Methods of enhancing plasma membrane particle functionality and uses thereof

Abstract

Provided herein are methods of producing temperature shifted PM21 plasma membrane particles, compositions comprising natural killer cells expanded by contact with temperature shifted PM21 particles, and uses thereof.

Description

[0001] METHODS OF ENHANCING PLASMA MEMBRANE PARTICLE FUNCTIONALITY AND USES THEREOF

[0002] TECHNICAL FIELD

[0003] [1] The disclosure relates to methods of producing plasma membrane particles having enhanced potency in the expansion of immune cells, as well as uses of the resultant immune cells, particularly expanded natural killer cells, in the treatment of cancer and infectious diseases.

[0004] BACKGROUND

[0005] [2] Natural killer (NK) cells are immune effector cells that are promising tools being developed by clinicians and researchers for use in the immunotherapy of cancer and various infectious diseases. Immunomodulatory regimes typically exhibit fewer side effects and adverse events than are observed with small molecule drugs, and they are less prone to tumor escape and drug resistance than traditional chemotherapeutics. In immunotherapy, cells derived from the human immune system are harnessed to target abnormal antigens expressed on target cells in patients. Adoptive cell transfer, a form of immunotherapy, involves the transfer of cells into a subject / patient and has shown promise for use in the treatment of diseases such as cancer. The cells may be from the patient (autologous) or from another individual (allogenic). For example, autologous immune enhancement therapy centers around removing a sample of a subject’s peripheral blood from which immune cells - such as cytotoxic T lymphocytes, natural killer (NK) cells, and the like - are obtained. The immune cells are expanded in vitro or ex vivo to achieve therapeutic levels of these cells, which then may be reinfused in the subject to trigger an immune response against targeted cells, such as cancer cells. These immune cells may be modified prior to reintroduction into a subject, such as genetically engineering them to express a chimeric antigen receptor or other feature to improve an immune response against cancer cells.

[0006] [3] One key component of preparing human cells for immunotherapy is expansion of the cells to generate a sufficient number to have a therapeutic effect in a subject. One known way to induce immune cell growth is to contact immune cells, such as NK cells, in vitro or ex vivo with feeder cells that express agents that stimulate NK cell replication. Growth promoting agents useful in initiating NK cell expansion include IL-12, IL-15, IL-18, IL-21, 4-1BBL, ICAM-1, 2B4, BCM1 / SLAMF2, CD155, CD112, CCR7, DAP12, and DAP10, which agents may be expressed alone or in combination in the feeder cells. The feeder cells may be derived from a cancer cell line, such as K562 cells. One known K562 cell line is K562-mbl5-41BBL, which is engineered to express membrane-bound IL- 15 and 4-1BBL. Another exemplary feeder cell line is K562-mb21-41BBL, a leukemia cell line engineered to express membrane-bound IL-21 and 4- 1BBL. NK cells contacted with K562-mb21-41 BBL feeder cells in vitro or ex vivo undergo cell replication in culture. However, NK cell products generated by the expansion of NK cells in the presence of feeder cells are at risk of contamination from the feeder cells despite end stage purification via various modalities. This limits the clinical desirability of immunotherapies employing NK cell products generated by contacting NK cells directly with feeder cells.

[0007] [4] Plasma membranes derived from feeder cells have proven to be an effective solution to this potential for feeder cell contamination immune cell products. Plasma membranes may be created from feeder cells expressing immune cell growth stimulating agents by known methods, such as those disclosed in WO 2014 / 005072. Plasma membranes produced from such feeder cells elicit immune cell replication upon co-culture in vitro, but the resulting populations of expanded immune cells are not at risk of contamination by cancerous feeder cells in the process. Plasma membranes generated from K562-mb21 feeder cells contain membrane-bound IL-21 and are denoted “PM21 particles.” Such PM21 particles may also contain one or more additional immune cell stimulatory agents, such as 4-1BBL, which arises when plasma membrane particles are derived from K562-mb21-41BBL feeder cells. PM21 particles containing membrane-bound IL-21 and membrane-bound 4-1BBL are a key reagent in the commercial production of natural killer (NK) cells for immunotherapy under regulatory compliant processes. Unfortunately, the generation of PM21 particles can be labor intensive, time consuming, and expensive, which are drawbacks to using them in manufacturing processes at commercial scale. Consequently, a need exists to improve the functionality of plasma membrane particles for expansion of immune cell populations to be formulated into cell products for the treatment of cancer and infectious diseases, and to efficiently and consistently manufacture these cell products at commercial scale.

[0008] SUMMARY

[0009] [5] PM21 particles, cell-free suspensions of plasma membrane-derived membrane particles, are used to stimulate the in vitro or ex vivo expansion of NK (Natural Killer) cells during the process of manufacturing NK cell products for therapeutic purposes. PM21 particles are prepared from lymphoblast cells isolated from a leukemia patient, specifically K562 cells. The PM21 particles can be generated by rupturing the plasma membrane of engineered K562 parental cells which were genetically modified to express 4-1BBL and membrane bound IL-21 (mb-IL21). 4- 1BBL is a type II protein of the Tumor Necrosis Factor (TNF) superfamily that exists as a trimeric transmembrane glycoprotein on antigen presenting cells. Binding of 4-1BBL with its receptor (4- IBB) on surface of activated immune cells (such as monocytes, B cells, NK cells, and neutrophils) delivers a costimulatory signal that promotes immune cell survival, expansion, and differentiation.

[0010] [6] The PM21 manufacturing process starts with the expansion of feeder cells genetically modified to express 4-1BBL and membrane bound IL-21. For this, a working cell bank vial of K562-mb21-41BBL was thawed, and the cells were serially expanded in shaker flasks under controlled conditions. The final cell expansion step was performed in a bioreactor, after which the cells were harvested, washed, and resuspended in homogenization buffer. Next, the feeder cell suspension was transferred into a Parr® cell disruption vessel which disrupts the plasma membranes of cells using nitrogen cavitation. The filled cavitation vessel was pressurized with nitrogen gas. The cell mixture was released slowly to lyse the feeder cells, resulting in the formation of the PM21 particles having membrane-bound IL-21 and transmembrane 4-1BBL within a homogenate.

[0011] [7] The homogenate was then centrifuged to remove large debris, followed by purification by three sequential ultracentrifugation steps. After the final step, the purified PM21 particles were resuspended and filled into vials, which were then frozen at < -65 °C, sterilized by electron beam (E-beam) irradiation, and stored for future use in the NK cell expansion process.

[0012] [8] Upon attempting to scale this process to a commercially effective production level of at least 100L, process engineers discovered that the levels of IL-21 expressed in the final PM21 cell products decreased from that observed at lower production volumes (e.g., 17L). An investigation into the root cause of this problem led to an innovative improvement to this process concerning the cell expansion step as performed in a stirred tank bioreactor operated in perfusion mode. Important process parameters, such as temperature, pH, and dissolved oxygen, are typically controlled at predetermined setpoints. During the 8-day expansion process, the feeder cell concentration increases from around 0.3xl06cells / ml to above 15xl06cells / ml. To determine possible sources of cell culture parameters responsible for the reduction of membrane-bound IL- 21 at commercial scale production levels, a test run was performed to vary culture conditions. Normally, feeder cell culture processes are performed at 37°C. Here it was surprisingly found that by reducing the culture temperature from 37°C to 32°C on day 6 or day 7 post inoculation of the stirred tank (STR) bioreactor, the amount of mb-IL21 and 4-1BBL proteins expressed on the purified PM21 particles were sharply increased over standard (constant 37°C) control conditions. In addition, the “improved” temperature shifted PM21 particles were also found to have enhanced potency, meaning fewer PM21 particles were needed during NK-cell stimulation steps to obtain the same NK-cell expansion as was achieved with PM21 particles derived from feeder cells cultured at a steady temperature of 37°C. Changes in other cell culture parameters did not affect the potency of PM21 particles derived from K562-mb21-41BBL feeder cells.

[0013] [9] The above-noted improvement is suitable for the manufacture of NK cell “drug” products (e.g. NK cells suitable for immunotherapy) in an effective variation of the standard workflow, eliminating the need for large amounts of PM21 particles when scaling to 100L - 200L culture volumes. The usefulness of this advancement lies in the increased potency of the innovative PM21 particles. Because three times fewer “higher” potency PM21 particles are needed to expand NK cells during commercial production than in the typical process, a decrease in the cost of goods sold (COGS) for final NK cell drug products can be realized due to the reduction in the amount of PM21 particles required to make each lot. By reducing the concentration of PM21 particles needed in a product manufacturing workflow, NK cell expansion from apheresis samples may be more effectively scalable in commercial production processes.

[0014]

[0010] In one embodiment, the disclosure provides a method of enhancing potency of plasma membrane particles comprising expanding feeder cells comprising membrane-bound IL-21 and 4-1BBL in a cell culture. The feeder cells are cultured at 37°C during Day 0 through Day 5 of expansion and thereafter cultured at 32°C from Day 6 until harvested. The expanded feeder cells are harvested when feeder cell concentration exceeds a threshold set point of cell viability and / or cell density. The expanded feeder cells are lysed in a pressurized vessel to form temperature enhanced plasma membrane particles containing IL-21 and 4-1BBL (referred to herein as PM21 particles). The temperature shifted PM21 particles exhibit a higher potency for NK cell proliferation than PM21 particles purified from an equivalent number of feeder cells expanded at a constant culture temperature of 37°C.

[0015]

[0011] In some aspects, disclosed is a method of the above-noted embodiment, wherein the temperature shifted PM21 particles contain higher levels of IL-21 and 4-1BBL than do PM21 particles purified from an equivalent number of feeder cells expanded at a constant culture temperature of 37°C.

[0016]

[0012] In some aspects, disclosed is a method of the above-noted embodiments, wherein the temperature shifted PM21 particles exhibit an EC50 value of less than 40 pg PM21 parti cles / mL for NK cell proliferation.

[0017]

[0013] In some aspects, disclosed is a method of the above-noted embodiments, wherein the feeder cells are expanded in a bioreactor.

[0018]

[0014] In some aspects, disclosed is a method of the above-noted embodiments, wherein the feeder cells comprise K562-mb21-41BBL cells.

[0019]

[0015] In some aspects, disclosed is a method of the above-noted embodiments, wherein the expanded feeder cells are harvested at Day 8.

[0020]

[0016] In some aspects, disclosed is a method of the above-noted embodiments, wherein the expanded feeder cells are harvested when feeder cell concentration exceeds 18-20 xlO6viable cells / mL.

[0021]

[0017] In some aspects, disclosed is a method of the above-noted embodiments, wherein the expanded feeder cells are harvested when cell viability falls below 70% and cell density is greater than 13xl06viable cells / mL.

[0022]

[0018] In some aspects, disclosed is a method of the above-noted embodiments, wherein cell culturing continues to Day 9 and the expanded feeder cells are harvested at a cell density below 13xl06viable cells / mL.

[0023]

[0019] In some aspects, disclosed is a method of the above-noted embodiments, wherein the PM21 particles are purified using a sucrose density gradient.

[0024]

[0020] In another embodiment, the disclosure provides for a population of expanded natural killer cells produced by contact with the temperature shifted plasma membrane particles comprising membrane-bound IL-21 and 4-1BBL (PM21 particles) created by the method according to any one of the above-noted embodiments.

[0025]

[0021] In some aspects, disclosed is a population of NK cells of any one of the above-noted embodiments, wherein NK cell expansion or proliferation is stimulated by contacting the NK cells ex vivo or in vitro with temperature shifted PM21 particles.

[0026]

[0022] In some aspects, the disclosure provides for a population of expanded natural killer cells according to the above-noted embodiments, wherein the NK cells are exposed to temperature shifted PM21 particles multiple times.

[0027]

[0023] In some aspects, disclosed is a population of expanded natural killer cells according to the above embodiment, wherein the temperature shifted PM21 particles further comprise at least one additional cytokine.

[0028]

[0024] In some aspects, disclosed is a population of expanded natural killer cells according to the above embodiments, wherein cytotoxicity of the NK cells expanded by contact with temperature shifted plasma membrane particles is greater than the cytotoxicity of NK cells expanded by contact with plasma membrane particles derived from feeder cells cultured at a consistent temperature of 37°C.

[0029]

[0025] In another embodiment, the disclosure provides for a pharmaceutical composition formulated to comprise an effective amount of the expanded natural killer cells produced using temperature shifted PM21 particles created by a method of any one of the above embodiments and a pharmaceutically acceptable carrier.

[0030]

[0026] In some aspects, disclosed is a pharmaceutical composition of the above-noted embodiments, wherein the composition further comprises an anti-infective agent or an anticancer agent.

[0031]

[0027] In some aspects, disclosed is a pharmaceutical composition of the above-noted embodiments, wherein the anti-cancer agent is selected from an antibody, a chemotherapeutic drug, and an immunotherapeutic agent.

[0032]

[0028] In some aspects, disclosed is a pharmaceutical composition of the above-noted embodiments, wherein the anti-infective agent is selected from an antiviral agent, an antibacterial agent, and an antifungal agent.

[0029] In some aspects, disclosed is a method of treating an infection or cancer in a subject in need thereof comprising administering to the subject an effective amount of the pharmaceutical composition of the above-noted embodiments.

[0033]

[0030] In some aspects, disclosed is a pharmaceutical composition of the above-noted embodiments for use in the treatment of an infection or cancer in a subject.

[0034]

[0031] In some aspects disclosed is the use of a population of expanded natural killer cells of the above-noted embodiments in the manufacture of a medicament for treating an infection or cancer.

[0035]

[0032] In some aspects, disclosed is a kit comprising a pharmaceutical composition according to the above-noted embodiments and instructions for use according to an above-noted method.

[0036]

[0033] In some aspects, the disclosure provides a use of any of the above-noted embodiments, wherein the expanded NK cells are combined with a pharmaceutically acceptable carrier to generate a medicament comprising NK cells formulated for treating an infection or cancer.

[0037]

[0034] In some aspects, the disclosure provides for a kit comprising a pharmaceutical composition as described in any of the above-noted embodiments and instructions for use according to the methods of any of the above-noted embodiments.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039]

[0035] Figure 1 shows a schematic overview of the workflow steps involved in producing purified plasma membrane particles comprising membrane-bound IL-21 (PM21 particles) expressing 4-1BBL suitable for use in stimulating the growth of natural killer (NK) cells. Multiple process parameters were varied at the cell culture step of Figure 1 using 2L stirred tank reactors to assess which impact the amount of proteins present in the final purified PM21 particles. Details of the process parameter variations are set forth in Table 1.

[0040]

[0036] Figure 2 depicts in graph format the effect of temperature shifted PM21 particles on NK cell proliferation in comparison to standard PM21 particles generated from growing K562-mb21- 4-1BBL feeder cells at a consistent temperature of 37°C. Temperature shifted PM21 particles stimulated NK cells to reach maximal growth levels at lower concentrations than control (standard) PM21 particles. The leftward shift in the cellular growth curve for temperature shifted PM21 particles demonstrates increased potency of these particles in comparison to control PM21 particles.

[0041]

[0037] Figure 3A-B shows the membrane-bound IL-21 (Figure 3 A) and 4-1BBL (Figure 3B) protein content of PM21 particles purified from K562-mb21-41BBL feeder cells grown under control (consistently at 37°C) and temperature shifted (37°C reduced to 32°C) conditions. One test set of feeder cells was temperature shifted on Day 6 of culture, and a second test set of feeder cells was temperature shifted on Day 7 of culture.

[0042]

[0038] Figure 4A-B shows the functionality of PM21 particles for K562-mb21-41BBL feeder cells grown under various culture conditions. Only shifting the culture temperature from 37°C to at 32°C on Day 6, and to a lesser extent on Day 7, substantially improved the ability of PM21 particles to stimulate NK cell growth at lower PM21 particle concentrations.

[0043] DETAILED DESCRIPTION

[0044] DEFINITIONS

[0045]

[0039] Unless otherwise noted, the terms used herein have definitions as ordinarily used in the art. Some terms are defined below, and additional definitions can be found within the rest of the detailed description.

[0046]

[0040] The term “a” or “an” refers to one or more of that entity, i.e., can refer to plural referents. As such, the terms “a,” “an,” “one or more,” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0047]

[0041] Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.

[0048]

[0042] As used herein, the term “in some embodiments,” “in certain embodiments,” “in other embodiments,” “in some other embodiments,” or the like, refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.

[0049]

[0043] As used herein, the terms “treat,” “treating” or “treatment of’ (and grammatical variations thereof) mean that the severity of the subject's condition is reduced, at least partially improved or stabilized and / or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and / or there is a delay in the progression of the disease or disorder.

[0050]

[0044] As used herein, the terms “prevent,” “preventing” and “prevention” (and grammatical variations thereof) refer to prevention and / or delay of the onset of a disease, disorder and / or a clinical symptom(s) in a subject and / or a reduction in the severity of the onset of the disease, disorder and / or clinical symptom(s) relative to what would occur in the absence of the compositions and / or methods described herein. The prevention can be complete, e.g., the total absence of the disease, disorder and / or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and / or clinical symptom(s) in the subject and / or the severity of onset is less than what would occur in the absence of the compositions and / or methods described herein.

[0051]

[0045] As used herein, a “therapeutically effective amount” is the amount of a pharmaceutical composition provided herein that is effective to treat or prevent a disease or disorder in a subject or to ameliorate a sign or symptom thereof. The “therapeutically effective amount” may vary depending, for example, on the disease and / or symptoms of the disease, severity of the disease and / or symptoms of the disease or disorder, the age, weight, and / or health of the patient to be treated, and the judgment of the prescribing physician.

[0052]

[0046] As used herein, an “effective” amount generally means an amount which results in a desired effect in a given system. The effect may be achieved in an in vitro or ex vivo system, such as the stimulation of cytokine formation. Or the effect may be in vivo, such as an amount sufficient to effectuate a localized or systemic response.

[0053]

[0047] As used herein, the term “formulated” refers to the multistep process in which active substances, such as an active pharmaceutical ingredient, are combined with other components to produce a final medicinal product. The process takes into consideration various factors, including particle size, polymorphism, pH, solubility, and the like. Pharmaceutical formulations can result in compositions taking various dosage forms designed for particular routes of administration, such as oral, intravenous, intramuscular, transdermal, topical, and inhalational.

[0054]

[0048] As used herein, the term “drug product” refers to a finished dosage form that contains one or more active ingredients generally - but not necessarily - in combination with one or more inactive ingredients. The active ingredient may be a chemical or biological substance that is intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease. Drug products may be manufactured in many dosage forms, including tablets, capsules, aerosols, liquids, gels, lozenges, and patches.

[0055]

[0049] As used herein, the term “allogeneic” means material derived from individuals of the same species that are sufficiently unlike genetically to interact antigenically. Allogeneic therapies can increase the risk of eliciting an immune response within a recipient patient.

[0056]

[0050] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range- from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0057]

[0051] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of’), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.

[0058]

[0052] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0059]

[0053] Improving manufacturing processes for cell-based immunotherapies is a continuing goal for pharmaceutical and biotechnology companies. Herein is described a streamlined manufacturing process employing conditions under which, contrary to conventional thinking, feeder cells were shifted to suboptimal temperature of 32°C during culturing. This temperature shift enabled the generation of PM21 particles exhibiting enhanced potency over control PM21 particles derived from feeder cells grown at a consistent temperature of 37°C. This enhanced potency was unexpected and NK cells exposed to temperature shifted PM21 particles surprisingly led to achieving optimal NK cell growth using substantially fewer stimulatory PM21 particles in test manufacturing runs. Utilization of more potent PM21 particles confers benefits on supply chain logistics and reduces Cost of Goods Sold (COGs). Such benefits potentially include manufacturing flexibility and decreased labor expended on making particles sufficient for an NK cell expansion run. This opens the potential for improving manufacturing efficiency on a commercial scale.

[0054] In line with the objective to improve the process of manufacturing PM21 particles, preparation of PM21 plasma membrane particles was performed according to the following exemplary embodiment. K562-mb21-41BBL cells were cultured in advanced RPMI media supplemented with 5% Gibco™ KnockOut™ serum replacement, 2mM glutamine and 2g / L Pluronic® F-68 non-ionic surfactant until sufficient cells were obtained to seed a stirred tank (STR) bioreactor at a working volume of 2L and a seeding cell density of 0.3xl06viable cells / ml. The STR was controlled at 37°C, 60% dissolved oxygen (DO), pH 6.9, and set at 220 rpm stirrer speed. After 1 day of cultivation, the bioreactor was perfused by the continuous addition of 2L / day of advanced RPMI media supplemented with 5% Gibco™ KnockOut™ serum replacement, 2% Hyclone™ Cell Boost™ 7a cell culture supplement solution, 0.2% Hyclone™ Cell Boost™ 7b cell culture supplement solution, 8mM glutamine and 2g / L Pluronic® F-68 non-ionic surfactant. The working volume of the STR was maintained at 2L by the removal of cell culture permeate through an ATF perfusion filter setup. On Day 4 of culture, the perfusion rate was increased to 4L / day. At Day 6 or Day 7 of cultivation, the temperature was decreased to 32°C. At Day 8 of cultivation, the feeder cells were harvested for processing into plasma membrane particles.

[0060]

[0055] The disclosure will be further clarified by the following examples, which are intended to be purely exemplary of the disclosure and in no way limiting.

[0061] EXAMPLES

[0062] EXAMPLE 1: Process steps in the manufacture of PM21

[0063]

[0056] The manufacturing steps depicted in the workflow cartoon of Figure 1 are explained in greater detail hereinbelow.

[0064]

[0057] Preculture

[0065]

[0058] A vial of K562 feeder cell line (K562 parental cells which were genetically modified to express 4-1BBL and membrane bound IL-21 (mb-IL21)) containing 1ml with 5xl06viable cells / mL was thawed and the cells were resuspended in preculture media. A new vial was thawed for each block run.

[0059] Cell culture in 2L STR

[0066]

[0060] A stirred tank bioreactor (STR) was inoculated using a K562-mb21-41BBL cell culture from a shaker flask at a target cell density of 0.3X106viable cells / mL. The STR was operated in perfusion mode, which was initiated at a perfusion rate of 1 working volume per day (Iwv / day) on Day 1 and increased to 2 working volumes per day (2 wv / day) on Day 4. The parameters were set according to those set forth in Table 1. Daily samples were taken to monitor cell growth, metabolite concentrations, pH, and osmolality. In this study, 16 different cell culture process conditions (Cond) were tested over two blocks (Block 1 : 1-8, Block 2: 9-16; see Table 1). A control condition (Cond 8 and 9) was taken in each Block (e.g. 100L STR) at commercial scale. Based on analytical data (e.g. IL-21 concentration), potential interesting conditions were selected for a confirmation experiment.

[0067]

[0061] Table 1

[0068]

[0062] The varying conditions listed in Table 1 include pH shift (n=6, different settings); dissolved oxygen (DO) shift (n=2); temperature shift (n=2; different settings); reduced perfusion rate (n=2; different settings); P2.0 media (n=l); P3.0 with Cell Boost supplement 6 (n=l); and P3.0 (n=2; 1 per Block). The experimental conditions were carried out at a 2L reduced scale model STR.

[0069]

[0063] Harvest

[0070]

[0064] The bioreactor was harvested when any of the criteria below were met:

[0071] • Feeder cell density in the bioreactor reached above 18-20xl06viable cells / ml.

[0072] • Feeder cell viability was below 70% and cell density >13xl06viable cells / ml.

[0073] • Day 9 and feeder cell density was >13xl06viable cells / ml.

[0074]

[0065] Concentration

[0075]

[0066] A Carr Biosystems UFmini® single use centrifuge was used to process cells from the STR at the end of the cell culture phase. A process cycle was run to harvest cells, wash them with cold, sterile homogenization buffer, and concentrate them resulting in a concentrated cell suspension (CCS), to which a protease inhibitor cocktail was added. The CCS was sampled and stored at 4°C until further processing. Between each condition, the single use centrifuge was flushed with homogenization buffer.

[0067] Cell Lysis

[0076]

[0068] A microfluidizer was used to process the CCS into homogenate (HMG). On the day of use, the microfluidizer was flushed with 70% ethanol followed by cold homogenization buffer. The CCS was processed through the microfluidizer at 900 PSI and HMG was collected. The HMG was sampled and stored at 4°C until further processing. Between each condition, the microfluidizer was flushed with homogenization buffer.

[0077]

[0069] Clarification

[0078]

[0070] The HMG was centrifuged (3000g for lOmin at 4°C), and the resulting supernatant was collected as clarified homogenate (CHMG). The clarified homogenate was then sampled and stored at 4°C until further processing.

[0079]

[0071] PM21 Purification

[0080]

[0072] The CHMG was further processed in three ultracentrifuge steps according to an approved purification procedure. The procedure involved steps: 1) first wash; 2) sucrose density gradient; and 3) second wash. Each step resulted in a pellet of PM21 particles. Prior to the first wash step, lOOmL of CHMG of each condition was diluted 2-fold with homogenization buffer. Following the second wash step, purified PM21 particles were collected as a centrifugation pellet.

[0081]

[0073] Fill and Finish

[0082]

[0074] The pellets obtained from the ultracentrifuge were resuspended in RPMI media at 6mL per gram of pellet to target PM21 particles with a final IL-21 and 4-1BBL protein concentration of approximately 5mg / mL. The particles were aliquoted in cryovials and one crystal vial. The crystal vial was sent for e-beam irradiation, which was required before performing a PM21 particle functionality assay.

[0083]

[0075] EXAMPLE 2: Characterization of Temperature Shifted PM21 Particles

[0084]

[0076] IL-21 and 4-1BBL Content of Temperature Shifted PM21 Particles

[0085]

[0077] PM21 particles were crafted from the K562-mb21-41BBL cells cultured under each of the 16 conditions set forth in Table 1. Results were collected for the effects of growing K562- mb21-41BBL feeder cells under varying pH, media, temperature, reduced perfusion rates and dissolved oxygen conditions on IL-21 and 4-1BBL protein content in PM21 particles purified from those feeder cells. Table 2 presents the IL-21 and 4-1BBL protein content per milligram total protein measured for the PM21 particles obtained for two of the 16 culture conditions set forth in Table 1. Culture conditions in which pH and temperature were shifted were selected as representative results for comparison purposes in Table 2. Total PM21 particle protein concentration was determined using a bicinchoninic acid assay. Table 2 shows that of all 16 conditions examined, only shifting the cell culture temperature for the K562-mb21-41BBL feeder cells at Day 6 and Day 7 substantially affected the IL-21 protein content of PM21 particles derived from these cells. Likewise, Table 2 shows that the 4-1BBL protein content of PM21 particles derived from K562-mb21-41BBL feeder cells temperature shifted at Day 6 and Day 7 was substantially elevated in comparison to all other conditions analyzed. Purified temperature shifted PM21 particles exhibited a 4.4-fold mean increase in membrane bound IL-21 content versus control levels, and an 8.9-fold mean increase in 4-1BBL content versus controls levels. By contrast, shifting the pH of the culture on Day 5, Day 6, or Day 7 did not result in any meaningful increases in IL-21 or 4-1BBL protein levels in PM21 particles purified from K562- mb21-41BBL feeder cells.

[0086]

[0078] Table 2

[0087]

[0079] To ensure that the observed increase in PM21 membrane-bound IL-21 and 4-1BBL protein content under temperature shifted conditions was reproducible, PM21 particles were again purified from K562-mb21-41BBL feeder cells subjected to a temperature reduction (37°C decreased to 32°C) at Day 6 and Day 7 of culture in a confirmatory repeat experiment. Figure 3 shows that the temperature shifted PM21 particles demonstrated elevated concentrations of IL-21 (Figure 3 A) and 4-1BBL (Figure 3B) as compared to control samples. The data points in blue represent the temperature shifted protein concentrations of the initial analysis depicted in Table 2, and the data points in red show the protein concentrations measured in the repeat experiment focused solely on the temperature shifted conditions. The temperature shifted PM21 particles exhibited a 4.4-fold mean increase in IL-21 protein in the initial analysis comparing 16 different test conditions (blue points), and a remarkably similar 5.2-fold mean increase (red points) in the repeated experiment. The temperature shift-dependent increase in 4-1BBL levels was also reproduced in the repeated experiment, although the mean increase was a more variable (8.9-fold versus 3.7-fold), but it fell within an acceptable assay variability (-20%).

[0088]

[0080] Functionality of Temperature Shifted PM21 Particles

[0089]

[0081] The potency of the purified temperature shifted PM21 particles was measured in a performance assay in which the ability of PM21 particles to induce NK cell proliferation was assessed. CD3-depleted peripheral blood mononuclear cells (PBMCs) were stimulated with purified, irradiated PM21 particles in a preset dilution range. At Day 7 post-stimulation, the proliferation of the NK cells was determined by the means of ATP detection (using the CellTiter- Glo® luminescent cell viability assay), measuring luminescence using a Molecular Devices Spectramax® spectrophotometer. The measurement of ATP for each test condition provides a measurement of cell viability. A higher ATP concentration indicates a larger number of living (viable) cells in a sample as compared to another sample or a control sample. The EC50 value of each test sample was calculated to enable comparisons to be made between each assay condition. The EC50 value indicates the half maximal effective concentration for PM21 particles derived from feeder cells cultivated under the various test conditions that induce a biological response (cell replication) halfway between a baseline value and a maximum value during 7 days of contact time between the PM21 particles and target cells. In this case, the EC50 value is the concentration of purified PM21 particles required to achieve 50% growth stimulation of targeted NK cells as represented via ATP detection.

[0082] Table 3 shows the EC50 value for each of the 16 conditions. For conditions 10 and 11, no curve fit could be found, as the PM21 particles appeared to be more potent than the other samples evaluated. This was indeed confirmed when the assay was repeated for conditions 9, 10 and 11 (Table 4). The results showed that temperature shifted PM21 particles exhibited an EC50 value of less than 40 pg PM21 parti cles / mL for NK cell proliferation.

[0090]

[0083] Table 3: EC50 value of produced PM21 (Assay Run 1)

[0091]

[0084] Table 4: EC50 value of produced PM21 (Assay Run 2)

[0092]

[0085] Figure 2 depicts PM21 particle functionality plotting target NK cell proliferation (measured as ATP detected via luminescence relative light units) against increasing concentrations of purified temperature shifted PM21 particles. Only 30% of the PM21 particles produced from feeder cells subjected to a temperature shift (37°C decreased to 32°C) were required to reach the EC50 value of the control condition. This is shown in Figure 2 as a growth curve positioned to the left of control and reference samples for the temperature shifted PM21 particles. Using NK cell growth as a surrogate marker, the temperature shifted PM21 particles demonstrated a higher level of potency as compared to control and reference PM21 particles. Thus, temperature shifted PM21 particles demonstrated a 3.3 -fold mean increase in functionality (e.g. achieving the same NK cell expansion over a 7-day culture period at a lower PM21 concentration). Figure 4 depicts the growth effects on NK cells contacted with PM21 particles purified from each of the 16 feeder cell culture conditions set forth in Table 1. Figure 4A depicts the growth curves elicited by NK cells contacted with PM21 particles purified from feeder cells subjected to Block 1 growth conditions (condition numbers 1-8). Figure 4B depicts the growth curves elicited by NK cells by NK cells contacted with PM21 particles purified from feeder cells subjected to Block 2 growth conditions (condition numbers 9-16). When comparing all of the growth curves in Figures 4A and 4B, only the temperature shifted PM21 particles moved the NK cell growth curves above that of the control, indicating maximal NK cell expansion was achieved at lower PM21 particle concentrations. Again, this demonstrates that only the temperature shifted PM21 particles can be characterized as having a higher potency than PM21 particles derived from K562-mb21-41BBL feeder cells grown under control conditions.

[0093]

[0086] Those of ordinary skill in the art will recognize or ascertain using no more than routine experimentation many equivalents to the disclosed embodiments of the methods and compositions discussed herein. Such equivalents are intended to be encompassed by the following claims.

[0094]

[0087] 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.

[0095]

[0088] 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 be 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.

[0096]

[0089] 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.

[0097]

[0090] 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.

[0098]

[0091] This application claims the benefit of, and relies on the filing date of, U.S. Provisional Application No. 63 / 700,226, filed 27 September 2024, the entire disclosure of which is herein incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A method of enhancing potency of plasma membrane particles comprising expanding feeder cells comprising membrane-bound IL-21 and 4-1BBL in a cell culture comprising a medium, wherein the feeder cells are cultured at 37°C during Day 0 through Day 5 of expansion and thereafter cultured at 32°C from Day 6 until harvested; harvesting the expanded feeder cells when feeder cell concentration exceeds a threshold set point of cell viability and / or cell density; and lysing the expanded feeder cells in a pressurized vessel to form temperature shifted plasma membrane particles containing IL-21 and 4-1BBL (PM21 particles); wherein the temperature shifted PM21 particles exhibit a higher potency for stimulating natural killer (NK) cell proliferation than PM21 particles purified from an equivalent number of feeder cells expanded at a constant culture temperature of 37°C.

2. The method of claim 1, wherein the temperature shifted PM21 particles contain higher amounts of IL-21 than do PM21 particles purified from an equivalent number of feeder cells expanded at a constant culture temperature of 37°C.

3. The method of claim 2, wherein the temperature shifted PM21 particles further contain higher amounts of 4-1BBL than do PM21 particles purified from an equivalent number of feeder cells expanded at a constant culture temperature of 37°C.

4. The method of claim 1 or claim 2, wherein the temperature shifted PM21 particles exhibit an EC50 value of less than 40 pg PM21 particles / mL for NK cell proliferation.

5. The method of any one of claims 1-4, wherein the feeder cells are expanded in a bioreactor.

6. The method of any one of claims 1-5, wherein the feeder cells comprise K562-mb21- 41BBL cells.

7. The method of any one of claims 1-6, wherein the expanded feeder cells are harvested at Day 8.

8. The method of any one of claims 1-7, wherein the expanded feeder cells are harvested when feeder cell concentration exceeds 18-20 xlO6viable cells / mL.

9. The method of any one of claims 1-7, wherein the expanded feeder cells are harvested when cell viability falls below 70% and cell density is greater than 13xl06viable cells / mL.

10. The method of any one of claims 1-6, wherein cell culturing continues to Day 9 and the expanded feeder cells are harvested at a cell density below 13xl06viable cells / mL.

11. The method of any one of claims 1-10, wherein the PM21 particles are purified using a sucrose density gradient.

12. A population of expanded natural killer (NK) cells produced by contacting NK cells with temperature shifted plasma membrane particles comprising membrane-bound IL-21 and 4-1BBL (PM21 particles) created by the method of any one of claims 1-12.

13. The population of claim 12, wherein NK cell expansion is stimulated by contacting the NK cells ex vivo or in vitro with the temperature shifted PM21 particles.

14. The population according to claim 13, wherein the NK cells are contacted by the temperature shifted PM21 particles multiple times.

15. The population according to any one of claims 12-14, wherein the temperature shifted PM21 particles further comprise at least one additional cytokine.

16. The population according to any one of claims 12-15, wherein cytotoxicity of the NK cells expanded by contact with the temperature shifted PM21 particles is greater than the cytotoxicity of NK cells expanded by contact with PM21 particles derived from feeder cells cultured at a consistent temperature of 37°C.

17. A pharmaceutical composition comprising: an effective amount of expanded natural killer cells produced using temperature shifted PM21 particles created by the method of any one of claims 1-11; and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition of claim 17, wherein composition further comprises an anti-infective agent or an anti-cancer agent.

19. The pharmaceutical composition of claim 18, wherein the anti-cancer agent is selected from an antibody, a chemotherapeutic drug, and an immunotherapeutic agent.

20. The pharmaceutical composition of claim 18, wherein the anti-infective agent is selected from an antiviral agent, an antibacterial agent, and an antifungal agent.

21. A method of treating an infection or a cancer in a subject in need thereof comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 17-20.

22. The pharmaceutical composition of any one of claims 17-20 for use in the treatment of an infection or a cancer in a subject.

23. Use of the population of expanded natural killer cells of any one of claims 12-16 in the manufacture of a medicament for treating an infection or cancer.

24. The use of claim 23, wherein the expanded NK cells are combined with a pharmaceutically acceptable carrier to generate the medicament.

25. A kit comprising the pharmaceutical composition of any one of claims 17-20 and instructions for use according to the method of claim 21.

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