the gRNA is at a concentration of 0. 15-4.00 pg gRNA per IxlO6cells,Attorney Docket No. :0171.0107-PCT and / or during electroporation, the NK cells are a concentration of 30-50x106viable cells / mL, and / or the cells are stimulated with PM21 particles 2 days after transfection (day 7 after thawing).

29. A method for treating a hematological cancer comprising administering to a patient in need thereof a pharmaceutical composition comprising a population of NK cells having reduced CD38 expression, wherein the population of NK cells was obtained from human Peripheral Blood Mononuclear Cells (PBMCs) and subjected to gene editing, and the CD38 gene in the population of NK cells has a disruption in exon 1 compared to parental NK cells prior to being subjected to the gene editing.

30. The method of claim 29, wherein the population of cells comprises at least 90% NK cells, and / or at least 90 % of the NK cells have reduced CD38 expression, and / or95% of the NK cells are viable, and / or the cells were obtained from previously frozen human PBMCs, and / or the disruption of the CD38 gene is exon 1 of genomic sequence of human CD38.

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