Method for producing clinical-grade lentiviral vectors

Abstract

This invention provides an improved method for the production and purification of clinical-grade retroviral vectors, such as lentiviral vectors. [Solution] A method for producing a lentiviral vector preparation, the method comprising: (a) a step of treating a filter-sterilized lentiviral vector preparation with a nuclease; and (b) a step of concentrating the nuclease-treated lentiviral vector preparation to produce a lentiviral vector preparation.

Description

[Technical Field]

[0001] • Cross-referencing of related applications: This application is a PCT application claiming priority to U.S. Provisional Application No. 63 / 065,225, filed on 13 August 2020, which is expressly incorporated herein by reference in its entirety. [Background technology]

[0002] ·background: Novel therapies using T cells engineered to express immune receptors are expected to be promising immunotherapies for a wide range of intractable diseases, including cancer and autoimmune diseases. Lentiviral vectors have been used for transgene delivery in many different applications because lentiviruses can infect non-dividing cells (Non-Patent Literature 1). Furthermore, lentiviral vectors enable highly stable, long-term expression of transgenes.

[0003] The method for producing lentiviral vectors is not simple and requires many steps. While many of these steps are successful to some extent, these steps are further complicated because clinical application of the vectors requires highly pure samples free from biological and non-biological contaminants. The vector purification process is a critical step in clinical gene therapy, and there is a need for novel methods to provide viral vectors more efficiently, safely, and on a large scale. This disclosure addresses this need. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Lewis and Emerman (1993) J. Virol. 68:510 [Overview of the initiative]

[0005] • Summary of disclosure: The present disclosure generally relates to methods for producing and purifying lentiviral vectors.

[0006] In some aspects, the present disclosure generally relates to a method of producing a lentiviral vector formulation, the method comprising: (a) treating a filter-sterilized lentiviral vector preparation with a nuclease, and (b) concentrating the nuclease-treated lentiviral vector preparation to produce a lentiviral vector formulation, wherein the concentrating step is the final step of the method. In some aspects, the method comprises a step of clarifying a cell culture supernatant. In some aspects, a first nuclease treatment is performed after the clarifying step. In some aspects, the nuclease has endonuclease activity. In some aspects, the nuclease has exonuclease activity. In some aspects, the nuclease is a modified nuclease derived from Serratia marcescens.

[0007] In some aspects, the first nuclease treatment precedes an ultrafiltration / diafiltration step. In some aspects, the ultrafiltration / diafiltration step comprises tangential flow filtration. In some aspects, the ultrafiltration / diafiltration step comprises hollow fiber filtration.

[0008] In some aspects, the lentiviral vector preparation is diluted prior to filter sterilization of the lentiviral vector preparation. In some aspects, the lentiviral vector preparation is diluted into a formulation buffer. In some aspects, during the ultrafiltration / diafiltration step, the lentiviral vector preparation is diluted into the formulation buffer.

[0009] In some embodiments, the method comprises a second nuclease treatment. In some embodiments, the second nuclease treatment is the penultimate step of the method. In some embodiments, the nuclease has endonuclease activity. In some embodiments, the nuclease has exonuclease activity. In some embodiments, the nuclease is a modified nuclease derived from Serratia marcescens.

[0010] In some embodiments, the present disclosure is generally directed to methods of producing a lentiviral vector preparation, the method comprising: (a) culturing a cell that produces the lentiviral vector, (b) recovering supernatant from the cultured cells, (c) clarifying the supernatant, (d) concentrating the clarified supernatant, (e) purifying the lentiviral vector from the concentrated supernatant to produce a lentiviral vector preparation, (f) filter sterilizing the lentiviral vector preparation, (g) treating the filter sterilized lentiviral vector preparation with one or more nucleases, and (h) concentrating the nuclease-treated lentiviral vector preparation to produce a final product, comprising.

[0011] In some embodiments, the present disclosure is generally directed to methods of producing a lentiviral vector preparation, the method comprising: (a) culturing a cell that produces the lentiviral vector, (b) recovering supernatant from the cultured cells, (c) clarifying the supernatant, (d) A step of concentrating the clarified supernatant, (e) A step of purifying the lentiviral vector from the concentrated supernatant to produce a lentiviral vector preparation, (f) A step of filter sterilizing the lentiviral vector preparation, and (g) A step of concentrating the nuclease-treated lentiviral vector preparation to produce the final product, Includes, The clarified supernatant and / or lentiviral vector preparation described above are treated with a nuclease. In some embodiments, the step of concentrating the clarified supernatant includes the step of exchanging the concentrated supernatant with a formulation buffer.

[0012] In some embodiments, the clarified supernatant and the lentiviral vector preparation are treated with a nuclease. In some embodiments, prior to (d), the clarified supernatant is treated with a nuclease. In some embodiments, after (c), the clarified supernatant is treated with a nuclease. In some embodiments, the nuclease has endonuclease activity. In some embodiments, the nuclease has exonuclease activity. In some embodiments, the nuclease is a modified nuclease derived from Serratia marcescens.

[0013] A method for producing a lentiviral vector formulation, comprising the following steps in chronological order: (a) A step of culturing cells that produce lentiviral vectors, (b) A step of recovering the supernatant containing the lentiviral vector, (c) A step of clarifying the supernatant, (d) A step of treating the clarified supernatant with a nuclease, (e) A step of concentrating the clarified supernatant treated with the nuclease, comprising replacing the concentrated supernatant with a formulation buffer, (f) A step of purifying the lentiviral vector from the concentrated supernatant and producing a lentiviral preparation, (g) A step of filter sterilizing the lentiviral vector preparation, (h) The step of treating the filter-sterilized lentiviral vector preparation with a nuclease, and (i) A step of concentrating the nuclease-treated lentiviral vector preparation to produce a final product.

[0014] In some embodiments, the present disclosure generally relates to a method for producing a viral vector formulation, the method being The process of culturing cells that produce viral vectors, A step of collecting the supernatant from the cultured cells, A step of clarifying the supernatant, A step of concentrating the clarified supernatant, A step of purifying a lentiviral vector from the concentrated supernatant to produce a viral vector preparation, The step of filter sterilizing the lentiviral vector preparation, and The process includes a step of concentrating the lentiviral vector preparation treated with the aforementioned nuclease to produce a final product, Here, the method preferably involves processing the vector in only a single nuclease step. The purification of the vector is (1) Via one or more chromatography steps, (2) via high-speed centrifugal separation or ultracentrifugal separation, (3) This may be done via a concentrated solution such as PEG or LENTI-X CONCENTRATOR. In some embodiments, the method comprises two separate nuclease treatment steps. In some embodiments, one of two separate nuclease treatment steps is: (1) One or more chromatography (2) High-speed centrifugal separation or ultracentrifugal separation, (3) This can be replaced with a vector purification step that includes purifying the vector via a concentrate such as PEG or LENTI-X CONCENTRATOR. In some embodiments, the method described herein comprises two separate nuclease treatment steps, a first nuclease treatment and a second nuclease treatment. In some embodiments, the first nuclease treatment is performed (1) One or more chromatography (2) High-speed centrifugal separation or ultracentrifugal separation, (3) This can be replaced with a vector purification step that includes purifying the vector via a concentrate such as PEG or LENTI-X CONCENTRATOR. In some embodiments, the second nuclease treatment is performed. (1) One or more chromatography (2) High-speed centrifugal separation or ultracentrifugal separation, (3) This step is replaced with a vector purification step that includes purifying the vector via a concentrate such as PEG or LENTI-X CONCENTRATOR.

[0015] The inventions described and claimed herein have many attributes and embodiments, including, but not limited to, those described, described, or referred to in this Brief Summary, the Brief Description of the Drawings, and the Detailed Description. Not intended to be exhaustive, the inventions described and claimed herein are included for illustrative purposes only and are not limited to, and are not limited to, the features or embodiments identified in this brief summary. Additional embodiments may be disclosed in the following detailed description. [Brief explanation of the drawing]

[0016] [Figure 1] Figure 1 is a schematic diagram of the virus purification process of this disclosure. The CC700 designation corresponds to a column containing CAPTOCORE 700, a multimodal chromatography resin used for the purification / polishing of viruses and other large biomolecules. [Figure 2] Figure 2 shows the results of a qPCR assay that detected and quantified the vesicular stomatitis virus glycoprotein (VSV-G) gene. Detection of VSV-G effectively measures the amount of residual VSV-G still present at multiple points in the vector manufacturing process. The amount of residual VSV-G generally serves as an indicator of the amount of plasmid contaminants remaining at the time of vector production. The figure graphs the step removal percentages observed after three steps in the vector manufacturing process. The "Harvest" sample was collected immediately after the clarification step. The "TFF1" sample was collected immediately after the first tangential flow filtration step. The "TFF2" sample was collected immediately after the final tangential flow filtration step, thus obtaining the final result of the lentiviral vector production and purification method. The data is normalized so that the amount of VSV-G in the harvest sample is 1, or 100%, the amount of VSV-G in the TFF1 sample is 0.16, or 16%, and the amount of VSV-G in the TFF2 sample is 0.009, or 0.09%. [Figure 3] Figure 3 shows the results of an assay to determine the effect of various lentiviral vector titers on the total yield of the lentiviral vector used in the virus purification method of this disclosure. [Modes for carrying out the invention]

[0017] • Detailed explanation: I. overview This application relates to a method for producing retroviral vectors such as lentiviral vectors, and formulations containing these vectors, particularly formulations that are isolated at high titers and are sufficiently free from contamination by cell debris, nucleic acid degradation molecules, and protein degradation substances. Producing purified formulations of retroviral vectors such as lentiviral vectors at high titers and free from contaminants is not a simple task. Surprisingly, the inventors have identified several embodiments of retroviral vector purification methods that result in increased titers of retroviral (e.g., lentiviral) vector stocks.

[0018] In particular, this disclosure is directed towards two aspects of the production of retroviral (e.g., lentiviral) vectors: (1) Dilution of the virus stock before sterilization and filtration, (2) Removal of residual and genomic DNA in the final vector production. Nucleic acids remaining in the final retroviral product are undesirable. Two aspects of the novel viral vector production method described herein not only produce an improved final product but also improve the overall yield.

[0019] The first aspect is directed to the concentration of retroviral (e.g., lentivirus) vectors, which has a significant impact on the titer of the vectors recovered in the purification method. Dilute samples of retroviral (e.g., lentivirus) vectors show a greater total recovery rate compared to more concentrated retroviral (e.g., lentivirus) vector samples. Surprisingly, in one embodiment, approximately 2 × 10 6 The infectivity titer of a retrovirus (e.g., lentivirus) vector, or 1.5 × 10⁻¹⁰ including this amount. 6 ~2.5×10 6 We discovered that this range resulted in the most efficient recovery of retroviral (e.g., lentiviral) vectors.

[0020] A second aspect of this disclosure is directed toward the efficiency of removing host cells and genomic DNA from retroviral (e.g., lentivirus) vector samples in one or more stages, which also significantly affects the titer of the recovered retroviral (e.g., lentivirus) vector. Specifically, it has been surprisingly found that the efficiency of host cell and genomic DNA removal is increased by adding at least one nuclease treatment to the diluted vector after the filter sterilization step and before the vector concentration step (e.g., using tangential flow filtration (TFF)). Any suitable nuclease treatment can be used, and exemplary treatments are described herein. In exemplary embodiments, to enhance the efficiency of residual nucleic acid removal, the inventors have shown that molecular sizes between approximately 10 kDa and approximately 1,000 kDa for hollow fibers or cassette TFFs can be used to enhance the efficiency of host cell and genomic DNA removal.

[0021] Figure 1 shows an illustrative flowchart of a lentiviral vector production method using the novel embodiments described herein.

[0022] The exemplary downstream process described herein includes two purification steps (e.g., using TFF), with a nuclease treatment step performed before each purification step (e.g., TFF). The inventors have surprisingly demonstrated that a vector purification method having two concentration steps, each preceded by a nuclease treatment step, results in further improvements in efficiency.

[0023] Furthermore, it has been found that performing a first nuclease step on the clarified supernatant before the initial concentration step helps remove residual DNA and prevents clogging of the filter (e.g., TFF). However, a second nuclease treatment performed before the final concentration step can effectively remove residual DNA without increasing the time required for downstream manufacturing steps. The incubation time and temperature for each nuclease treatment may vary to maximize the process time and efficiency of our purification method.

[0024] The inventors have demonstrated that a high molecular weight pore cutoff of approximately 750 kDa to 1,000 kDa effectively removes nucleases, thus eliminating concerns about residual nucleases in the final product.

[0025] In further embodiments, the nuclease process is carried out at a lower temperature, such as room temperature or, for example, 30°C, which is a typical temperature at which the nuclease process is carried out. For example, as further described herein, the nuclease process may be carried out at a “low temperature” of about 2 to about 8°C. Other exemplary temperatures at which the nuclease process may be carried out are described herein.

[0026] Introducing these additional steps into the lentiviral vector purification process inevitably increases the time required to produce viable vectors, adding to an already time-consuming process of approximately three days. Adding about an extra day to an already time-consuming process would likely deter others from pursuing this path.

[0027] Vector purification is a crucial step in establishing clinical gene therapy, and there is a need for novel methods to provide viral vectors more efficiently, safely, and with high titers. This disclosure addresses this need.

[0028] II. Retrovirus vectors In some aspects, viruses are retroviral vectors. In some embodiments, the virus is a recombinant retroviral vector containing the heterologous transgene or nucleic acid sequence of interest. In some embodiments, xenogenes or nucleic acid sequences may be used in a therapeutic setting for the purpose of gene therapy. In some embodiments, a retroviral vector containing the desired xenogene or nucleic acid sequence may be used to transduce immune cells.

[0029] In some embodiments, the vector is targeted to a desired cell type. In some embodiments, the vector is targeted to a desired cell type to which the vector fuses during the process of vector-mediated gene transfer. In a further embodiment, the desired cell type is an immune cell. In some embodiments, the desired cell type is a T cell, a B cell, a dendritic cell, or an antigen-presenting cell.

[0030] In some embodiments, the target xenogene or nucleic acid sequence may have therapeutic or diagnostic applications. Suitable target xenogenes or nucleic acid sequences may include, but are not limited to, sequences encoding enzymes, cytokines, chemokines, hormones, antibodies, and antioxidant molecules. In some embodiments, the target xenogene or nucleic acid sequence may be an engineered immunoglobulin-like molecule, an immunomodulatory molecule, an antisense RNA, a microRNA, an shRNA, an siRNA, a ribozyme, a gene editing system (e.g., CRISPR / CAS, zincfin nuclease, TALENs), an antigen receptor (e.g., a chimeric antigen receptor, a T cell receptor), an antigen, a toxin, a transdomain-negative variant of a target protein, a tumor suppressor protein, a growth factor, a membrane protein, a reporter protein (e.g., a fluorescent protein), or a derivative thereof.

[0031] In some forms, retroviral vectors are lentiviral vectors. In some embodiments, lentiviral vectors are derived from bovine immunodeficiency virus, Caprin arthritis encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus, human immunodeficiency virus, human immunodeficiency virus 1, human immunodeficiency virus 2, Jembrana disease virus, Pluma lentivirus, Simian immunodeficiency virus, or Bisnamedi virus.

[0032] In some embodiments, lentiviral vectors are pseudotypes, meaning they contain envelope glycoproteins derived from different viruses. In some embodiments, the lentiviral vectors are pseudotyped with envelope glycoproteins derived from vesicular stomatitis virus (VSV-G), measles virus, modified measles virus, baboon endogenous virus, and Divonian leukemia virus. In some embodiments, lentiviral vectors are pseudotyped using modified envelope glycoproteins. In some embodiments, lentiviral vectors are pseudotyped using chimeric envelope glycoproteins.

[0033] In some embodiments, a lentiviral vector is modified such that one or more protein-coding regions necessary for replication are removed from the lentiviral vector, and thus the vector becomes non-replicating. In some embodiments, the target heterologous transgene or nucleic acid sequence replaces or substitutes a portion of the viral genome. In some embodiments, the target heterologous transgene or nucleic acid sequence can be added to the viral genome, thereby enabling sufficient replication of the vector. In some embodiments, the vector is a non-integrated vector, as described in USPat.Appln.Pub.No.US20090014754A1. In some embodiments, lentiviral vectors are modified such that one or more protein-coding regions necessary for replication are removed from the lentiviral vector, as described in USPat.Appln.Pub.No.US20090075370A1.

[0034] III. Retrovirus vector production system Retroviral vectors can be grown in producer cells or packaging cells. Producer cells or packaging cells can be any cells that can grow and subsequently harvest retroviral vectors. In some embodiments, the producer cells or packaging cells may be a stable producer cell line or host cell line for growing large quantities of viral particles for subsequent purification. In some embodiments, producer cells or packaging cells are selected from human cells. In a further embodiment, the human cells are HEK293, HEK293T, HEK293FT, Te671, HT1080, or CEM. In some embodiments, producer cells or packaging cells are selected from mouse cells. In a further aspect, the mouse cells are NIH-3T3. In some embodiments, producer cells or packaging cells are selected from mustelidae cells. In a further embodiment, weasel cells are Mpf. In some embodiments, producer cells or packaging cells are selected from canine cells. In a further embodiment, canine cells are D17. In some embodiments, the producer cells or packaging cells are HEK293 cells. In some embodiments, transient transfection may be used to generate lentiviral vectors in producer cells and / or packaging cells.

[0035] As used herein, the terms “packaging cell” and “producer cell” refer to cells that contain the elements necessary for the production of recombinant retroviruses or lentiviruses lacking a viral genome. Typically, such packaging cells contain one or more producer plasmids that can express viral structural proteins (such as codon-optimized gag-pol and env) but do not contain a packaging signal. Preferably, the producer / packaging cells are obtained from mammalian cells, preferably primate cells such as human cells. In some embodiments, the human cells are human embryonic kidney (HEK) cells. Any type of cell capable of supporting the replication of recombinant retroviruses or lentiviruses can be used to propagate the recombinant virus.

[0036] In some embodiments, the packaging / producer cell lines have been modified to remove the 3' LTR of the provirus to improve safety. Furthermore, the gag-pol and env genes have been modified to be introduced onto separate plasmids and can be sequentially introduced into the cell line to further avoid recombination. In some embodiments, recombinant viruses and transgenes are introduced into a packaging / producer cell line as a third-generation lentiviral system. This lentiviral system is introduced into the cell as four separate plasmids: a plasmid encoding gag-pol, a plasmid encoding the viral rev gene, an envelope plasmid encoding an envelope glycoprotein (e.g., VSV-G), and a transplasmid encoding the target transgene or nucleic acid sequence.

[0037] Cells transfected with a retroviral vector system are cultured to increase the cell and virus count and / or viral titer by means and methods well known to those skilled in the art, and this includes, but is not limited to, providing the cells with appropriate nutrients in a suitable culture medium. The method may include growth on a surface, growth in a suspension, or a combination thereof. Culture can be carried out, for example, in tissue culture flasks, dishes, roller bottles, or bioreactors using batch, fed-batch, continuous systems, hollow fiber systems, etc. However, in order to achieve large-scale (continuous) production of viruses by cell culture, it is preferred in this art to have cells that can grow in a suspension state. Appropriate conditions for culturing cells are known (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and RI Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9)).

[0038] In some embodiments, cells are cultured in tissue culture flasks and then cultured in multilayer culture chambers to generate recombinant virus particle producer cells. In some embodiments, producer cells are adherent cells that propagate viral particles. In some embodiments, producer cells are non-adherent cells that transmit viral particles.

[0039] In some embodiments, the cells are grown in a medium sufficiently suitable for culturing the selected cell type and producing lentiviral vectors. In some embodiments, the culture medium is a compound medium or a minimal medium. In some embodiments, the culture medium is supplemented with antibiotics, mammalian blood serum (such as fetal bovine serum), pH indicators, etc. In some embodiments, the culture medium is serum-free.

[0040] IV. Purification of retroviral vectors An exemplary vector purification process is illustrated in Figure 1. In some aspects, the purification process begins with clarifying the used cell culture medium / supernatant. In a further embodiment, after clarification, (1) One or more concentration steps, (2) One or more nuclease treatment steps, (3) One or more refining steps, (4) One or more sterilization and filtration steps follow. In a further embodiment, after clarification, (1) One or two (or more) concentration steps, (2) 1 or 2 (or more) nuclease treatment steps, (3) 1 or 2 (or more) purification steps, (4) One or two (or more) sterilization and filtration steps follow, The process may or may not be carried out in the order of steps (1) to (4). In a further embodiment, clarification is followed by one or more dilution steps, which may or may not be performed immediately after clarification and before the subsequent steps.

[0041] In some embodiments, the purification process is carried out in chronological order. (1) A process to clarify the used culture medium / supernatant, (2) First concentration step, (3) Nuclease process, (4) Purification process, (5) Sterilization filtration process, and (6) A process comprising or consisting of a second concentration step. In some embodiments, the purification process is carried out in chronological order. (1) Process of clarifying used culture medium / supernatant, (2) Nuclease process, (3) First concentration step, (4) Purification process, (5) Sterilization filtration process, and (6) A process comprising or consisting of a second concentration step. In some embodiments, the purification process is carried out in chronological order. (1) Process of clarifying used culture medium / supernatant, (2) First nuclease step, (3) First concentration step, (4) Purification process, (5) Sterile filtration process, (6) Second nuclease step, and (7) A second concentration step, or a step comprising a second concentration step. In some embodiments, the purification step may include one or more dilution steps.

[0042] In some embodiments, the first nuclease treatment is carried out before the first concentration step. In some embodiments, the second nuclease treatment is carried out before the second concentration step. In some embodiments, the first and second nuclease steps are performed before the final concentration step. In some embodiments, the first and second nuclease steps are not performed sequentially. In some embodiments, the first and second purification steps are not performed consecutively. In some embodiments, the first and second purification steps are not performed consecutively. In some embodiments, the first and second purification steps are not performed consecutively. In some embodiments, the first and second concentration steps are not performed simultaneously. In some embodiments, the first and second nuclease steps are not performed consecutively. In some embodiments, the first and second purification steps are not performed consecutively.

[0043] In some embodiments, the concentration step is the final step of the method. In some embodiments, when the first and second nuclease steps are performed, the first nuclease step is performed before the first concentration step, and the second nuclease step is performed before the second concentration step. In some embodiments, the dilution step is performed after either or both of the first nuclease steps. In some embodiments, the nuclease step is performed before the clarification step. In some embodiments, the sterilization filtration and concentration steps are carried out in a closed system such that the filtrate flows directly from the sterilization filter to the concentration device.

[0044] In some embodiments, the sterilization filtration step is not the final step of the purification process. In some embodiments, the sterile filtration step is not the second-to-last step of the purification process. In some embodiments, the second nuclease treatment step is not performed before the sterilization filtration step. In some embodiments, when the sterile filtration step is the final step of the purification process, the vector yield is reduced compared to a control in which the sterile filtration step is not performed last, or compared to the stepwise convention as defined in Figure 1.

[0045] In some embodiments, the concentration step is an ultrafiltration step, and when used for buffer exchange, is sometimes called diafiltration. In some embodiments, an ultrafiltration / diafiltration step enriches the vector. In some embodiments, ultrafiltration / dialysis filtration may take the form of tangential flow filtration (TFF). In some embodiments, the ultrafiltration / dialysis filtration membrane is selected to have a pore size small enough to hold the vector and large enough to effectively remove impurities.

[0046] During the process of culturing cells transfected with a lentiviral vector, the lentiviral vector accumulates in the used culture medium in which the cells constituting the culture medium are cultured. In some embodiments, intact cells remaining in the culture medium are lysed, thereby releasing any remaining lentiviral vectors.

[0047] In some embodiments, used culture medium / cell culture supernatant is clarified. Clarification is the removal of cell debris from the supernatant as a means of initiating the isolation of lentiviral vectors.

[0048] In some embodiments, clarification of the cell culture supernatant is carried out by a filtration step to remove cell debris and other impurities. Suitable filters include cellulose filters, regenerated cellulose fibers, cellulose fibers combined with inorganic filter aids (e.g., diatomaceous earth, perlite, fumed silica), cellulose filters combined with inorganic filter aids and organic resins, or any combination thereof, and polymer filters (e.g., nylon, polypropylene, polyethersulfone, etc.) that can achieve effective removal and acceptable recovery. Generally, a multi-stage process is preferred, but not essential. An exemplary two- or three-step process involves removing large precipitates and cellular debris with a coarse filter, followed by a second polishing filter with a nominal pore size of 0.2 microns or more and less than 1 micron. The optimal combination may be a function of the precipitate size distribution and other variables. Furthermore, single-stage operations using filters with relatively small pore sizes or centrifugal separation can also be used for clarification. More generally, any clarification approach, including but not limited to dead-end filtration, microfiltration, centrifugal separation, or the incorporation of filter aids (e.g., diatomaceous earth) in combination with dead-end filtration or deep filtration, shall provide a filtrate of sufficient clarity to avoid contaminating the membrane and / or resin in subsequent stages and shall be permitted for use in the clarification process of the present invention.

[0049] In some embodiments, clarification is carried out using a filter. In some embodiments, the filter has a pore size ranging from approximately 0.1 μm to approximately 1.5 μm. In some embodiments, the filter has pore sizes ranging from approximately 0.2 μm to approximately 1.5 μm. In some embodiments, the filter has pore sizes ranging from approximately 0.45 μm to approximately 0.8 μm. In some embodiments, the filter has pore sizes ranging from approximately 0.45 μm to approximately 1.5 μm. In some embodiments, the filter has pore sizes of approximately 0.1 μm, 0.2 μm, 0.22 μm, 0.45 μm, 0.65 μm, 0.8 μm, 1.0 μm, 1.2 μm, 1.3 μm, or 1.5 μm. In some embodiments, the maximum pore size is 0.1 μm, 0.2 μm, 0.22 μm, 0.45 μm, 0.65 μm, 0.8 μm, 1.0 μm, 1.2 μm, 1.3 μm, or 1.5 μm.

[0050] In some embodiments, the clarified supernatant is treated with a nuclease to degrade contaminating nucleic acids, particularly DNA and / or RNA, from producer cells. In some embodiments, the nuclease is either DNAse or RNAse. In some embodiments, nucleases are both DNAse and RNAse.

[0051] In some embodiments, the improved final product of this disclosure contains an undetectable amount of nuclease. Any suitable nuclease may be used in the methods described herein. In some embodiments, the nuclease is BENZONASE nuclease (EP0229866 and U.S. Patent No. 5,173,418), which degrades all forms of DNA and RNA, including single-stranded, double-stranded, linear, and circular. BENZONASE nuclease is commercially available from Merck KGaA. BENZONASE is a genetically engineered endonuclease derived from Serratia marcescens. This protein is a dimer of 30 kDa subunits with two essential disulfide bods. This endonuclease attacks and degrades all forms of DNA and RNA (single-stranded, double-stranded, linear, and circular) and is effective under a wide range of operating conditions. BENZONASE has both DNAse and RNAse activity and no proteolytic activity.

[0052] In some embodiments, the nuclease is DENARASE nuclease. DENARASE nuclease is commercially available from c-LECTA GmbH. In some embodiments, nucleases are DNases and / or RNases commonly used in the art for the purpose of removing unwanted or contaminated DNA and / or RNA from preparations. DENARASE is described in US2012 / 0135498, where it is included as sequence identifier number 3. DENARASE is a genetically engineered endonuclease derived from Serratia marcescens. DENARASE possesses both DNAse and RNAse activity and does not have proteolytic activity.

[0053] In some embodiments, the purification protocol includes one or more nuclease treatments. In some embodiments, each nuclease treatment involves only a single type of nuclease, for example, only denarase. In some embodiments, each nuclease treatment may include one or more nucleases. In some embodiments, the first nuclease treatment comprises only one type of nuclease, and the second nuclease treatment comprises one or more different types of nucleases. In some embodiments, the first nuclease treatment includes one or more different types of nucleases, and the second nuclease treatment includes only one type of nuclease.

[0054] In some embodiments, one or more nucleases are endonucleases. In some embodiments, one or more nucleases are exonucleases. In some embodiments, one or more nucleases include both endonucleases and exonucleases. In some embodiments, endonucleases or exonucleases are modified enzymes isolated from bacteria or fungi. In some embodiments, the endonuclease or exonuclease is a recombinant enzyme that still possesses endonuclease and / or exonuclease activity.

[0055] In some embodiments, one or more of the virus purification methods described herein are performed after one or more nuclease treatments. In some embodiments, one or more nuclease treatments are performed as post-treatments prior to the final ultrafiltration / diafiltration step. In some embodiments, one or more of the virus purification methods described herein are performed instead of nuclease treatment. In some embodiments, nuclease treatment is omitted from the virus purification method.

[0056] In some embodiments, the nuclease treatment step is performed at about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, or about 15°C. In some embodiments, the nuclease treatment is immediately before or immediately after the clarification step. In some embodiments, the nuclease treatment is the penultimate step of the purification protocol.

[0057] In some embodiments, the dilution step dilutes a lentiviral vector sample to about 1×10 5 , about 2×10 5 , about 3×10 5 , about 4×10 5 , about 5×10 5 , about 6×10 5 , about 7×10 5 , about 8×10 5 , about 9×10 5 , about 1×10 6 , about 2×10 6 , about 3×10 6 , about 4×10 6 , about 5×10 6 , about 6×10 6 , about 7×10 6 , about 8×10 6 , about 9×10 6 , about 1×10 7 , about 2×10 7 , about 3×10 7 , about 4×10 7 , about 5×10 7 , about 6×10 7 , about 7×10 7 , about 8×10 7 , or about 9×10 7 infectious titer units per ml. In some embodiments, the dilution step dilutes a lentiviral vector sample to a maximum of 5×10 6 infectious titer units per ml.

[0058] In some embodiments, diluting the amount of retroviral (e.g., lentivirus) vector before one or more nuclease treatment steps increases the titer of the retroviral (e.g., lentivirus) vector in the final product. In some embodiments, this increase is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, or at least about 400%, or up to about 400%, compared to a control purification assay in which the titer of the retroviral (e.g., lentivirus) vector was not diluted.

[0059] In some embodiments, the methods described herein for the purification of retroviral (e.g., lentiviral) vectors are (1) Plasmids used in the production of retroviral vectors, and / or (2) This results in the removal of impurities remaining from the cells used in the production of the retroviral vector. In some embodiments, the impurities are cell debris, residual cellular DNA, and / or residual plasmid DNA. In some embodiments, measuring the presence of VSV-G provides an indicator of the presence of residual plasmid DNA.

[0060] In some embodiments, the methods described herein produce an improved final product, which is a purified sample of a retroviral (e.g., lentivirus) vector. In some embodiments, this improvement involves the removal of residual cellular DNA from the cells used to produce the vector. In some embodiments, this improvement involves the removal of residual plasmid DNA used to shuttle retroviral nucleic acids to cells used to manufacture the vector. For several purposes, residual cellular DNA is residual nuclear DNA.

[0061] In some aspects, the improved final product exhibits residual cellular DNA of less than approximately 0.00001%, less than approximately 0.00005%, less than approximately 0.0001%, less than approximately 0.0005%, less than approximately 0.001%, less than approximately 0.005%, less than approximately 0.01%, less than approximately 0.05%, less than approximately 0.1%, less than 0.5%, or less than approximately 1% compared to the amount of cellular DNA present in the sample after clarification.

[0062] In some aspects, the improved final product exhibits total residual DNA of approximately less than 0.00001%, less than 0.00005%, less than 0.0001%, less than 0.0005%, less than 0.001%, less than 0.005%, less than 0.01%, less than 0.05%, less than 0.1%, less than 0.5%, or about 1% compared to the amount of residual DNA present in the clarified sample.

[0063] In some embodiments, after clarification, the vector suspension is subjected to a concentration step via ultrafiltration / dialysis filtration. In some embodiments, ultrafiltration / dialysis filtration may occur once during the purification process. In some embodiments, ultrafiltration / diafiltration may be performed multiple times during the purification process. Ultrafiltration / diafiltration is used to concentrate the vector by forcing the diluent through the filter in such a way that the diluent is removed from the vector preparation, while the vector cannot pass through the filter and thus remains in the vector preparation in a concentrated form. In some embodiments, the ultrafiltration / dialysis filtration process is tangential flow filtration (TFF), as described, for example, in the Millipore catalogue entitled "Pharmaceutical Process Filtration Catalogue" pp. 177-202 (Bedford, Massachusetts, 1995 / 96). TFF is widely used in the bioprocess industry for cell harvesting, clarification, purification, and concentration of products containing viruses. The system consists of three different process streams: feed solution, permeate, and retenate. Depending on the application, filters with different pore sizes may be used. In this invention, the retenate contains the product (retrovirus or lentiviral vector). Here, the specific ultrafiltration membrane selected has a pore size small enough to hold the vector and large enough to effectively remove impurities. Depending on the manufacturer and membrane type, for retroviral vectors, a nominal molecular weight cutoff (NMWC) of 100-1000 kDa is appropriate, such as a 300 kDa or 500 kDa NMWC membrane. In a preferred embodiment, the hollow fiber is used in the first or all TFF steps of the method. In some embodiments, hollow fibers are used in all TFF processes. In some embodiments, the molecular weight cutoff is between approximately 10 and approximately 1,000 kDa. In some embodiments, the molecular weight cutoff is between approximately 10 kDa and 750 kDa, approximately 10 and 500 kDa, approximately 10 kDa and 250 kDa, approximately 10 kDa and 100 kDa, approximately 100 kDa and 1,000 kDa, approximately 100 kDa and 750 kDa, approximately 100 kDa and 500 kDa, approximately 100 kDa and 250 kDa, approximately 250 kDa and 1,000 kDa, approximately 250 kDa and 750 kDa, approximately 250 kDa and 500 kDa, approximately 500 kDa and 1,000 kDa, approximately 500 kDa and 750 kDa, or 750 kDa and 1,000 kDa. In a preferred embodiment, the hollow fiber is a 750 kDa hollow fiber.

[0064] In some embodiments, the vector is purified via a high-speed centrifugation process to concentrate the vector. In some embodiments, high-speed centrifugation is greater than at least 10,000 × g. In some embodiments, ultracentrifugation is used to purify vectors. In some embodiments, the vector is purified through gradient preparations such as sucrose and iodixanol. In some embodiments, the vector is subjected to one or more solutions or compounds capable of concentrating the vector, such as polyethylene glycol (PEG) or LENTI-X CONCENTRATOR. In some embodiments, the vector, after being subjected to one or more concentrated solutions or compounds, is centrifuged and the vector is pelletized. In some embodiments, the pellet is resuspended in a buffer solution, thus producing a vector suspension.

[0065] In some embodiments, the viral vector purification process includes a column purification step. The column purification step may include column chromatography. This step separates the viral vector particles from cell debris and other contaminants for further purification of the viral vector particles, as is known in the art. In some embodiments, this step may be ion exchange column purification, for example, anion exchange or cation exchange. In some embodiments, column chromatography can be size exclusion chromatography. In some embodiments, column chromatography can be affinity chromatography. In some embodiments, column chromatography can be immobilized metal ion affinity chromatography. In some embodiments, column chromatography may include one or more chromatographic strategies. In some embodiments, chromatography is performed in an open chromatography system. In some embodiments, chromatography is performed in a closed chromatography system.

[0066] In some embodiments, the methods of the present disclosure do not utilize ion exchange chromatography. In some embodiments, the methods of the present disclosure do not utilize anion exchange chromatography. In some embodiments, the methods for purifying lentiviral vectors described herein do not utilize ion exchange chromatography or anion exchange chromatography.

[0067] After performing any or all of the above steps, the vector is filter-sterilized. Filter sterilization is a common process for pharmaceutical-grade materials and is known to those skilled in the art. Filter sterilization removes any remaining contaminants in the viral vector preparation. The level of contaminants after filter sterilization must be such that the vector preparation is for clinical use. In some embodiments, filter sterilization is carried out under sterile conditions. Suitable filters are well known to those skilled in the art. In some embodiments, the sterilization filter has a pore size of 0.22 μm.

[0068] In some embodiments, lentiviral vector preparations are purified by chromatography after filter sterilization and before ultrafiltration / dialysis filtration. To reduce potential contamination events, chromatography is preferably carried out using a closed-system process. In some embodiments, the chromatography is selected from ion exchange chromatography, multimodal chromatography, size exclusion chromatography, and affinity chromatography. In some embodiments, chromatography may be repeated two or more times with the same type of chromatography for each repeat, or with a different type of chromatography for at least one of the repeats.

[0069] V. target cells In some embodiments, the viral vector of the present invention may be used to modify target cells. In some embodiments, a viral vector can introduce a target transgene or nucleic acid sequence into immune cells or populations of immune cells. In some embodiments, the target transgene or nucleic acid sequence encodes an exogenous antigen receptor. In some embodiments, exogenous antigen receptors are chimeric antigen receptors (CARs) or T cell receptors (TCRs). In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells may be immune cells or their precursor cells. In some embodiments, the immune cell or its precursor cell may be a T cell. In some aspects, T cells can be cytotoxic T cells, regulatory T cells, or NKT cells. In an exemplary embodiment, the T cells are CD8+ T cells and / or CD4+ T cells.

[0070] In some embodiments, immune cells or populations of immune cells are harvested from apheresis samples from patients. In some embodiments, apheresis samples are cryopreserved before collecting immune cells or populations of immune cells. In some embodiments, the apheresis sample is a fresh apheresis sample from a patient that has not been cryopreserved. In some embodiments, immune cells or populations of immune cells are obtained from an apheresis sample during a process or protocol that includes an enrichment step. In some embodiments, the modified cells are self-cells. In some embodiments, modified cells are allogeneic cells.

[0071] In some embodiments, immune cells or populations of immune cells modified by the viral vector of the present invention can be used to treat, prevent, or ameliorate a disease or disorder. In some aspects, a disease or disorder may include, but is not limited to, malignancy disorders, including cancer, the growth, metastasis, and angiogenesis of benign and malignant tumors; arthritis; rheumatoid arthritis; allergic reactions; asthma; and autoimmune diseases, including systemic lupus erythematosus.

[0072] VI. Pharmaceutical composition The pharmaceutical compositions of the present invention may contain the gene-modified immune cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, adjuvants, or excipients. Such compositions may consist of buffers such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, the compositions of the present invention are preferably formulated for intravenous administration. In some embodiments, pharmaceutical compositions containing retroviral (e.g., lentiviral) vectors are suitable for administration to patients. In some embodiments, pharmaceutical compositions comprising retroviral (e.g., lentiviral) vectors are suitable for administration to cells, which are administered to patients.

[0073] VII. definition The following terms are expected to be well understood by those skilled in the art, but the following definitions are provided to facilitate the explanation of the subject matter currently disclosed.

[0074] The terms "a" or "an" can refer to one or more entities, that is, to multiple referents. Therefore, the terms "a" or "an," "one or more," and "at least one" are used interchangeably in this specification. Furthermore, a reference to "an element" using the indefinite article "a" or "an" does not exclude the possibility of two or more elements existing unless the context explicitly requires the existence of only one of those elements.

[0075] Throughout this specification, any reference to “one embodiment,” “an embodiment,” “one aspect,” or “an aspect” means that a particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of this disclosure. Therefore, wherever the expression “one embodiment” or “an embodiment” appears throughout this specification, it does not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic can be combined in any suitable way in one or more embodiments.

[0076] Where used herein, the terms “about” or “approximately” preceding a number indicate a value that is plus or minus 10% of that value.

[0077] As will be understood by those skilled in the art, all scopes disclosed herein, in order to provide all purposes, particularly written explanations, also encompass all possible subranges and combinations thereof. Any enumerated range can be readily recognized as being sufficiently explainable and possible to decompose the same range into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily decomposed into lower thirds, middle thirds, upper thirds, etc. Also, as will be understood by those skilled in the art, all language such as “up to,” “at least,” “greater than,” and “less than” includes the number mentioned and means a range that can subsequently be decomposed into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual numerical value. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells means a group having 1, 2, 3, 4, or 5 cells, etc.

[0078] Various aspects of this disclosure can be presented in range format. It should be understood that descriptions in range format are merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the invention. Therefore, a range description should be considered to specifically disclose not only the individual numerical values ​​within that range, but also all possible sub-ranges. For example, a description of the range from 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numerical values ​​within those ranges, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, 6. This applies regardless of the breadth of the range.

[0079] In general, the nomenclature used herein in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization is well known and commonly used in the art. The methods and techniques provided herein are generally carried out in accordance with conventional methods well known in the art, unless otherwise specified, and as described in the various general and more specific literature cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out in accordance with the manufacturer's specifications, as commonly achieved in the art or as described herein. The nomenclature used herein in relation to analytical chemistry, organic synthesis chemistry, and medicinal chemistry, as well as laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, preparation, formulation, and delivery of pharmaceuticals, and in the treatment of patients.

[0080] As used herein, “control” refers to an alternative sample used in an experiment for comparative purposes. A control may be “positive” or “negative.” As used herein, “control sample” or “reference sample” refers to a sample or reference that serves as a counter for comparison with an experimental sample. For example, the experimental sample may contain compounds A, B, and C in a vial, while the control may be the same type of sample, processed in the same way as the experimental sample but lacking one or more of compounds A, B, or C.

[0081] This disclosure provides novel methods and compositions for the production and purification of viral vectors. In a particular embodiment, a method for producing a lentiviral vector formulation provided herein includes the steps of treating a filter-sterilized lentiviral vector formulation with a nuclease, and concentrating the nuclease-treated lentiviral vector formulation to produce a lentiviral vector formulation, wherein the concentration step is the final step in the method. In a particular embodiment, a method for producing a lentiviral vector formulation provided herein includes the following steps in chronological order: (i) A step of culturing cells that produce lentiviral vectors, (ii) A step of recovering the supernatant containing the lentiviral vector, (iii) A step of clarifying the supernatant, (iv) A step of concentrating the clarified supernatant (including replacing the concentrated supernatant with a formulation buffer), (v) A step of purifying the lentiviral vector from the concentrated supernatant to produce a lentiviral vector preparation, (vii) A step of treating a filter-sterilized lentiviral vector preparation with a nuclease, and (viii) A step of concentrating the nuclease-treated lentiviral vector preparation to produce the final product.

[0082] Furthermore, unless otherwise specified, the experiments described herein utilize conventional molecular and cell biological and immunological techniques within the scope of those skilled in the art. Such techniques are well known to those skilled in the art and are well described in the literature. For example, see Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987–2008), including all supplements, Molecular Cloning; A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook and Harlow et al., Antibodies; A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0083] As used herein, the term “polynucleotide” is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed to monomeric “nucleotides.” Monomeric nucleotides can be hydrolyzed to nucleosides. Polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning and synthesis of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and polymerase chain reactions, etc.

[0084] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein consisting of two or more amino acids linked to each other by peptide bonds. As used herein, this term refers to both short chains, commonly called peptides, oligopeptides, and oligomers in the art, and long chains, commonly called proteins in the art, of which there are many types. Examples of “polypeptides” include biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0085] As used herein, the term “composition” is intended to encompass any product containing a specified component (e.g., a viral vector provided herein) in a specified amount, as well as any product directly or indirectly resulting from combining the specified components in a specified amount.

[0086] As used herein, the term “vector” means a composition of matter comprising isolated nucleic acids that can be used to deliver isolated nucleic acids into the interior of cells. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and lentiviral vectors.

[0087] As used herein, the term “expression vector” means a vector containing recombinant polynucleotides that include an expression regulatory sequence operatively ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be supplied in a host cell or an in vitro expression system. Expression vectors include all known in the art, such as cosmids, plasmids (e.g., naked or liposome-containing) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus), incorporating recombinant polynucleotides.

[0088] As used herein, the term “expression” is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0089] As used herein, the term “lentivirus” refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells. They are one of the most efficient methods of gene transfer because they can deliver large amounts of genetic information into the host cell's DNA. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means of achieving significant levels of gene transfer in vivo.

[0090] As used herein, the term “lentiviral vector” refers to a vector derived from at least a portion of a lentiviral genome, and in particular includes self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, but are not limited to, the “LENTIVECTOR” gene delivery technology from Oxford BioMedica and the “LENTIMAX” vector system from Lentigen. Non-clinical lentiviral vectors are also available and will be known to those skilled in the art.

[0091] As used herein, the term “residual nucleic acid” means non-retroviral (e.g., non-lentiviral) nucleic acids present in the samples of this disclosure after clarification of retroviral (e.g., lentiviral) vectors. Similarly, the term “residual DNA” means non-retroviral (e.g., non-lentiviral) nucleic acids present in the samples of this disclosure after clarification of retroviral (e.g., lentiviral) vectors. Residual nucleic acids refer to DNA and RNA. Residual nucleic acids mean genetic material corresponding to nuclear DNA and / or plasmids that remain in the samples described herein after clarification of vectors.

[0092] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence that codes for a protein or RNA" can include introns, insofar as a nucleotide sequence that codes for a protein may contain introns in some version.

[0093] This technology is not limited to the specific embodiments described herein, which are intended as single examples of individual embodiments of the technology. Many modifications and variations of this technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatus within the scope of this technology, in addition to those listed herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of this technology. It should be understood that this technology is not limited to specific methods, reagents, compounds, compositions, or biological systems, which, of course, can be changed. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them. [Examples]

[0094] · Example 1 • Manufacturing and purification of retroviral vectors HEK293T cells were grown in tissue culture flasks and then placed in a 10-layer cell factory for up to 14 days. On day 14, the cells were transfected with a plasmid encoding a third-generation lentiviral vector system, and the culture medium containing the viral vector was collected two days later, on day 16.

[0095] The cell culture medium was clarified. After clarification, prior to the first tangential flow filtration (TFF), the clarified medium was treated with DENARASE nuclease (c-LECTA GmbH; Leipzig, Germany) to remove residual cell genomic DNA and prevent filter clogging. Next, the clarified medium treated with the vector-containing nuclease was concentrated by ultrafiltration / dialysis filtration using TFF in formulation buffer (Tris, salt, sugar, and near-neutral pH). A 750 kDa hollow fiber column was used for the TFF. Then, the formulation buffer containing the vector was passed through a CAPTOCORE 700 resin (GE) column to remove host cell proteins, serum proteins, nucleic acids, and residual nucleases. Next, the resulting preparation was diluted, sterilized by filtration, and then treated a second time with DENARASE nuclease. This effectively removed any remaining contaminating DNA. The vector preparation was then passed through a 750 kDa hollow fiber again as a final ultrafiltration step to remove the nuclease and produce the final product. This process is shown in Figure 1.

[0096] To track the removal of residual DNA throughout the process, quantitative PCR (qPCR) was used at different steps of the method to detect the presence of the bullous stomatitis virus glycoprotein gene (VSV-G). Samples were collected after clarification of the cell culture supernatant, after the initial TFF step, and with the final product. Figure 2 shows that the amount of DNA remaining in the final product from vector collection was reduced to almost undetectable levels by the end of the method.

[0097] In particular, Figure 2 shows the results of a qPCR assay that detected and quantified the vesicular stomatitis virus glycoprotein (VSV-G) gene. Detection of VSV-G effectively measures the amount of residual VSV-G still present at multiple time points in the vector manufacturing process. The amount of residual VSV-G generally serves as an indicator of the amount of residual plasmid contaminants left over from vector production. Table 1 below identifies the process removal percentages observed after three steps in the vector manufacturing process. Figure 2 is a graphical representation of the "rVSV DNA persistence rate" data shown in Table 1. The clarified harvest sample was collected immediately after clarification. The "TFF 1" sample was collected immediately after the first tangential flow filtration step. The "Final Product" sample was collected immediately after the final tangential flow filtration step, thus yielding the final result of the lentiviral vector manufacturing and purification method.

[0098] [Table 1]

[0099] Since the second nuclease step is the second to last step in the process, there was a question as to how much residual nuclease would remain in the formulation after the TFF step, and consequently in the final product. The detection and quantification of residual endonucleases are performed using the MIILLIPORESIGMA BENZONASE ELISA Kit II, #1016810001. This is an enzyme-linked immunosorbent assay (ELISA) using antibodies specific to benzonase endonuclease. In the evaluation of the BENZONASE ELISA Kit II, the assay was tested using GMP-grade DENARASE samples. In this evaluation, DENARASE was detected by measuring multiple dilutions of DENARASE using a BENZONASE-specific antibody. Table 2 shows that the level of residual DENARASE nuclease after the TFF process is almost below the detection limit, indicating that only trace amounts of residual endonuclease are present in the final product.

[0100] [Table 2]

[0101] · Example 2 • Lentivirus titer We performed lentiviral vector titration using the human T-cell lymphoblastic lymphoma cell line SupT1, and measured the percentage of SupT1 cells expressing the transgene by flow cytometry.

[0102] Four samples were evaluated for the amount of lentivirus in each sample (SupT1 cells by flow cytometry). The samples were clarified, filtered, and subjected to the isolation and purification of lentiviruses according to the method described herein. For each sample, the method was used to evaluate lentiviruses with a titer of 1.75 × 10⁶. 6 ~4.84×10 6 The amount of lentivirus diluted before sterilization filtration varied accordingly. Figure 3 shows each lentivirus titer and the corresponding final yield. Based on Figure 3, it is clear that there is no standard dose (titer) dependent curve between titer and yield. Instead, the optimal titer is ~2 × 10⁻⁶ 6 Therefore, 1.75 × 10 6 At a titer of 2.55 × 10⁶, the final yield appears to decrease slightly. However, a significant decrease in the final yield of lentiviral vectors occurred at higher titers (for example, as shown in Figure 3, at 2.55 × 10⁶). 6 and 4.84 × 10 6 ).

[0103] · Example 3 • Residual DNA assay: The detection and quantification of residual vesicular stomatitis virus glycoprotein gene (VSV-G) were performed using quantitative PCR (QPCR) assays with primers and probes that specifically target VSV-G. The results shown in Table 3 summarize the findings from the three large-scale experiments.

[0104] • Assay of residual denases: The detection and quantification of residual endonucleases were performed using Millipore Sigma BENZONASE ELISA Kit II, #1016810001, an enzyme-linked immunosorbent assay (ELISA) using an antibody specific to BENZONASE endonuclease. In the evaluation of the BENZONASE ELISA Kit II, the assay was tested using GMP-grade DENARASE samples. In this evaluation, DENARASE was detected by measuring multiple dilutions of DENARASE using a BENZONASE-specific antibody. The results for residual DENARASE are shown in Table 3.

[0105] [Table 3]

[0106] · Example 4 • Scaled-up manufacturing The methods for producing clinical-grade lentiviral vectors described herein are performed on a clinical scale under Good Manufacturing Practice (GMP) conditions as published by the U.S. Food and Drug Administration. The clinical scale is 7 × 10⁶ 6 TU / ml ~ 8 × 10 7 This is defined as administering 20 to 60 patients per batch within the range of TU / ml, where TU is the transduction unit.

[0107] The methods described herein as exemplary may be suitably implemented in the absence of any element, elements, limitations, or limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein are for illustrative purposes only, not limiting ones, and the use of such terms and expressions is not intended to exclude any equivalent of any of the features or parts thereof shown or described. It should be recognized that various modifications are possible within the scope of the disclosures. Therefore, while this disclosure is specifically disclosed by preferred embodiments and any features, modifications and changes to the disclosures embodied herein can be relied upon by those skilled in the art, and such modifications and changes should be understood to be within the scope of this disclosure.

[0108] This disclosure is described extensively and generally herein. Each group of species and subgenera that falls under the general disclosure also forms part of the method. This includes the general description of the method with a proviso or negative limitation that removes any subject from a genus, whether or not the excised material is specifically described herein. The Art is not limited to the specific embodiments described herein, which are intended as single examples of individual embodiments of the Art. Many modifications and variations of the Art can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatus within the scope of the Art, in addition to those listed herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the Art. It should be understood that the Art is not limited to specific methods, reagents, compounds, compositions, or biological systems, which, of course, can change. Furthermore, it should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them.

[0109] Those skilled in the art will readily understand that this disclosure is well suited to achieving its purpose and obtaining the purposes and benefits mentioned therein, as well as what is inherent therein. Modifications and other uses therein will arise for those skilled in the art. These modifications are included within the spirit of this disclosure and are defined by the claims which define the non-limiting aspects of this disclosure.

[0110] Furthermore, if the characteristics or aspects of the disclosure are described in relation to the Markush Group, a person skilled in the art will recognize that the disclosure also describes any individual member or subgroup of any member of the Markush Group.

[0111] All references, articles, publications, patents, patent gazettes, and patent applications cited in this book are incorporated in their entirety by reference for all purposes.

[0112] However, the references, articles, publications, patents, patent publications, and patent applications cited herein shall not, and should not, be considered an acknowledgment or any form of suggestion that they constitute valid prior art or part of common technical knowledge in any country worldwide.

Claims

1. A method for producing a lentiviral vector preparation, the method is: (a) A step of treating a filter-sterilized lentiviral vector preparation with a nuclease, and (b) A step of concentrating the nuclease-treated lentiviral vector preparation to produce a lentiviral vector formulation, The method wherein the concentration step is the final step of the method.

2. The method according to claim 1, further comprising a step of clarifying the cell culture supernatant.

3. The method according to claim 2, wherein a first nuclease treatment is performed after the clarification step.

4. The method according to claim 3, wherein the nuclease has endonuclease activity.

5. The method according to claim 3, wherein the nuclease has exonuclease activity.

6. The method according to any one of claims 4 or 5, wherein the nuclease is a modified nuclease derived from Serratia marcescens.

7. The method according to any one of claims 3 to 6, wherein the first nuclease treatment precedes the ultrafiltration / dialysis filtration step.

8. The method according to claim 7, wherein the ultrafiltration / dialysis filtration step includes tangential flow filtration.

9. The method according to claim 7 or 8, wherein the ultrafiltration / dialysis filtration step includes hollow fiber filtration.

10. The method according to any one of the preceding claims, wherein the lentiviral vector preparation is diluted before filter sterilization of the lentiviral vector preparation.

11. The method according to claim 10, wherein the lentiviral vector preparation is diluted in a formulation buffer.

12. The method according to claim 11, wherein the lentiviral vector preparation is diluted in the formulation buffer during the ultrafiltration / diafiltration step according to any one of claims 7 to 9.

13. The method according to claim 1, wherein the method comprises a second nuclease treatment.

14. The method according to claim 13, wherein the second nuclease treatment is the second to last step of the method.

15. The method according to claim 14, wherein the nuclease has endonuclease activity.

16. The method according to claim 14, wherein the nuclease has exonuclease activity.

17. The method according to claim 15 or 16, wherein the nuclease is a modified nuclease derived from Serratia marcescens.

18. A method for producing a lentiviral vector preparation, the method is: (a) A step of culturing cells that produce lentiviral vectors, (b) A step of collecting the supernatant from the cultured cells, (c) A step of clarifying the supernatant, (d) A step of concentrating the clarified supernatant, (e) A step of purifying the lentiviral vector from the concentrated supernatant to produce a lentiviral vector preparation, (f) A step of filter sterilizing the lentiviral vector preparation, (g) The step of treating the filter-sterilized lentiviral vector preparation with one or more nucleases, and (h) A step of concentrating the nuclease-treated lentiviral vector preparation to produce the final product, Methods that include...

19. A method for producing a lentiviral vector preparation, the method is: (a) A step of culturing cells that produce lentiviral vectors, (b) A step of collecting the supernatant from the cultured cells, (c) A step of clarifying the supernatant, (d) A step of concentrating the clarified supernatant, (e) A step of purifying the lentiviral vector from the concentrated supernatant to produce a lentiviral vector preparation, (f) A step of filter sterilizing the lentiviral vector preparation, and (g) A step of concentrating the nuclease-treated lentiviral vector preparation to produce the final product, Includes, The clarified supernatant and / or lentiviral vector preparation is treated with a nuclease, in a method.

20. The method according to claim 18 or 19, wherein the step of concentrating the clarified supernatant includes the step of exchanging the concentrated supernatant with a formulation buffer.

21. The method according to claim 19, wherein the clarified supernatant and the lentiviral vector preparation are treated with a nuclease.

22. The method according to any one of claims 19 to 21, wherein the clarified supernatant is treated with a nuclease prior to (d).

23. The method according to any one of claims 19 to 21, wherein, after (c), the clarified supernatant is treated with a nuclease.

24. The method according to any one of claims 18 to 23, wherein the nuclease has endonuclease activity.

25. The method according to any one of claims 18 to 23, wherein the nuclease has exonuclease activity.

26. The method according to claim 24 or 25, wherein the nuclease is a modified nuclease derived from Serratia marcescens.

27. A method for producing a lentiviral vector formulation, comprising the following steps in chronological order: (a) A step of culturing cells that produce lentiviral vectors, (b) A step of recovering the supernatant containing the lentiviral vector, (c) A step of clarifying the supernatant, (d) A step of treating the clarified supernatant with a nuclease, (e) A step of concentrating the clarified supernatant treated with the nuclease, comprising replacing the concentrated supernatant with a formulation buffer, (f) A step of purifying the lentiviral vector from the concentrated supernatant and producing a lentiviral preparation, (g) A step of filter sterilizing the lentiviral vector preparation, (h) The step of treating the filter-sterilized lentiviral vector preparation with a nuclease, and (i) A step of concentrating the nuclease-treated lentiviral vector preparation to produce a final product.

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