Manufacture of viral particles

Abstract

Methods for large scale production of viral particles, as well as compositions and methods for using the viral particles, are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 342,975 filed on May 17, 2022, U.S. Provisional Application No. 63 / 371,756 filed on August 17, 2022, U.S. Provisional Application No. 63 / 371,864 filed on August 18, 2022, and U.S. Provisional Application No. 63 / 440,093 filed on January 19, 2023, and the contents of the above provisional applications are incorporated by reference in their entirety. Incorporation by Reference to the Sequence Listing

[0002] This application is filed with a sequence listing in electronic form. The sequence listing is provided as a file named 260132000440SeqList.xml, created on May 17, 2023, and is 207,707 bytes in size. The information in the electronic format of the sequence listing is incorporated by reference in its entirety. Technical Field

[0003] The present disclosure provides methods for large-scale production of viral particles, as well as compositions and methods for using the viral particles. Background Art

[0004] Gene therapy has shown significant clinical success in treating cancer and other diseases. However, access to these life-saving therapies has been limited due to significant challenges in cost, supply chain, and manufacturing. Retroviruses are typically used as delivery agents for transferring one or more target nucleotides to one or more target sites. Among retroviruses, the lentiviral vector system has attracted considerable interest because lentiviruses are able to infect non-dividing cells. In addition, lentiviral vectors allow for stable, long-term expression of target genes. In most small-scale applications, the vector can be concentrated and purified by relatively simple methods using centrifugation techniques. However, there are significant challenges in scaling up the purification method for large-scale production for clinical use.

[0005] In particular, when considering the production of viral vectors for human use, the vector purification process is directly related to safety in terms of purity. There remains a need for a large-scale retroviral vector purification method that produces a high purity product. Summary of the invention

[0006] The present disclosure is based at least in part on the discovery of a method for making viral particles (e.g., lentiviral particles) for in vivo administration to a subject. Specifically, as shown herein, a mixture of host cells and viral particles is subjected to at least two filtration steps to reduce contaminants (e.g., host cell DNA and proteins) before concentrating the mixture via chromatography and ultrafiltration. Without wishing to be limited by theory, the filtration steps described herein reduce contaminants to an amount sufficient for in vivo administration. Therefore, in some aspects, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising: (i) filtering the suspension mixture comprising the host cell population and the lentiviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture through a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate through a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate through a third filter, thereby producing a filtered lentiviral particle preparation, wherein the third filter has a lower retention threshold than the second filter; and (ii) concentrating the filtered lentiviral particle preparation, wherein concentrating comprises chromatography and ultrafiltration.

[0007] In other aspects, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising (i) contacting the host cell population in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the host cell population of step (i) for a period of time sufficient to produce a suspension mixture comprising the host cell population and lentiviral particles; (iii) filtering the suspended mixture to remove contaminants, the filtering comprising: (a) contacting the mixture with an endonuclease, (b) filtering the mixture through a first filter to obtain a first filtrate, wherein the first filter is a depth filter (c) filtering the first filtrate through a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (d) filtering the second filtrate through a third filter, thereby producing a filtered lentiviral particle preparation, wherein the third filter has a lower retention threshold than the second filter; and (iv) concentrating the filtered lentiviral particle preparation, wherein concentrating comprises chromatography and ultrafiltration.

[0008] In some aspects, the host cell includes a human cell. In some aspects, the human cell includes a HEK293 cell, a HEK293T cell, a HEK293F cell, a HEK293FT cell, a Te671 cell, a HT1080 cell or a CEM cell. In some aspects, the cell includes a HEK293 cell. In some aspects, the cell includes a HEK293T cell.

[0009] In some aspects, the first filter has a retention threshold of 1-60 μm. In some aspects, the first filter has a retention threshold of 60 μm. In some aspects, the second filter has a retention threshold of 0.4-4 μm. In some aspects, the second filter has a retention threshold of 0.45 μm. In some embodiments, the third filter has a retention threshold of 0.45 μm ± 0.2 μm. In some aspects, the third filter has a retention threshold of 0.2-0.3 μm. In some aspects, the third filter has a retention threshold of 0.2 μm. In some aspects, the first filter has a retention threshold of 60 μm, the second filter has a retention threshold of 0.45 μm, and the third filter has a retention threshold of 0.2 μm.

[0010] In some aspects, the second filter and the third filter are two layers within a dual-layer filter assembly. In some aspects, the third filter is a dual-layer filter comprising a first layer filter and a second layer filter, wherein the second layer filter has a smaller retention threshold than the first layer filter. In some aspects, the retention threshold of the first filter is 60 μm, the retention threshold of the second filter is 0.45 μm, the retention threshold of the first layer filter is 0.45 μm, and the retention threshold of the second layer filter is 0.2 μm.

[0011] In some aspects, an endonuclease is present in steps (i)(a)-(i)(c). In some aspects, an endonuclease is present in steps (iii)(b)-(iii)(d).

[0012] In some aspects, the chromatography is an anion exchange chromatography. In some embodiments, the AEX chromatography includes eluting the lentiviral particles with a salt buffer. In some embodiments, the salt buffer comprises NaCl. In some embodiments, the concentration of the NaCl is about 0.5M to 3M. In some embodiments, the concentration of the NaCl is about 0.5M to 1M. In some embodiments, the NaCl is or is about 0.75M. In some embodiments, the concentration of the NaCl is about 1M to 3M. In some embodiments, the concentration of the NaCl is about 1.5M to 2.5M. In some embodiments, the NaCl is about 2M.

[0013] In some aspects, the chromatogram is carried out before ultrafiltration. In some aspects, the ultrafiltration is ultrafiltration / diafiltration (UF / DF). In some aspects, the UF / DF is carried out by one or more tangential flow filtration (TFF) steps. In some aspects, the filter of the one or more TFFs is a hollow fiber filter. In some aspects, the nominal molecular weight cutoff (NMWC) of the hollow fiber filter is or is about 500kDa. In some aspects, the TFF comprises a first tangential flow filtration (TFF) step and a second tangential flow filtration (TFF) step. In some aspects, the first TFF is carried out with a first hollow fiber filter and the second TFF is carried out with a second hollow fiber filter. In some aspects, the first hollow fiber filter and the second hollow fiber filter have the same nominal molecular weight cutoff (NMWC). In some aspects, the NMWC is 500kDa. In some aspects, compared with the second hollow fiber filter, the first hollow fiber filter has a larger nominal molecular weight cutoff (NMWC). In some aspects, the first hollow fiber filter is 500kDa. In some aspects, compared with the second hollow fiber filter, the first hollow fiber filter has a larger surface area. In some aspects, the first hollow fiber filter is between 790cm2 and 1600cm2. In some aspects, compared with the second hollow fiber filter, the first hollow fiber filter accommodates a larger volume. In some aspects, the volume of the first hollow fiber filter is 300mL.

[0014] In some aspects, the method includes sterilizing and filtering the filtered preparation after concentration, thereby producing a sterilized preparation. In some aspects, sterilizing and filtering includes filtering the filtered preparation with a fourth filter. In some aspects, the fourth filter has a retention threshold of 0.2 μm. In some aspects, the method includes preparing the sterilized preparation in a buffer, thereby producing a drug substance.

[0015] In some aspects, the method is performed at a pH of 6-8. In some aspects, the lentiviral formulation is for in vivo administration to a subject.

[0016] In some respects, compared with the amount of the pollutant in the second filtrate, the amount of the pollutant in the preparation of the filtration is reduced. In some respects, the amount of the pollutant in the preparation of the sterilization is in an acceptable level for in vivo administration to a subject. In some respects, the pollutant includes host cells, host cell DNA (hcDNA) and / or host cell proteins (HCP). In some respects, the amount of hcDNA accounts for less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.5% of the preparation of the sterilization. In some respects, the amount of hcDNA in the preparation of the sterilization is reduced by more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99%. In some aspects, the amount of HCP accounts for less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.5% of the sterilized formulation. In some aspects, the amount of HCP in the sterilized formulation is reduced by greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99%.

[0017] In some aspects, the amount of hcDNA in the filtered preparation is less than about 2500ng / 1E9 TU. In some aspects, the amount of hcDNA in the filtered preparation is at least about 80 times lower than the amount of hcDNA in the suspended mixture. In some aspects, the amount of hcDNA in the filtered preparation is at least about 5 times lower than the amount of hcDNA in the second filtrate. In some aspects, the amount of HCP after chromatography is less than about 3000μg / 1E9 TU. In some aspects, the amount of HCP after chromatography is at least about 40 times lower than the amount of HCP before chromatography. In some aspects, the amount of HCP after chromatography is at least about 99% lower than the amount of HCP before chromatography. In some aspects, after the first UF / DF step, the amount of HCP is less than about 1500μg / 1E9 TU. In some aspects, after the second UF / DF step, the amount of HCP is undetectable.

[0018] In some aspects, the suspension mixture comprises culture medium, and condition is that the culture medium does not contain serum and / or animal by-products. In some aspects, the cultivation of step (ii) lasts 40-48 hours. In some aspects, the filtration and the concentration are carried out in the time period of 5-8 hours. In some aspects, the volume of the suspension mixture is 3-50 liters. In some aspects, the volume of the suspension mixture is 5L to 200L. In some aspects, the volume of the suspension mixture is 100L to 200L. In some aspects, the volume of the suspension mixture is for or about 180L to for or about 200L.

[0019] In some aspects, the lentiviral particle comprises at least one payload. In some aspects, the payload is at least one nucleic acid. In some aspects, the at least one nucleic acid is a non-coding nucleic acid, optionally wherein the non-coding nucleic acid is siRNA, miRNA or shRNA. In some aspects, the at least one nucleic acid is a polynucleotide encoding a polypeptide of interest. In some aspects, the at least one plasmid is a polynucleotide encoding a polypeptide of interest. In some aspects, the polypeptide of interest is a chimeric antigen receptor (CAR). In some aspects, the CAR is specific to tumor-associated antigens. In some aspects, the tumor-associated antigen is CD19, BCMA, GPRC5D, ROR1, FcRL5, alpha-fetoprotein or Her2. In some aspects, the CAR is a universal CAR. In some aspects, the universal CAR comprises a tag binding domain. In some aspects, the tag is fluorescein. In some aspects, the CAR comprises a hapten binding domain.

[0020] In some aspects, the lentiviral particles are included in a surface engineered fusion protein exposed on the surface of the lentiviral particles, optionally wherein the surface engineered protein is embedded in a lipid bilayer. In some aspects, the surface engineered protein is composed of a single binding domain protein that binds to a target molecule on a target cell. In some aspects, the surface engineered protein is composed of a multi-binding domain protein, wherein each binding domain binds to a target molecule on a target cell, optionally wherein each binding domain binds to a different target molecule. In some aspects, the single binding domain protein or the multi-binding domain protein is an immune cell activation protein. In some aspects, the surface engineered protein is a fusion protein comprising an immune cell activation protein and a viral envelope protein.

[0021] In some aspects, the lentiviral particles include a viral envelope containing an immune cell activation protein and a viral envelope protein. In some aspects, the at least one plasmid is a plasmid encoding an immune cell activation protein and a plasmid encoding a viral envelope protein. In some aspects, the immune cell activation protein is specifically bound to the following proteins CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244, TCR alpha chain, TCR beta chain, TCR zeta chain, TCR gamma chain, TCR delta chain, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit or NKp80.

[0022] In some aspects, the immune cell activation protein comprises at least one binding domain, and the at least one binding domain binds to a target molecule selected from a T cell activation receptor, a costimulatory molecule or an adhesion molecule. In some aspects, the immune cell activation protein comprises a single binding domain, and the single binding domain binds to a target molecule selected from a T cell activation receptor, a costimulatory molecule or an adhesion molecule. In some aspects, the immune cell activation protein comprises multiple binding domains, and the multiple binding domains bind to two or more target molecules selected from T cell activation receptors, costimulatory molecules and adhesion molecules. In some aspects, the immune cell activation protein comprises multiple binding domains, and the multiple binding domains each bind to different target molecules, and the target molecules are T cell activation receptors, costimulatory molecules and adhesion molecules. In some aspects, the immune cell activation protein comprises at least one binding domain in conjunction with at least one costimulatory molecule. In some aspects, the T cell activation receptor is CD3; the costimulatory molecule is CD28, CD137 or CD134; and / or the adhesion molecule is CD58 or CD2. In some aspects, each of the at least one binding domain is independently selected from an antibody or antigen binding fragment, or an extracellular domain of a natural ligand of the target molecule. In some aspects, the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a Cocal virus G protein.

[0023] In some aspects, the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein or a Kokar virus G protein. In some aspects, the viral envelope protein comprises at least one co-stimulatory molecule. In some aspects, the at least one plasmid is a plasmid encoding a co-stimulatory molecule. In some aspects, the at least one co-stimulatory molecule is CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, OX40, 4-1BB, CD40L or any combination thereof. In some aspects, the at least one plasmid is a plasmid encoding a helper virus protein. In some aspects, the helper virus protein is rev and / or gagpol.

[0024] In some aspects, the host cell population is contacted with a plasmid mixture comprising (i) a plasmid encoding a gene of interest; (ii) a plasmid encoding a rev viral protein; (iii) a plasmid encoding a gagpol viral protein; and (iv) a plasmid encoding a viral envelope protein. In some aspects, the plasmid mixture comprises (v) a plasmid encoding an immune cell activation protein, (vi) a plasmid encoding a co-stimulatory molecule, or any combination of (vii) (v)-(vi).

[0025] In some aspects, the present disclosure provides a lentiviral formulation produced by the methods described herein. In some aspects, the lentiviral formulation has an infectious titer of 2.0 to 6 x 10 8 TU / mL. In some aspects, the infectious titer of the formulation is 2.5 to 4.7 x 10 8In some aspects, the total number of infection units in the preparation is 4x1010TU to 8x1010TU. In some aspects, the total number of infection units in the preparation is 5x1010TU to 7x1010TU, optionally or about 6x1010TU. In some aspects, the preparation comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.5% HCP. In some aspects, the preparation comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.5% hcDNA. In some aspects, the formulation comprises greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% reduction in HCP. In some aspects, the formulation comprises greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% reduction in hcDNA, optionally compared to the filtered formulation before the concentration. In some aspects, the formulation comprises greater than 99% reduction in hcDNA and greater than 99% reduction in HCP, optionally compared to the filtered formulation before the concentration. In some aspects, the formulation comprises less than 1% hcDNA and less than 1% HCP. In some aspects, the lentiviral formulation comprises a titer of 2.5 to 4.7x10 8 TU / mL of lentiviral vector, wherein the formulation comprises less than 1% hcDNA and less than 1% HCP. In some aspects, the volume of the formulation is 1 mL to 500 mL, optionally 10 mL to 100 mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Various objects and advantages of the present disclosure, as well as a more complete understanding of the present disclosure, may be more clearly and more readily appreciated by referring to the following detailed description and appended claims, taken in conjunction with the accompanying drawings, in which:

[0027] Figure 1A-Figure 1C Schematic diagrams depicting upstream manufacturing processes, downstream manufacturing processes, or both upstream and downstream manufacturing processes of lentiviral particles are provided. Figure 1A The upstream process for viral particle production using host cells in suspension is shown, and Figure 1B The downstream process of viral particle purification is shown. Figure 1C An exemplary lentiviral production process is shown.

[0028] Figure 2A box plot showing the scale-up of the upstream process is provided. Consistent product titers were achieved when the bioreactor was scaled up from 3 to 10 to 40 liters.

[0029] Figure 3 The number of surface engineered lentiviral vectors (LVs) produced by HEK 293 cells or HEK 293T harvested in bioreactors capable of accommodating 3 L, 10 L, 40 L, or 50 L of cell culture medium is shown. The surface of LVs produced by HEK 293 cells was engineered to express a single domain fusion protein that is expressed in Figure 3 The surface of LV produced by HEK 293T cells was engineered to express a multidomain fusion protein that is expressed in Figure 3 It is called LV-5.

[0030] Figure 4A-4B Provided are schematic diagrams depicting host cell proteins (HCPs) and host cell DNA (hcDNA) present in the viral particle formulations after upstream and downstream processes provided herein. LV-1 refers to a lentivirus expressing a single domain fusion protein. LV-5 refers to a lentivirus expressing a multi-domain fusion protein.

[0031] Figure 5 A table showing the operating conditions for seven different downstream processes is provided.

[0032] Figure 6 Tables showing viral particle titers and impurities are provided. Titers are measured by ddPCR, p24, and particles per transducing unit (TU). Impurities include residual HCP, hcDNA, E1ADNA, benzonase, and endotoxin.

[0033] Figure 7 Bar graphs are provided showing the amount of host cell DNA after a second filtration step and a third filtration step with or without the use of an endonuclease in a downstream process.

[0034] Figure 8 A bar graph is provided showing the amount of host cell protein after harvest clarification, ion exchange chromatography and subsequent ultrafiltration / diafiltration in downstream processes.

[0035] Fig. 9 Bar graphs showing residual DNA and host cell protein per dose calculated for individual steps in process No. 6 are provided.

[0036] Fig.10A schematic diagram showing candidate downstream processes is provided. TFF refers to tangential flow filtration. Ultrafiltration / diafiltration (UF / DF) is performed in two steps (referred to as TFF-A and TFF-B).

[0037] Figure 11A-11B A schematic diagram depicting the amount of host cell protein after downstream processing is provided. Fig.11A Box plots showing HCP (μg / TU) in clarified harvest, chromatographically purified harvest and UF / DF purified harvest are provided. Protein was measured by ELISA assay in four different runs (labeled). Fig. 11B Box plots showing host cell protein in the clarified harvest, chromatographically purified harvest, and UF / DF purified harvest as measured by LC-MS are provided.

[0038] Fig.12 A schematic diagram depicting host cell DNA present after downstream processing is provided.Host cell DNA was measured by qPCR in four different runs (labeled).

[0039] Fig.13 A schematic diagram is provided depicting the yield of lentiviral particles in the following lentiviruses, wherein the yield is depicted as the percentage of the amount of virus loaded onto the column in the chromatography step after elution at 0.75 M NaCl compared to elution at 2 M NaCl: lentivirus expressing a single domain protein (LV-1), lentivirus expressing three different single domain proteins + GFP (LV-2), lentivirus expressing three different single domain proteins + CAR (LV-3), lentivirus expressing a multi-domain fusion protein + CAR (LV-4), or lentivirus expressing a multi-domain fusion protein (LV-5). DETAILED DESCRIPTION

[0040] In some embodiments, the disclosure provides methods for preparing infectious viral particle formulations suitable for pharmaceutical product manufacture and / or in vivo use. In some embodiments, the disclosure provides methods for preparing viral particle formulations for direct in vivo administration. In some embodiments, preparing a viral particle formulation includes subjecting a mixture of host cells and viral particles to at least two filtration steps to clarify the mixture. In some embodiments, preparing a viral particle formulation includes subjecting a mixture of host cells and viral particles to at least two filtration steps to clarify the mixture, and then concentrating the clarified mixture via chromatography and ultrafiltration.

[0041] In some embodiments, preparing a viral particle formulation comprises subjecting a mixture of host cells and viral particles to three filtration steps to clarify the mixture. In some embodiments, preparing a viral particle formulation comprises subjecting a mixture of host cells and viral particles to three filtration steps to clarify the mixture, and then concentrating the clarified mixture via chromatography and ultrafiltration.

[0042] Embodiments of the present disclosure may employ conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, and immunology, which are within the capabilities of ordinary technicians in the field. Such techniques are explained in the literature. See, e.g., J. Sambrook, EF Fritsch and T. Maniatis (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel; FM et al. (1995 and regular supplements) Current Protocols in Molecular Biology, Chapters 9, 13 and 16, John Wiley & Sons, New York, NY; B. Roe, J. Crabtree and A. Kahn (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; JM Polac and James O'D. McGee (1990) In Situ Hybridization: Principles and Practice; Oxford University Press; MJ Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and DMJ Lilley and JEDahlberg (1992) Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA; Methods in Enzymology, Academic Press, edited by Roe, Simon, Protein Purification Techniques. 2nd edition Oxford: Oxford University Press, 2001; Sofer, Gail and Hagel, Lars. Handbook of Process Chromatography: A Guide to Optimization, Scale-up, and Validation.London and San Diego: Academic Press, 1997; Janson, Jan-Christer and Ryden, Lars, eds. Protein Purification: Principles, High Resolution Methods, and Applications. 2nd edition New York: John Wiley & Sons, Inc., 1998, Masters, John RW, ed. Animal Cell Culture: A Practical Approach. 3rd edition Oxford: Oxford University Press, 2000. Each of these general texts is incorporated herein by reference. I. Methods for Producing Viral Particles

[0043] Scaling up viral particle production for large-scale manufacturing for clinical use presents challenges in terms of purity and yield. The present disclosure aims to overcome this challenge by providing a scalable method of manufacturing viral particles that are pure enough for direct injection in vivo in human subjects.

[0044] In some embodiments, the present disclosure provides a scalable, suspension cell culture-based manufacturing process.Some embodiments of the methods of the present disclosure provide consistent cell culture titers for bioreactor scale-up from 3L to 10L to 40L.

[0045] In some embodiments, upstream and downstream processes in the production and purification of viral vector particles for in vivo use are described. The disclosure also provides methods for effectively removing host cell DNA and proteins, which are critical quality attributes directly related to drug product safety. The disclosure provides methods that can be transferred to clinical and commercial scale manufacturing of viral particles for in vivo administration. A. Upstream Process

[0046] In some embodiments, the present disclosure provides a method for preparing a viral particle formulation.

[0047] In some embodiments, the method for preparing a viral particle formulation includes seed culture, bioreactor growth, transfection with a vector, and vector production, referred to herein as an upstream process. The goal of the upstream process is to increase cell yield. In some embodiments, the upstream process includes Figure 1A Upstream process steps shown.

[0048] In some embodiments, the methods described herein include upstream processes to produce viral particles. In some embodiments, the upstream processes include (i) host cell amplification; (ii) plasmid transfection; (iii) harvesting viral particles; or (iv) any combination of (i)-(iii). In some embodiments, the methods described herein produce retroviral particles. In some embodiments, the methods described herein produce lentiviral particles.

[0049] In some embodiments, the present disclosure provides a method for preparing a viral particle preparation, the method comprising (i) contacting a host cell population in suspension with at least one plasmid encoding a viral protein; (ii) culturing the host cell population of step (i) for a period of time sufficient to produce a suspension mixture comprising the host cell population and viral particles; (iii) filtering the suspension mixture to remove contaminants, the filtration comprising: (a) contacting the mixture with a nuclease, (b) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (c) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (d) filtering the second filtrate with a third filter to produce a filtered viral particle preparation, wherein the third filter has a smaller retention threshold than the second filter; and (iv) concentrating the filtered viral particle preparation, wherein the concentration comprises chromatography and ultrafiltration.

[0050] In some embodiments, the present disclosure provides a method for preparing a retroviral particle formulation, the method comprising (i) contacting a host cell population in suspension with at least one plasmid encoding a retroviral protein; (ii) culturing the host cell population of step (i) for a period of time sufficient to produce a suspension mixture comprising the host cell population and retroviral particles; (iii) filtering the suspension mixture to remove contaminants, the filtration comprising: (a) contacting the mixture with a nuclease, (b) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (c) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (d) filtering the second filtrate with a third filter to produce a filtered retroviral particle formulation, wherein the third filter has a smaller retention threshold than the second filter; and (iv) concentrating the filtered viral particle formulation, wherein the concentration comprises chromatography and ultrafiltration.

[0051] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising (i) contacting a host cell population in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the host cell population of step (i) for a period of time sufficient to produce a suspension mixture comprising the host cell population and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, the filtration comprising: (a) contacting the mixture with an endonuclease, (b) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (c) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (d) filtering the second filtrate with a third filter to produce a filtered lentiviral particle formulation, wherein the third filter has a smaller retention threshold than the second filter; and (iv) concentrating the filtered lentiviral particle formulation, wherein the concentration comprises chromatography and ultrafiltration. 1. Host Cell Expansion

[0052] The viral vector can be propagated appropriately in a cell (also referred to as a "host cell"). According to the present disclosure, the cell can be any cell in which the desired viral vector can be propagated. By using a stable production / packaging cell line, a certain number of viral vector particles can be propagated (e.g., to prepare a suitable titer of viral vector) for subsequent purification.

[0053] As used herein, the term "producer cell" or "host cell" refers to a cell that contains all the elements required to produce lentiviral vector particles. As used herein, the term "packaging cell" refers to a cell that contains those elements required to produce infectious recombinant viruses that are lacking in the RNA genome. Typically, such packaging cells contain one or more production plasmids that are capable of expressing viral structural proteins (such as codon-optimized gag-pol and env), but they do not contain packaging signals.

[0054] In some embodiments, the host cells / packaging cells of the present disclosure are derived from mammalian cells and. In the practice of the methods of the present disclosure, any cell type capable of supporting viral replication is acceptable.

[0055] In some embodiments, host cells can be selected from any cell that allows the production of enveloped viruses. In some embodiments, host cells are selected from human cells (HEK293, HEK293T, HEK293F, HEK293FT, Te671, HT1080, CEM), murine cells (NIH-3T3), Mustelidae cells (Mpf) and canine cells (D17) (Miller and Chen 1996; Miller 2001; Merten 2004; Rodrigues et al. 2011; Stacey and Merten 2011). Other non-limiting examples of host cells include, but are not limited to, Vero, MDBK, BK-21, CV-1 cells and mammalian fibroblasts or cultured epithelial cells. In some embodiments, host cells can be easily obtained from commercial sources (e.g., ATCC, Rockwell, Maryland).

[0056] In some embodiments, the host cell / packaging cell of the present disclosure is derived from a primate cell, such as a human embryonic kidney cell. In some embodiments, the cell can be derived from an existing cell line, for example, from a HEK 293 cell line. In some embodiments, the host cell is a HEK 293 cell line. In some embodiments, the cell can be derived from an existing cell line, for example, from a HEK 293T cell line. In some embodiments, the host cell is a HEK 293T cell line. In some embodiments, the cell is suitable for suspension culture.

[0057] In some embodiments, host cells are cultured in a medium suitable for growing mammalian cells and suitable for producing enveloped viruses. The medium may be supplemented with additives well known in the art, such as antibiotics and serum (especially fetal bovine serum, etc.) added at appropriate concentrations. It is worth noting that the medium used may contain serum or may not contain serum. The culture medium for mammalian cells is well known in the art, including but not limited to DMEM (Double Modified Eagle Medium), RPMI1640 or a mixture of various culture media including, for example, DMEM / F12, or a serum-free medium (such as Freestyle (Life Technologies) or 293 (Sigma-Aldrich), and LV-MAX TM Culture medium (ThermoFisherScientific #A3583402).

[0058] In some embodiments, the host cells are cultured in a serum-free medium. In some embodiments, the host cells are cultured in a medium free of animal products.

[0059] In some embodiments, the host cells are first diluted into a suitable culture medium and then centrifuged to remove the culture medium. In some embodiments, a seed culture expansion of the host cells is performed after removing the culture medium. In some embodiments, a seed culture expansion is performed to achieve a target cell number. In some embodiments, a range of target cell numbers are achieved. In some embodiments, different target cell numbers correspond to containers of different sizes.

[0060] In some embodiments, seed expansion is first performed in multiple vessels until a sufficient number of cells are obtained for inoculation of a bioreactor.

[0061] In some embodiments, seed expansion includes target inoculation cell density and target passage cell density. In some embodiments, the seed culture expansion of host cells is carried out to achieve the target inoculation cell density. In some embodiments, the seed culture expansion of host cells is carried out to achieve the target passage cell density. In some embodiments, seed expansion is started by inoculating a container with the target inoculation cell density. In some embodiments, the target passage cell density is achieved after cultivating host cells in a container for host cell expansion. In some embodiments, once the target passage cell density is achieved, the host cell is passaged to another container, and one or more (for example, continuously) is performed. In some embodiments, each passage to a new container is carried out with the target inoculation density, and cultivation is maintained under the conditions for achieving the target passage density. In some embodiments, seed expansion is carried out to achieve the target cell quantity for bioreactor inoculation.

[0062] In some embodiments, cells are seeded in one or more containers for cultivation in culture medium until the target cell density is achieved. In some embodiments, the container includes a tissue culture bottle, a culture dish or a roller bottle. In some embodiments, the container is a tissue culture bottle. In some embodiments, the tissue culture bottle is a shake bottle. In some embodiments, the tissue culture bottle is suitable for suspension cells. In some embodiments, the tissue culture bottle is an untreated flask. In some embodiments, the container is a bioreactor. In some embodiments, the bioreactor is suitable for suspension cells.

[0063] In some embodiments, seed culture expansion is continuously performed in multiple containers. In some embodiments, multiple containers include different containers. In some embodiments, different containers have different sizes. In some embodiments, the size of the container can accommodate a volume between 0.125L and 50L (such as 0.125L to 10L or 0.125L to 5L), and in a series of containers for expansion, each container has an incremental size compared to the previous container. In some embodiments, the size of the container can accommodate a volume between 0.125L and 5L, and in a series of containers for expansion, each container has an incremental size compared to the previous container. In some embodiments, the container can accommodate a volume of 0.125L, 0.25L, 0.5L, 1L, 1.6L, 2L or 5L. In some embodiments, multiple containers include two or more shaking flasks. In some embodiments, multiple containers are used for continuous passage of cells. In some embodiments, cells are cultured at 2x10 5 cells / mL to 5x10 5 cells / mL (e.g., 3x10 5 cells / mL to 5x10 5 Cells are seeded in containers (e.g., shake flasks) at a target seeding density between 3.5 and 6 x 10 cells / mL and serially passaged until the cells reach a target seeding density between 3.5 and 6 x 10 6 cells / mL (e.g. 4.0 to 6x10 6 In some embodiments, the method for seed expansion before inoculation of the bioreactor involves 2, 3, 4, 5 or more passages of the cells. In some embodiments, the method for seed expansion involves passage of the cells at least 5 times. In some embodiments, the cells are passaged into containers of increasing size, such as containers that can accommodate 0.125L, 0.5L, 1L, 1.6L, and 5L volumes.

[0064] In some embodiments, the seed culture expansion is carried out in multiple shake flasks. In some embodiments, the seed culture expansion is carried out in two or more, three or more, or four or more shake flasks. In some embodiments, the seed culture expansion is carried out in at least two, at least three, at least four, or at least five shake flasks.

[0065] In some embodiments, seed culture expansion is initiated by inoculating the host cells that achieve the target inoculation cell density into the first shake flask. In some embodiments, the first shake flask contains a volume of 0.125 L. In some embodiments, the target inoculation cell density of the first shake flask is not less than 5x10 5 cells / mL.

[0066] In some embodiments, once the inoculated cells achieve the target passage cell density in the first shake flask, the host cells are passaged to the second shake flask at the target inoculation cell density. In some embodiments, the second shake flask holds a volume of 0.5 L. In some embodiments, the target inoculation cell density of the second shake flask is about 2x10 5 cells / mL to 5x10 5 cells / mL, such as or about 3x10 5 cells / mL to 5x10 5 In some embodiments, the target inoculation cell density of the second shake flask is not less than 5x10 5 In some embodiments, the target inoculation cell density of the second shake flask is about 5x10 5 In some embodiments, the target cell density for the first passage flask is about 3.5 to 6 x 10 6 In some embodiments, the target cell density for the first shake flask is 4x10 6 In some embodiments, the target cell density for the first shake flask is 5x10 6 In some embodiments, the target cell density for the first shake flask is 6 x 10 6 cells / mL.

[0067] In some embodiments, once the inoculated cells achieve the target passage cell density in the second shake flask, the host cells are passaged to a third shake flask at the target inoculation cell density. In some embodiments, the third shake flask holds a volume of 1 L. In some embodiments, the target inoculation cell density of the third shake flask is about 2 x 10 5 cells / mL to 5x10 5 cells / mL, such as or about 3x10 5 cells / mL to 5x10 5 In some embodiments, the target inoculation cell density of the third shake flask is not less than 5x10 5 In some embodiments, the target inoculation cell density of the third shake flask is about 5x10 5 In some embodiments, the target cell density of the second shake flask is about 3.5 to 6 x 10 6 In some embodiments, the target cell density for the second shake flask is 4x10 6 In some embodiments, the target cell density for the second shake flask is 5x10 6 In some embodiments, the target cell density for the second shake flask is 6 x 106 cells / mL.

[0068] In some embodiments, once the inoculated cells achieve the target passage cell density in the third shake flask, the host cells are passaged to the fourth shake flask at the target inoculation cell density. In some embodiments, the fourth shake flask holds a volume of 1.6 L. In some embodiments, the target inoculation cell density of the fourth shake flask is about 2x10 5 cells / mL to 5x10 5 cells / mL, such as or about 3x10 5 cells / mL to 5x10 5 In some embodiments, the target inoculation cell density of the fourth shake flask is not less than 5x10 5 In some embodiments, the target inoculation cell density of the fourth shake flask is about 5x10 5 In some embodiments, the target cell density of the third shake flask is about 3.5 to 6 x 10 6 In some embodiments, the target cell density of the third shake flask is 4x10 6 In some embodiments, the target cell density of the third shake flask is 5x10 6 In some embodiments, the target cell density of the third shake flask is 6x10 6 cells / mL.

[0069] In some embodiments, once the inoculated cells achieve the target passage cell density in the fourth shake flask, the host cells are passaged to the fifth shake flask at the target inoculation cell density. In some embodiments, the fifth shake flask holds a volume of 5 L. In some embodiments, the target inoculation cell density of the fifth shake flask is about 2 x 10 5 cells / mL to 5x10 5 cells / mL, such as or about 3x10 5 cells / mL to 5x10 5 In some embodiments, the target inoculation cell density of the fifth shake flask is not less than 5x10 5 In some embodiments, the target inoculation cell density of the fifth shake flask is about 5x10 5 In some embodiments, the target cell density of the fourth flask is about 3.5 to 6 x 10 6 In some embodiments, the target cell density of the fourth shake flask is 4x10 6 In some embodiments, the target cell density of the fourth flask is 5x106 In some embodiments, the target cell density of the fourth flask is 6x10 6 cells / mL.

[0070] In some embodiments, when the host cells in the fifth shake flask achieve the target cell density, the seed culture expansion is complete. In some embodiments, the target cell density of the fifth shake flask is about 3.5 to 6 x 10 6 In some embodiments, the target cell density of the fifth shake flask is 4x10 6 In some embodiments, the target cell density of the fifth flask is 5x10 6 In some embodiments, the target cell density of the fifth flask is 6x10 6 In some embodiments, the target cell density of the fifth flask is about 6 to 7 x 10 9 In some embodiments, when the seed culture expansion is completed, the host cells are passaged into the bioreactor at the target inoculation cell density. In some embodiments, the target inoculation cell density of the bioreactor is not less than 5x10 5 In some embodiments, the target inoculation cell density of the bioreactor is about 2 x 10 5 cells / mL to 5x10 5 cells / mL, such as or about 3x10 5 cells / mL to 5x10 5 In some embodiments, the target inoculation cell density of the second shake flask is about 5x10 5 cells / mL.

[0071] In some embodiments, the bioreactor can accommodate a volume between 3L and 200L. In some embodiments, the bioreactor can accommodate a volume of 3L, 10L, 40L, 50L or 200L. In some embodiments, the bioreactor can accommodate a volume of 40L. In some embodiments, the bioreactor can accommodate a volume of 200L. In some embodiments, the volume of the bioreactor is about 20mL to about 1500mL, such as about 20mL to about 1000mL, about 20mL to about 500mL, about 20mL to about 200mL, about 20mL to about 100mL or about 20mL to about 40mL. In some embodiments, the volume of the bioreactor is about 40mL to about 200mL. In some embodiments, the volume of the bioreactor is or about 40mL. In some embodiments, the volume of the bioreactor is or about 50mL. In some embodiments, the volume of the bioreactor is or about 100mL. In some embodiments, the volume of the bioreactor is or about 200mL.

[0072] In some embodiments, the host cells are inoculated and grown in each of one or more bioreactors under dissolved oxygen and pH control. In some embodiments, the host cells are inoculated in a first bioreactor and grown under dissolved oxygen and pH control. In some embodiments, the host cells are transferred from the first bioreactor to a second bioreactor and grown under dissolved oxygen and pH control. In some embodiments, the bioreactor is suitable for transfection.

[0073] For example, in some embodiments, cells are first inoculated into a shake flask (e.g., a 125 mL shake flask) and then transferred to one or more larger shake flasks (e.g., 500 mL), with the volume of each shake flask gradually increasing until a sufficient number of cells is achieved for bioreactor inoculation. In some embodiments, a sufficient number of cells for bioreactor inoculation is 6 to 7×10 9 In some embodiments, the sufficient cell density for inoculating the bioreactor is 3.5 to 6 x 10 6 cells / mL.

[0074] In some embodiments, the concentration of at least 5x10 5 In some embodiments, the cells are inoculated into a 125 mL flask. In some embodiments, the cells are inoculated into a 500 mL shake flask. In some embodiments, the cells are inoculated into a 1 L shake flask. In some embodiments, the cells are inoculated into a 1.6 L shake flask. In some embodiments, the cells are inoculated into a 5 L shake flask.

[0075] In some embodiments, the bioreactor is inoculated after seed culture expansion up to 5 L shake flasks.

[0076] In some embodiments, a range of target cell numbers is achieved by culturing in each of a plurality of containers. In some embodiments, different target cell numbers correspond to containers of different sizes.

[0077] In some embodiments, a range of target cell densities are achieved by culturing in each of a plurality of containers. In some embodiments, different target cell densities correspond to containers of different sizes.

[0078] In some embodiments, expansion is continued in one or more bioreactors until about 4×10 6 cells / mL to 6x10 6 In some embodiments, the bioreactor is sized at or about 40 mL to 200 mL. In some embodiments, the bioreactor is sized at or about 40 mL. In some embodiments, the bioreactor is sized at or about 50 mL. In some embodiments, the bioreactor is sized at or about 100 mL. In some embodiments, the bioreactor is sized at or about 200 mL.

[0079] In some embodiments, about 5×10 5 In some embodiments, the second target cell density is 4 to 6 x 10 cells / mL. 6 In some embodiments, the second target cell density is achieved in a container of 125-500 mL. In some embodiments, the third target cell number is 6 to 7×10 9 In some embodiments, about 5×10 5 In some embodiments, the third target cell number is achieved at a cell density of 1 cell / mL. In some embodiments, the third target cell number is achieved in a container of 1-5L. In some embodiments, the third target cell number is suitable for inoculation in a container of about 50L, optionally wherein the container is a bioreactor. In some embodiments, the bioreactor is a first bioreactor, and the cells are transferred to a second bioreactor for full-scale amplification until the final target cell density is achieved. In some embodiments, when the final target cell density is achieved, the host cells are transfected with a plasmid.

[0080] In some embodiments, the host cells are inoculated and grown in a first bioreactor under dissolved oxygen and pH control. In some embodiments, the first bioreactor is about 50 L. In some embodiments, the target cell density in the first bioreactor is 4 to 6 x 10 6 cells / mL.

[0081] In some embodiments, once the target cell density is reached in the first bioreactor, the host cells are transferred to a second bioreactor. In some embodiments, the second bioreactor is about 200 L. In some embodiments, the initial cell density in the second bioreactor after the transfer is 1-5×10 5 In some embodiments, the host cells are grown in the second bioreactor until a final target cell density is reached. In some embodiments, the final target cell density in the second bioreactor is 4 to 6 x 10 6 In some embodiments, the final target cell density in the second bioreactor is about 5x10 6 In some embodiments, the final target cell density in the second bioreactor is suitable for transfection.

[0082] In some embodiments, about 5×10 5 The cells are cultured in the first container until a density of 4 to 6 x 10 cells / mL is achieved. 6 In some embodiments, the cells from the first container are transferred to the second container and the cells are cultured in the second container until they have expanded to 6 to 7 x 10 9 In some embodiments, each of the first container and the second container is a container of increasing size. In some embodiments, the first container and the second container are shake flasks. In some embodiments, the volume of the first container (e.g., shake flask) is 125-500mL. In some embodiments, the volume of the second container (e.g., shake flask) is 1-5L. In some embodiments, the first shake flask holds a volume of 125mL. In some embodiments, the first shake flask holds a volume of 500mL. In some embodiments, the second shake flask holds a volume of 1L. In some embodiments, the second shake flask holds a volume of 1.6L. In some embodiments, the second shake flask holds a volume of 5L.

[0083] In some embodiments, the target number of cells expanded from the seed culture in the shake flask is used to inoculate the bioreactor. In some embodiments, cells from the target number of cells in the second container are inoculated into the first bioreactor and the cells are cultured in the bioreactor until a cell count of 4 to 6 x 10 6 In some embodiments, cells from the first bioreactor are inoculated into a second bioreactor at scale and the cells are grown in the second bioreactor until a target cell density of 4 to 7 x 10 cells / mL is achieved. 6 In some embodiments, the final target cell density in the second bioreactor is about 5x10 cells / mL. 6 In some embodiments, the first bioreactor has a smaller volume than the second bioreactor. In some embodiments, the volume of the first bioreactor is 3-50L. In some embodiments, the volume of the second bioreactor is 40-200L. In some embodiments, the first bioreactor holds a volume of 3L. In some embodiments, the first bioreactor holds a volume of 10L. In some embodiments, the first bioreactor holds a volume of 50L. In some embodiments, the first bioreactor holds a volume of 40L. In some embodiments, the second bioreactor holds a volume of 200L.

[0084] In some embodiments, when the final target cell density is achieved, the host cells are transfected with a plasmid. 2. Plasmid Transfection

[0085] In some embodiments, the methods described herein include transfecting host cells with at least one plasmid to produce viral particles. In some embodiments, the methods described herein include contacting a population of host cells with at least one plasmid encoding a viral particle for a period of time sufficient to produce viral particles. In some embodiments, contacting a population of host cells with at least one plasmid encoding a viral protein is performed by transfection.

[0086] In some embodiments, once the target transfection density is reached, transfection is performed in a bioreactor. In some embodiments, the target transfection density is the final target density achieved by a seed expansion method as described above for host cell expansion. In some cases, fresh medium can be added to the bioreactor to dilute the final target density to the target transfection density. In some embodiments, the target transfection density is about 0.5 to about 10x10 6 In some embodiments, the target transfection density is about 1 to about 3 x 10 6 In some embodiments, the target transfection density is about 3 to 6 x 106 In some embodiments, the target transfection density is about 4 to about 7 x 10 6 In some embodiments, the target transfection density is about 1x10 6 cells / mL, about 2x10 6 cells / mL, about 3x10 6 cells / mL, about 4x10 6 cells / mL, about 5x10 6 cells / mL or approximately 6x10 6 cells / mL or any value between any of the foregoing values. In some embodiments, the target transfection density is 4x10 6 cells / mL.

[0087] In some embodiments, the target transfection density depends on or is dependent on the host cell. In some cases, certain host cells have higher productivity. For example, HEK293T cells can exhibit a higher productivity than HEK293 cells, so that the target transfection density when using HEK293 cells can be 3 times or lower, such as 2-3 times lower, than the target transfection density when using HEK293T cells. The ability to achieve the desired yield using lower cell density improves the process by providing a lower residual impurity burden for downstream steps.

[0088] In some embodiments, the host cell is a HEK293T cell. In some embodiments, the target transfection density is about 1 to about 3 x 10 6 In some embodiments, the target transfection density is about 1x10 6 In some embodiments, the target transfection density is about 2x10 6 In some embodiments, the target transfection density is about 3x10 6 cells / mL.

[0089] In some embodiments, the host cell is a HEK293 cell. In some embodiments, the target transfection density is about 3 to 6 x 10 6 In some embodiments, the target transfection density is about 4x10 6 In some embodiments, the target transfection density is about 5x10 6 In some embodiments, the target transfection density is about 6x10 6 cells / mL.

[0090] In some embodiments, the contents of the bioreactor are diluted with fresh medium to achieve a target transfection density. In some embodiments, the target transfection density is 4×10 6 In some embodiments, a bolus of glucose is added to a bioreactor with fresh culture medium to a target glucose level. In some embodiments, glucose is supplemented in the culture medium for transfection. In some embodiments, the target glucose level in the culture medium for transfection is 1g / L to 10g / L, such as 2.5g / L to 7.5g / L. In some embodiments, the target glucose level is or is about 5.5g / L. In some embodiments, glucose is supplemented to target 5.5g / L at a certain concentration.

[0091] In some embodiments, transfection is performed shortly after the target transfection density is reached. The transfection method can be performed using techniques well known in the art. For example, the transfection process can use calcium phosphate or commercially available formulations such as Lipofectamine TM 2000CD (Invitrogen, CA) or polyethyleneimine (PEI).

[0092] Commercially available instruments and reagents can be used for transfection. In some embodiments, the instruments and reagents used for transfection are designed for large-scale virus production. Illustrative examples of such commercially available reagents include

[0093] In some embodiments, at least one plasmid used for transfection comprises a non-coding nucleic acid. In some embodiments, the non-coding nucleic acid is a siRNA, miRNA, or shRNA.

[0094] Exemplary target genes include any payload as described in Section II.A.2.bv. In some embodiments, at least one plasmid for transfection comprises a polynucleotide encoding a target polypeptide or gene (e.g., also referred to as a payload gene). In some embodiments, at least one plasmid comprising a polynucleotide encoding a target gene or polypeptide is a transfer plasmid. In some embodiments, the target gene is not expressed by the host cell, and the transfer plasmid is incorporated into the viral particle as a nucleic acid. In some embodiments, the target polypeptide is a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is specific for tumor-associated antigens. In some embodiments, the tumor-associated antigen is CD19, BCMA, GPRC5D, ROR1, FcRL5, alpha-fetoprotein, or Her2. In some embodiments, the chimeric antigen receptor is specific for a tag. In some embodiments, the tag is a hapten. In some embodiments, exemplary haptens include: DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin and digoxigenin, as well as fluorescein and its derivatives (including FITC (fluorescein isothiocyanate), NHS-fluorescein, and pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives), knottin, centyrin and DARPin.

[0095] In some embodiments, at least one plasmid used for transfection encodes a viral envelope protein. In some embodiments, the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a Cocal virus G protein. In some embodiments, the viral envelope protein is a Cocal virus G protein.

[0096] In some embodiments, the Cocal virus G envelope protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the Cocal virus G envelope protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the Cocal virus G envelope protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the Cocal virus G envelope protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the Cocal virus G envelope protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the Cocal virus G envelope protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the Cocal virus G envelope protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the Cocal virus G envelope protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the Cocal virus G envelope protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the Cocal virus G envelope protein comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the Cocal virus G envelope protein comprises the amino acid sequence of SEQ ID NO: 124. In some embodiments, the Cocal virus G envelope protein consists of the amino acid sequence of SEQ ID NO: 124.

[0097] In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the Cocal virus G envelope protein is encoded by nucleotides comprising the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the Cocal virus G envelope protein is encoded by nucleotides consisting of the nucleotide sequence of SEQ ID NO: 125.

[0098] In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the Cocal virus G envelope protein is encoded by a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the Cocal virus G envelope protein is encoded by nucleotides comprising the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the Cocal virus G envelope protein is encoded by nucleotides consisting of the nucleotide sequence of SEQ ID NO: 126.

[0099] The envelope expression cassette can include one of a variety of envelopes, such as VSV-G or various mouse retrovirus envelopes (such as 4070A). In some embodiments, the viral envelope protein is a fusion protein with an immune cell activation protein. The immune activation protein can be a single binding domain protein or a multi-binding domain protein that binds to one or more target molecules on immune cells to stimulate or activate immune cell activity. In some embodiments, the immune cell activation protein contains at least one binding domain, and the at least one binding domain binds to a primary T cell receptor (e.g., CD3), a costimulatory molecule or an adhesion molecule. In some embodiments, the viral envelope protein includes a binding domain that binds to at least one costimulatory molecule.

[0100] In some embodiments, at least one plasmid for transfection encodes a protein that binds a co-stimulatory molecule. In some embodiments, at least one co-stimulatory molecule is CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, OX40, 4-1BB, CD40L, or any combination thereof. In some embodiments, the co-stimulatory molecule is any co-stimulatory molecule described in Section II.A.1.b.

[0101] In some embodiments, at least one plasmid used for transfection encodes an immune cell activation protein. In some embodiments, the immune cell activation protein is a protein that specifically binds to the following: CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244, TCR alpha chain, TCR beta chain, TCR zeta chain, TCR gamma chain, TCR delta chain, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit or NKp80. In some embodiments, the costimulatory molecule is any costimulatory molecule described in section II.A.1.a.

[0102] In some embodiments, at least one plasmid encoding for transfection binds the protein of adhesion molecule. In some embodiments, at least one adhesion molecule is CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, B7-H6, SLAMF2, B7-H2, B7-H5, B7-H3, B7x and TMIGD2 or any combination thereof. In some embodiments, adhesion molecule is any adhesion molecule described in chapter II.A.1.c.

[0103] In some embodiments, to improve safety, the host cell is engineered in which the 3'LTR of the original virus is deleted. In such cells, two recombination events are required to produce wild-type virus. In some embodiments, further safety improvements involve the introduction of gag-pol gene and env gene on separate constructs. These constructs can be introduced sequentially to prevent recombination during transfection. In these split construct cell lines, further reduction of recombination can be achieved by changing codons. Based on the redundancy of the genetic code, this technology is intended to reduce the homology between separate constructs (e.g., between overlapping regions in the gag-pol and env open reading frames).

[0104] In some embodiments, at least one plasmid used for transfection encodes a helper virus protein. In some embodiments, the helper virus protein is rev and / or gagpol.

[0105] In some embodiments, the gag protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:46. In some embodiments, the gag protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:46. In some embodiments, the gag protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:46. In some embodiments, the gag protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:46. In some embodiments, the gag protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:46. In some embodiments, the gag protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:46. In some embodiments, the gag protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:46. In some embodiments, the gag protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:46. In some embodiments, the gag protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the gag protein comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the gag protein comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the gag protein consists of the amino acid sequence of SEQ ID NO: 46.

[0106] In some embodiments, the pol protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:49. In some embodiments, the pol protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:49. In some embodiments, the pol protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:49. In some embodiments, the pol protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:49. In some embodiments, the pol protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:49. In some embodiments, the pol protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:49. In some embodiments, the pol protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:49. In some embodiments, the pol protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:49. In some embodiments, the pol protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 49. In some embodiments, the pol protein comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 49. In some embodiments, the pol protein comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, the pol protein consists of the amino acid sequence of SEQ ID NO: 49.

[0107] In some embodiments, the gag and pol proteins are encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:52. In some embodiments, the gag and pol proteins are encoded by a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:52. In some embodiments, the gag and pol proteins are encoded by a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:52. In some embodiments, the gag and pol proteins are encoded by a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:52. In some embodiments, the gag and pol proteins are encoded by a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:52. In some embodiments, the gag and pol proteins are encoded by a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:52. In some embodiments, the gag and pol proteins are encoded by a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:52. In some embodiments, the gag and pol proteins are encoded by a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 52. In some embodiments, the gag and pol proteins are encoded by a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 52. In some embodiments, the gag and pol proteins are encoded by a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 52. In some embodiments, the gag and pol proteins are encoded by the nucleotide sequence of SEQ ID NO: 52. In some embodiments, the gag and pol proteins consist of the nucleotide sequence of SEQ ID NO: 52.

[0108] In some embodiments, the rev protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the rev protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the rev protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the rev protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the rev protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the rev protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the rev protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the rev protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the rev protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the rev protein comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the rev protein comprises the amino acid sequence of SEQ ID NO: 127. In some embodiments, the rev protein consists of the amino acid sequence of SEQ ID NO: 127.

[0109] In some embodiments, the rev protein is encoded by a nucleotide sequence comprising a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the rev protein is encoded by a nucleotide sequence comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the rev protein is encoded by a nucleotide sequence comprising a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the rev protein is encoded by a nucleotide sequence comprising a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the rev protein is encoded by a nucleotide sequence comprising a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the rev protein is encoded by a nucleotide sequence comprising a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the rev protein is encoded by a nucleotide sequence comprising a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the rev protein is encoded by a nucleotide sequence comprising a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the rev protein is encoded by a nucleotide sequence comprising a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the rev protein is encoded by a nucleotide sequence comprising a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the rev protein is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the rev protein is encoded by a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 128.

[0110] In some embodiments, a plasmid mixture is used for transfection.In some embodiments, the plasmid mixture comprises a plasmid encoding a gene of interest, a plasmid encoding a rev viral protein, and a plasmid encoding a gagpol viral protein.

[0111] In some embodiments, the plasmid mixture comprises a plasmid encoding a gene of interest, a plasmid encoding a rev viral protein, a plasmid encoding a gagpol viral protein, and a plasmid encoding a viral envelope protein.

[0112] In some embodiments, the plasmid mixture comprises a plasmid encoding a gene of interest, a plasmid encoding a rev viral protein, a plasmid encoding a gagpol viral protein, a plasmid encoding a viral envelope protein, and a plasmid encoding an immune cell activation protein.

[0113] In some embodiments, the plasmid mixture comprises a plasmid encoding a gene of interest, a plasmid encoding a rev viral protein, a plasmid encoding a gagpol viral protein, and a plasmid encoding a viral envelope protein.

[0114] In some embodiments, the plasmid mixture comprises a plasmid encoding a gene of interest, a plasmid encoding a rev viral protein, a plasmid encoding a gagpol viral protein, a plasmid encoding a viral envelope protein, and a plasmid encoding a co-stimulatory molecule.

[0115] In some embodiments, the plasmid mixture comprises a plasmid encoding a gene of interest, a plasmid encoding a rev viral protein, a plasmid encoding a gagpol viral protein, a plasmid encoding a viral envelope protein, and a plasmid encoding an immune cell activation protein.

[0116] In some embodiments, the plasmid mixture comprises a plasmid encoding a gene of interest, a plasmid encoding a rev viral protein, a plasmid encoding a gagpol viral protein, a plasmid encoding a viral envelope protein, a plasmid encoding an immune cell activation protein, and a plasmid encoding a co-stimulatory molecule.

[0117] In some embodiments, the plasmid mixture comprises a plasmid encoding a gene of interest, a plasmid encoding a rev viral protein, a plasmid encoding a gagpol viral protein, a plasmid encoding a viral envelope protein, and a plasmid encoding a co-stimulatory molecule.

[0118] In some embodiments, the plasmid mixture comprises a plasmid encoding a gene of interest, a plasmid encoding a rev viral protein, a plasmid encoding a gagpol viral protein, a plasmid encoding a viral envelope protein, a plasmid encoding an immune cell activation protein, and a plasmid encoding a co-stimulatory molecule.

[0119] In some embodiments, transfection is performed using a 5-plasmid system. In some embodiments, the 5-plasmid system comprises a plasmid expressing a target gene, a plasmid expressing a lentiviral gagpol gene, a plasmid expressing a lentiviral rev gene, a plasmid expressing a viral envelope protein, and a plasmid expressing an immune activation protein.

[0120] In some embodiments, transfection is performed using a plasmid system comprising more than 5 plasmids. In some embodiments, the plasmid system comprises a plasmid expressing a gene of interest, a plasmid expressing a lentiviral gagpol gene, a plasmid expressing a lentiviral rev gene, a plasmid expressing a viral envelope protein, a plasmid expressing an immune activation protein, and at least one additional plasmid expressing a protein (e.g., a co-stimulatory molecule or other viral particle surface molecules described herein) for incorporation into a viral envelope.

[0121] In some embodiments, transient transfection is used to produce the virus-producing cells of the present disclosure. Transient transfection avoids the long time required to produce a stable vector-producing cell line and can be used when the vector genome or lentiviral packaging components are toxic to the cells. In the process of using transiently transfected cells, any reagent that allows plasmid transfection can be used. Illustrative examples include calcium phosphate or polyethyleneimine, but other reagents can be considered by those skilled in the art (Ansorge et al. 2010). Those skilled in the art can adjust conditions (e.g., the number of plasmids, the ratio between plasmids, the ratio between plasmids and transfection agents, the type of culture medium, etc.) and transfection time according to the characteristics of the virus produced and / or the characteristics of the transgene introduced into the transfer plasmid. 3. Harvesting of Viral Particles

[0122] In some embodiments, cells transfected with viral vectors (also referred to herein as vector-producing cells) are cultured to increase cell and virus numbers and / or viral titers. Cell culture can be accomplished by methods well known to those skilled in the art, and includes, but is not limited to, providing nutrients to the cells, such as in an appropriate culture medium. The method may include growth adhering to a surface, growth in suspension, or a combination thereof.

[0123] In some embodiments, in tissue culture bottles, culture dishes, roller bottles, or in bioreactors, batches, fed-batch, continuous systems, hollow fibers, etc. are used to culture to achieve large-scale production of viruses by cell culture. In some embodiments, vector production cells can grow in suspension. Suitable conditions for culturing cells are known to those skilled in the art (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, ed. (1973); and RI Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9)).

[0124] In some embodiments, the vector producing cells are cultured to a target density suitable for harvesting. In some embodiments, harvesting is performed 40-50 hours after transfection. In some embodiments, harvesting is performed about 48 hours after transfection.

[0125] In some embodiments, the endonuclease is added to the vector production cell before harvesting. In some embodiments, the endonuclease is added to the vector production cell 1-5 hours before harvesting. In some embodiments, the endonuclease is added to the vector production cell about 2 hours before harvesting. In some embodiments, the endonuclease is a nuclease. In some embodiments, the endonuclease is a salt-active nuclease. A variety of endonucleases are known and can be used. In some embodiments, the endonuclease is an all-purpose nuclease. In some embodiments, the endonuclease is a denarase.

[0126] In some embodiments, endonuclease is added to the bioreactor to achieve a concentration of 1U / mL to 100U / mL, such as 1U / mL to 50U / mL, 1U / mL to 20U / mL, 1U / mL to 10U / mL, 10U / mL to 100U / mL, 10U / mL to 50U / mL, 10U / mL to 20U / mL, 20U / mL to 100U / mL, 20U / mL to 50U / mL, or 50U / mL to 100U / mL. In some embodiments, the concentration is between 5U / mL and 20U / mL. In some embodiments, the concentration is 20U / mL.

[0127] In some embodiments, MgCl2 supplement is added to the vector production cells before harvesting. In some embodiments, MgCl2 supplement is added to the vector production cells 1-5 hours before harvesting. In some embodiments, MgCl2 supplement is added to the vector production cells about 2 hours before harvesting. In some embodiments, MgCl2 is added to the bioreactor to achieve the following concentration: between 0.5mM and 5mM, such as 0.5mM to 4mM, 0.5mM to 2mM, 0.5mM to 1mM, 1mM to 5mM, 1mM to 4mM, 1mM to 2mM, 2mM to 5mM, 2mM to 4mM or 4mM to 5mM. In some embodiments, the concentration is between 0.5mM and 4mM. In some embodiments, the concentration is 2mM.

[0128] In some embodiments, the harvested cell culture may also be referred to as a suspension mixture. In some embodiments, the volume of the suspension mixture is about 3-50 liters. In some embodiments, the volume of the suspension mixture is about 50-500 liters. In some embodiments, the volume of the suspension mixture is about 200 liters.

[0129] In some embodiments, the upstream process produces a suspension mixture having an infectious titer of 0.5 x 10 5 TU / mL to 5x10 6 TU / mL of harvest. In some embodiments, the infectious titer comprises 1x10 5 TU / mL to 3x10 5 TU / mL, 2x10 5 TU / mL up to 4x10 5 TU / mL, 3x10 5 TU / mL to 5x10 5 TU / mL, 4x10 5 TU / mL up to 6x10 5 TU / mL, 5x10 5 TU / mL up to 7x10 5 TU / mL, 6x10 5 TU / mL up to 8x10 5 TU / mL, 7x10 5 TU / mL up to 9x10 5 TU / mL, 8x10 5 TU / mL to 1x10 6 TU / mL, 9x10 5 TU / mL to 2x10 6 TU / mL, 1x10 6 TU / mL to 3x10 6 TU / mL, 2x10 6 TU / mL up to 4x10 6 TU / mL, 3x10 6 TU / mL to 5x10 6 TU / mL. In some embodiments, the infectious titer is 2x10 6 TU / mL up to 4x10 6 TU / mL.

[0130] In some embodiments, the infectious titer comprises at least about 2 x 10 6 TU / mL. In some embodiments, the infectious titer comprises at least about 3x10 6 TU / mL. In some embodiments, the infectious titer comprises at least about 4x10 6TU / mL. In some embodiments, the infectious titer is 2x10 6 TU / mL. In some embodiments, the infectious titer is 3x10 6 TU / mL. In some embodiments, the infectious titer is 4x10 6 TU / mL.

[0131] In some embodiments, the suspension mixture is about 3 L to 200 L. In some embodiments, the suspension mixture comprises a volume of between about 1 to 10 L, 5 to 15 L, 10 to 20 L, 15 to 25 L, 20 to 30 L, 25 to 35 L, 30 to 40 L, 35 to 45 L, 40 to 50 L, 45 to 55 L, 50 to 60 L, 55 to 65 L, 60 to 70 L, 65 to 75 L, 70 to 80 L, 75 to 85 L, 80 to 90 L, 85 to 95 L, 90 to 100 L, 95 to 105 L, or 100 to 200 L. In some embodiments, the suspension mixture comprises at least 3 L, 10 L, 40 L, 50 L, 100 L, or 200 L, or any value between any of the foregoing values. In some embodiments, the suspension mixture comprises at least 3 L, 10 L, 40 L, 50 L, 100 L, or 200 L. In some embodiments, the suspension mixture is about 40 L. In some embodiments, the suspension mixture is about 200 L. In some embodiments, the suspension mixture is about 160 L to 200 L, such as at or about 160 L, 170 L, 180 L, 190 L, or 200 L, or any value between any of the foregoing values.

[0132] In some embodiments, the upstream process produces a suspension mixture having an infectious titer of 2×10 9 TU to 2x10 11 In some embodiments, the infection titer is 4x10 9 TU to 12x10 9 TU, 8x10 9 TU up to 16x10 9 TU, 12x10 9 TU to 2x10 10 TU, 16x10 9 TU to 2.4x10 10 TU, 2x10 10 TU to 2.8x10 10 TU, 2.4x10 10 TU to 3.2x10 10 TU, 2.8x10 10 TU to 3.6x10 10 TU, 3.2x10 10 TU to 4x10 10TU, 3.6x10 10 TU to 8x10 10 TU, 4x10 10 TU to 12x10 10 TU, 8x10 10 TU up to 16x10 10 TU, 12x10 10 TU to 2x10 11 TU. In some embodiments, the upstream process produces a suspension mixture having an infectious titer of 8×10 10 TU up to 16x10 10 TU's harvest.

[0133] In some embodiments, the infectious titer comprises at least about 8 x 10 10 TU. In some embodiments, the infectious titer comprises at least about 12 x 10 10 TU. In some embodiments, the infectious titer comprises at least about 16 x 10 10 TU. In some embodiments, the infectious titer is 8x10 10 TU. In some embodiments, the infectious titer is 12x10 10 TU. In some embodiments, the infectious titer is 16x10 10 TU.

[0134] In some embodiments, the upstream process produces a suspension mixture having an infectious titer of 1×10 10 TU to 1x10 12 In some embodiments, the infection titer is 2x10 10 TU to 6x10 10 TU, 4x10 10 TU to 8x10 10 TU, 6x10 10 TU to 10x10 10 TU, 8x10 10 TU to 12x10 10 TU, 10x10 10 TU to 14x10 10 TU, 12x10 10 TU up to 16x10 10 TU, 14x10 10 TU to 18x10 10 TU, 16x10 10 TU to 2x10 11 TU, 18x10 10 TU to 4x10 11 TU, 2x10 11TU to 6x10 11 TU, 4x10 11 TU to 8x10 11 TU, 6x10 11 TU to 10x10 11 TU. In some embodiments, the upstream process produces a suspension mixture having an infectious titer of 4×10 11 TU to 8x10 11 TU's harvest.

[0135] In some embodiments, the infectious titer comprises at least about 4×10 11 TU. In some embodiments, the infectious titer comprises at least about 6×10 11 TU. In some embodiments, the infectious titer comprises at least about 8x10 11 TU. In some embodiments, the infectious titer is about 4x10 11 TU. In some embodiments, the infectious titer is about 6x10 11 TU. In some embodiments, the infectious titer is about 8x10 11 TU. B. Downstream Processes

[0136] In some embodiments, the present disclosure provides a method for preparing a viral particle formulation.

[0137] In some embodiments, the method for preparing a viral particle formulation includes the introduction of nucleases, harvest clarification, harvest concentration and purification, and harvest formulation and concentration, referred to herein as downstream processes. The goal of the downstream process is to increase the yield of viral particles and to increase the purity of viral particles. In some embodiments, the downstream process includes Figure 1B The downstream process shown in .

[0138] In some embodiments, the present disclosure provides a method for preparing a viral particle formulation from a suspended mixture of a host cell group and viral particles. In some embodiments, the suspended mixture is prepared from the upstream process as described in the above article section. In some embodiments, the preparation of the viral particle formulation includes (i) clarifying the mixture of host cells and viral particles to produce a filtered viral particle formulation, and (ii) concentrating the filtered formulation to produce a concentrated viral particle formulation. In some embodiments, the method includes (iii) sterile filtering the concentrated viral particle formulation to produce a viral particle formulation. In some embodiments, the viral particle formulation is suitable for in vivo administration. In some embodiments, the process of preparing the viral particle formulation purifies the viral vector to remove impurities.

[0139] In some embodiments, the present disclosure provides a method for preparing a viral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and viral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter to produce a filtered viral particle preparation, wherein the third filter has a smaller retention threshold than the second filter; and (ii) concentrating the filtered viral particle preparation, wherein the concentration comprises chromatography and ultrafiltration.

[0140] In some embodiments, the present disclosure provides a method for preparing a retroviral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and retroviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter to produce a filtered retroviral particle preparation, wherein the third filter has a smaller retention threshold than the second filter; and (ii) concentrating the filtered retroviral particle preparation, wherein concentrating comprises chromatography and ultrafiltration.

[0141] In some embodiments, the present disclosure provides a method for preparing a retroviral (e.g., lentiviral) formulation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and lentiviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter to produce a filtered lentiviral particle formulation, wherein the third filter has a smaller retention threshold than the second filter; and (ii) concentrating the filtered lentiviral particle formulation, wherein concentrating comprises chromatography and ultrafiltration. 1. Clarification of Bioreactor Harvest

[0142] In some embodiments, the methods described herein include filtering the mixture of host cell populations and viral particles to remove contaminants. This filtering step is also referred to herein as "clarifying" or "clarification". In some embodiments, clarifying or clarification includes using a filter. In some embodiments, the filter is a depth filter.

[0143] In some embodiments, the suspended mixture of host cells and viral particles is clarified to remove host cells, cell debris and precipitated impurities from the suspended mixture from the bioreactor harvest while retaining viral vector particles. In some embodiments, a filter can be used or clarified by centrifugation. In some embodiments, centrifugation can include density gradient centrifugation, ultracentrifugation and differential centrifugation. In some embodiments, clarification or clarification includes centrifugation and deep filtration. In some embodiments, deep filtration or membrane filtration is used to clarify. In some embodiments, one or more filters are hollow fiber filters.

[0144] In some embodiments, a series of filters with gradually decreasing aperture are used to carry out the clarification process step. In some embodiments, at least one filter is a depth filter, and the depth filter captures pollutants in its structure and can be used for collecting things (pool) with high-throughput filtration turbidity. In some embodiments, at least one filter is a membrane filter, and the membrane filter captures pollutants greater than aperture on the described surface of membrane usually. In some embodiments, the treatment step comprises a series of filters, and wherein the first filter is a depth filter, and then filters with a membrane filter. In some embodiments, using a depth filter to reduce the total number of large particles in the collection allows to improve the flux through the membrane filter with a smaller aperture.

[0145] In some embodiments, the clarification process uses primary, secondary, and tertiary filtration.

[0146] In some embodiments, clarifying or clarification comprises centrifugation. In some embodiments, centrifugation can comprise density gradient centrifugation, ultracentrifugation and differential centrifugation. In some embodiments, clarifying or clarification comprises centrifugation and deep filtration.

[0147] In some embodiments, filtering the mixture includes (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter to produce a filtered viral particle preparation, wherein the third filter has a smaller retention threshold than the second filter.

[0148] In some embodiments, the method comprises filtering a mixture of a population of host cells and retroviral particles. In some embodiments, filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter to produce a filtered retroviral particle preparation, wherein the third filter has a smaller retention threshold than the second filter.

[0149] In some embodiments, the method comprises filtering a mixture of a population of host cells and lentiviral particles. In some embodiments, filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter to produce a filtered lentiviral particle preparation, wherein the third filter has a smaller retention threshold than the second filter.

[0150] In some embodiments, the first filter has a retention threshold of about 1-60 μm, about 5-60 μm, about 10-60 μm, about 15-60 μm, about 20-60 μm, about 25-60 μm, about 30-60 μm, about 35-60 μm, about 40-60 μm, about 45-60 μm, about 50-60 μm, about 55-60 μm, or above 60 μm. In some embodiments, the first filter has a retention threshold of about 1 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, about 90 μm, about 95 μm, or about 100 μm. In some embodiments, the first filter has a retention threshold of 60 μm.

[0151] In some embodiments, the first filter is a depth filter. In some embodiments, the first filter is a commercially available depth filter. Illustrative examples of commercially available first filters include, but are not limited to Depth filter

[0152] In some embodiments, the second filter has a retention threshold of about 0.2-4 μm, about 0.3-4 μm, about 0.4-4 μm, about 0.5-4 μm, about 0.6-4 μm, about 0.7-4 μm, about 0.8-4 μm, about 0.9-4 μm, about 1-4 μm, about 2-4 μm, or about 3-4 μm. In some embodiments, the second filter has a retention threshold of about 0.4-3 μm, about 0.4-2 μm, about 0.4-1 μm, about 0.4-0.9 μm, about 0.4-0.8 μm, about 0.4-0.7 μm, about 0.4-0.6 μm, or about 0.4-0.5 μm. In some embodiments, the second filter has a retention threshold of about 0.2 μm, about 0.25 μm, about 0.3 μm, about 0.35 μm, about 0.4 μm, about 0.45 μm, about 0.5 μm, about 0.55 μm, about 0.6 μm, about 0.65 μm, about 0.7 μm, about 0.75 μm, about 0.8 μm, about 0.85 μm, about 0.9 μm, about 0.95 μm, about 1 μm, about 2 μm, about 3 μm, or about 4 μm. In some embodiments, the second filter has a retention threshold of 0.45 μm.

[0153] In some embodiments, the second filter is a commercially available filter. Illustrative examples of commercially available second filters include, but are not limited to PP3 filter (Sartorius).

[0154] In some embodiments, the third filter has a retention threshold of about 0.2-1 μm, about 0.2-0.9 μm, about 0.2-0.8 μm, about 0.2-0.7 μm, about 0.2-0.6 μm, about 0.2-0.5 μm, about 0.2-0.4 μm, or about 0.2-0.3 μm. In some embodiments, the third filter has a retention threshold of about 0.2 μm, about 0.25 μm, about 0.3 μm, about 0.35 μm, about 0.4 μm, about 0.45 μm, or about 0.5 μm. In some embodiments, the third filter has a retention threshold of 0.2 μm.

[0155] In some embodiments, the third filter is a commercially available filter. Illustrative examples of commercially available third filters include, but are not limited to 2HF filter (Sartorius).

[0156] In some embodiments, the first filter has a retention threshold of 60 μm, the second filter has a retention threshold of 0.45 μm, and the third filter has a retention threshold of 0.2 μm.

[0157] In some embodiments, the second filter and the third filter are two layers in a double-layer filter assembly. In some embodiments, the third filter is a double filter comprising a first layer filter and a second layer filter. In some embodiments, each layer of the double-layer filter assembly has a different retention threshold. In some embodiments, the first layer has the following retention threshold: about 0.4 μm, about 0.45 μm, about 0.5 μm, about 0.55 μm, about 0.6 μm, about 0.65 μm, about 0.7 μm, about 0.75 μm, about 0.8 μm, about 0.85 μm, about 0.9 μm, about 0.95 μm, about 1 μm, about 2 μm, about 3 μm or about 4 μm. In some embodiments, the second layer has the following retention threshold: about 0.2 μm, about 0.25 μm, about 0.3 μm, about 0.35 μm, about 0.4 μm, about 0.45 μm or about 0.5 μm. In some embodiments, the first layer has a retention threshold of about 0.45 μm and the second layer has a retention threshold of about 0.2 μm.

[0158] In some embodiments, the dual-layer filter assembly is commercially available. Illustrative examples of commercially available dual-layer filter assemblies include, but are not limited to 2 (pore size: 0.45|0.2μm), 2XLG (pore size: 0.8|0.2μm) and 2XLI (pore size: 0.35|0.2μm).

[0159] In some embodiments, the present disclosure provides a method for preparing a viral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and viral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a double-layer filter assembly comprising a second filter and a third filter to produce a filtered viral particle preparation, wherein the second filter has a smaller retention threshold than the first filter, and the third filter has a smaller retention threshold than the second filter.

[0160] In some embodiments, the present disclosure provides a method for preparing a retroviral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and retroviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a double-layer filter assembly comprising a second filter and a third filter to produce a filtered retroviral particle preparation, wherein the second filter has a smaller retention threshold than the first filter, and the third filter has a smaller retention threshold than the second filter.

[0161] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and lentiviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a double-layer filter assembly comprising a second filter and a third filter to produce a filtered lentiviral particle formulation, wherein the second filter has a smaller retention threshold than the first filter, and the third filter has a smaller retention threshold than the second filter.

[0162] In some embodiments, the present disclosure provides a method for preparing a viral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and viral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a double-layer filter assembly comprising a second filter and a third filter to produce a filtered viral particle preparation, wherein the third filter has a smaller retention threshold than the second filter.

[0163] In some embodiments, the present disclosure provides a method for preparing a retroviral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and retroviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a double-layer filter assembly comprising the second filter and a third filter to produce a filtered viral particle preparation, wherein the third filter has a smaller retention threshold than the second filter.

[0164] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and lentiviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a double-layer filter assembly comprising the second filter and a third filter to produce a filtered lentiviral particle formulation, wherein the third filter has a smaller retention threshold than the second filter.

[0165] In some embodiments, the present disclosure provides a method for preparing a viral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and viral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter to produce a filtered viral particle preparation, wherein the third filter is a double-layer filter assembly comprising a first filter and a second filter, wherein the second filter has a smaller retention threshold than the first filter.

[0166] In some embodiments, the present disclosure provides a method for preparing a retroviral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and retroviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter to produce a filtered retroviral particle preparation, wherein the third filter is a double-layer filter assembly comprising a first filter and a second filter, wherein the second filter has a smaller retention threshold than the first filter.

[0167] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and lentiviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter to produce a filtered lentiviral particle formulation, wherein the third filter is a double-layer filter assembly comprising a first filter and a second filter, wherein the second filter has a smaller retention threshold than the first filter.

[0168] In some embodiments, the present disclosure provides a method for preparing a viral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and viral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter to produce a filtered viral particle preparation, wherein the third filter is a double-layer filter assembly comprising a first layer filter and a second layer filter, wherein the retention threshold of the first layer filter is the same as that of the second filter, and wherein the second layer filter has a smaller retention threshold than the first layer filter.

[0169] In some embodiments, the present disclosure provides a method for preparing a retroviral particle formulation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and retroviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter to produce a filtered retroviral particle formulation, wherein the third filter is a double-layer filter assembly comprising a first layer filter and a second layer filter, wherein the retention threshold of the first layer filter is the same as that of the second filter, and wherein the second layer filter has a smaller retention threshold than the first layer filter.

[0170] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and lentiviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter to produce a filtered lentiviral particle formulation, wherein the third filter is a double-layer filter assembly comprising a first filter and a second filter, wherein the retention threshold of the first filter is the same as that of the second filter, and wherein the second filter has a smaller retention threshold than the first filter.

[0171] In some embodiments, one or more filters are hollow fiber filters (hereinafter also referred to as hollow fiber modules). In some embodiments, the nominal molecular weight cutoff (NMWC) of the hollow fiber filter is between 100kDa and 1000kDa. In some embodiments, the NMWC is between 100kDa and 500kDa. In some embodiments, the NMWC is between 300kDa and 750kDa. In some embodiments, the NMWC is between 500kDa and 1000kDa.

[0172] In some embodiments, the NMWC is 500 kDa. In some embodiments, the NMWC is 300 kDa. In some embodiments, the NMWC is 100 kDa.

[0173] In some embodiments, the pore size of the hollow fiber filter is between 0.1 μm and 0.65 μm. In some embodiments, the pore size is 0.1 μm. In some embodiments, the pore size is 0.2 μm. In some embodiments, the pore size is 0.45 μm. In some embodiments, the pore size is 0.65 μm.

[0174] In some embodiments, the length of the hollow fiber filter is 20 cm, 41.5 cm, 65 cm, 50 cm, 68 cm or 108 cm. In some embodiments, the lumen of the hollow fiber filter is 0.50 mm, 0.63 mm, 0.75 mm, 1 mm, 2 mm or 3 mm.

[0175] In some embodiments, the surface area of ​​the hollow fiber filter is 235 cm 2 With 1600cm 2 In some embodiments, the surface area is 235 cm 2 In some embodiments, the surface area is 790 cm 2 In some embodiments, the surface area is 1600 cm 2 .

[0176] In some embodiments, the hollow fiber filter holds a volume of 300 mL.

[0177] In some embodiments, the hollow fiber filter is a commercially available hollow fiber filter. Illustrative examples of commercially available hollow fiber filters include, but are not limited to Hollow fiber filter (Repligen).

[0178] In some embodiments, filtering is carried out under sterilizing conditions. In some embodiments, each filtering step is carried out in a closed system. In some embodiments, one or more filters can be operably connected in series in a closed system. In some embodiments, the first filter is connected in series with a bioreactor. In some embodiments, the second filter or the third filter is connected in series with the first filter, wherein all filters are operably connected. In some embodiments, filtration is carried out in a closed system, and is operably connected in series with a bioreactor, wherein the suspended harvest from the bioreactor is directly pumped through the first filter, and optionally further directly pumped through the second filter and the third filter, all carried out in a closed system. In some embodiments, the closed system does not allow any material to be transferred out of the system, such as not allowing material to be exposed to air or external environment.

[0179] In some embodiments, the endonuclease is added before filtering the suspended mixture. In some embodiments, the endonuclease is added before filtering the mixture with the first filter. In some embodiments, the endonuclease is added before filtering the mixture with the second filter. In some embodiments, the endonuclease is added before filtering the mixture with the third filter. In some embodiments, the endonuclease is added after filtering the mixture with the third filter. In some embodiments, the endonuclease is added after filtering the mixture with the third filter and before concentrating the filtered preparation.

[0180] In some embodiments, the endonuclease is present during the step of filtering the mixture with a third filter and concentrating the filtered preparation. In some embodiments, the endonuclease is present during the step of filtering the mixture with a second filter and concentrating the filtered preparation. In some embodiments, the endonuclease is present during the step of filtering the mixture with a first filter and concentrating the filtered preparation.

[0181] In some embodiments, residual host cell DNA (hcDNA) is measured after the filtration step. hcDNA can be measured by qPCR and normalized to the amount of viral particles. In some embodiments, hcDNA is measured in units / dose, where a dose is defined using transduction units (TU) of the virus. In some embodiments, hcDNA is measured in ng / dose, where a dose is 1E9 TU. A unit of 1E9 is equivalent to 1x10 9 same.

[0182] In some embodiments, after the third filter, the hcDNA is less than about 5000ng / 1E9 TU, less than about 4500ng / 1E9 TU, less than about 4000ng / 1E9 TU, less than about 3500ng / 1E9 TU, less than about 3000ng / 1E9 TU, less than about 2500ng / 1E9 TU, less than about 2000ng / 1E9 TU, less than about 1500ng / 1E9 TU, or less than about 1000ng / 1E9TU. In some embodiments, after the third filter, the hcDNA is less than about 2500ng / 1E9 TU. In some embodiments, after the third filter, the hcDNA is undetectable.

[0183] In some embodiments, the hcDNA after the third filter is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold lower than the hcDNA at harvest (in the suspension mixture). In some embodiments, the hcDNA after the third filter is at least 80-fold lower than the hcDNA at harvest.

[0184] In some embodiments, the hcDNA after the third filter is at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times, or at least 10 times lower than the hcDNA after the second filter. In some embodiments, the hcDNA after the third filter is at least 5 times lower than the hcDNA after the second filter. 2. Chromatography

[0185] In some embodiments, the method of the present disclosure includes concentrating the filtered viral particle preparation. In some embodiments, concentration can be performed by chromatography. In some embodiments, chromatography also acts on removing impurities, such as HCP and HcDNA, from the filtered particle preparation. In some embodiments, the method of the present disclosure includes clarifying the suspended mixture by using a series of filters as described above, followed by a chromatography step. In some embodiments, the chromatography is used to further remove impurities, such as HCP and hcDNA, and concentrate the viral particles in the preparation from the filtered particle preparation.

[0186] Chromatographic techniques well known to those skilled in the art can be used in the methods of the present disclosure. These techniques may involve separation of the vector particles from the cellular environment and, if necessary, further purification of the vector particles. One or more of a variety of chromatographic methods can be used for purification. In some embodiments, chromatography is performed after the harvest is clarified.

[0187] In some embodiments, residual host cell proteins (HCP) are measured after the chromatogram. HCP can be measured by ELISA and normalized relative to the amount of viral particles. In some embodiments, HCP is measured in units / dose, wherein a dose is defined using a transduction unit (TU) of the virus. In some embodiments, HCP is measured in μg / dose, wherein a dose is 1E9 TU.

[0188] In some embodiments, after chromatography, HCP is less than about 5000 μg / 1E9 TU, less than about 4500 μg / 1E9 TU, less than about 4000 μg / 1E9 TU, less than about 3500 μg / 1E9 TU, less than about 3000 μg / 1E9 TU, less than about 2500 μg / 1E9 TU, less than about 2000 μg / 1E9 TU, less than about 1500 μg / 1E9 TU, or less than about 1000 μg / 1E9 TU. In some embodiments, after chromatography, HCP is less than about 3000 μg / 1E9 TU.

[0189] In some embodiments, the HCP after chromatography is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, or at least 50-fold lower than before chromatography. In some embodiments, the HCP after chromatography is at least about 40-fold lower than before chromatography.

[0190] Illustrative chromatography techniques are described below. a. Ion exchange chromatography

[0191] In some embodiments, chromatography includes ion exchange chromatography. Ion exchange chromatography utilizes such fact that charged species (such as biomolecules and viral vectors) can reversibly bind to a stationary phase (such as a film, or the filler in a column), and the stationary phase has a group with opposite charges fixed on its surface. There are two types of ion exchangers. Anion exchangers are following stationary phases, and they carry groups with positive charges and therefore can be combined with species with negative charges. Cation exchangers carry groups with negative charges and therefore can be combined with species with positive charges. The pH of culture medium has an important influence on this, because it can change the charge on the species. Therefore, for species such as proteins, if pH is higher than π, then net charge will be negative, and if lower than π, then net charge will be positive.

[0192] The displacement (elution) of the bound species can be affected by using a suitable buffer. Generally speaking, the ionic concentration of the buffer is increased until the species is displaced by competition of the buffer ions for ionic sites on the stationary phase. Alternative elution methods require changing the pH of the buffer until the net charge of the species is no longer conducive to binding the stationary phase. An illustrative example is to reduce the pH until the species has a net positive charge and will no longer bind to an anion exchanger.

[0193] If the impurity is uncharged, or if the impurity carries a charge of the opposite sign to the charge of the desired species but the same sign as the charge on the ion exchanger, some purification can be achieved. This is because uncharged species and species with the same sign as the charge on the ion exchanger will generally not bind. For different bound species, the strength of binding varies according to a variety of factors, such as charge density and the distribution of charge on the various species. Therefore, by applying an ion or pH gradient (either as a continuous gradient, or as a series of steps), the desired species can be eluted separately from the impurities.

[0194] In some embodiments, ion exchange chromatography is performed after harvest clarification. In some embodiments, anion exchange chromatography is performed after harvest clarification.

[0195] In some embodiments, the chromatography is anion exchange (AEX) chromatography. In some embodiments, AEX is performed with a membrane having a surface coating with positively charged functional groups, thereby allowing transmembrane convection flow of negatively charged viral vectors, which can bind to the positively charged coating surface on the membrane. In some embodiments, the nominal pore size of the membrane is about 0.8 μM.

[0196] In some embodiments, AEX uses commercially available membranes. Illustrative commercially available membranes for AEX include, but are not limited to Q chromatography membrane. In certain embodiments, the AEX membrane binds the lentivirus, while impurities do not. The lentivirus can then be eluted by increasing the salt (e.g., NaCl) concentration.

[0197] In some embodiments, the use of AEX allows larger impurities and impurities with different binding abilities to the viral vector (primarily positively charged impurities) to flow through the membrane during loading. Loosely bound impurities are then removed with a wash step. In some embodiments, the wash step is performed using a salt buffer that typically includes the same pH and conductivity as the filtered formulation added as bulk load.

[0198] In some embodiments, ion exchange chromatography (such as anion exchange chromatography) includes a step of eluting a viral vector bound to a membrane. In some embodiments, elution can be performed by increasing the transmembrane salt concentration to cause the viral vector to dissociate from the membrane. In some embodiments, a low concentration of salt (e.g., NaCl) is used during the balance, loading, and washing steps of anion exchange chromatography, and then the salt (e.g., NaCl) concentration in the buffer added for elution is increased.

[0199] In some embodiments, the methods provided herein involve eluting surface engineered lentiviral particles by adding a high salt buffer, such as a high salt buffer containing sodium chloride (NaCl), potassium chloride (KCl), sodium acetate (NaOAc) or tetramethylammonium chloride. In some embodiments, the method of eluting surface engineered lentiviral particles includes NaCl.

[0200] In some embodiments, the NaCl concentration in the salt buffer used for elution is about 0.5 M to 3 M. In some embodiments, the NaCl concentration in the salt buffer used for elution is about 0.75 M to 2 M. In some embodiments, the NaCl concentration in the salt buffer used for elution is about 0.75 M. In some embodiments, the NaCl concentration in the salt buffer used for elution is about 1 M. In some embodiments, the NaCl concentration in the salt buffer used for elution is about 2 M.

[0201] In some embodiments, the present disclosure surprisingly demonstrates herein that the concentration of the salt buffer used for elution can affect the yield of the lentivirus produced by the process. In particular, the results show that the yield percentage tends to decrease with changes in surface engineering and / or transgenic payload. In some embodiments, when the surface engineered protein is larger or the transgenic payload sequence is longer, using a higher salt concentration for elution can achieve improved lentiviral yields.

[0202] In an exemplary method, lentiviral particles produced to express surface engineered proteins containing single domain fusion proteins can be eluted in a buffer containing 0.5M to 1.5M NaCl, such as 0.5M to 0.75M NaCl. In some embodiments, lentiviral particles produced to express surface engineered proteins containing single domain fusion proteins can be eluted in a buffer containing 0.75M NaCl.

[0203] In another exemplary method, the lentiviral particles produced to express a surface engineered protein containing a multi-domain fusion protein can be eluted in a buffer containing 1.5M NaCl to 2.5M NaCl (e.g., 1.75M to 2.25M NaCl). In some embodiments, the lentiviral particles produced to express a multi-domain fusion protein are eluted in a buffer containing 2M NaCl. b. Size Exclusion Chromatography

[0204] In some embodiments, chromatography includes size exclusion chromatography. Size exclusion chromatography is a technique for separating species according to species size. Typically, size exclusion chromatography is performed using a column filled with particles having pores with a clear size. For chromatographic separation, particles with a pore size appropriate to the size of the species in the mixture to be separated are selected. When the mixture is applied to the column as a solution (or suspension, in the case of viruses) and then eluted with a buffer, the largest particles will be eluted first because they are limited (or unable) to enter the pores. Smaller particles will be eluted later because they can enter the pores, thereby taking a longer path to pass through the column.

[0205] Therefore, when considering the use of size exclusion chromatography to purify a viral vector, it is expected that the vector will elute before smaller impurities such as proteins.

[0206] In some embodiments, size exclusion chromatography is performed after clarification of the harvest. c. Hydrophobic Interaction Chromatography (HIC)

[0207] In some embodiments, the chromatography includes hydrophobic interaction chromatography. Species such as proteins have hydrophobic regions on their surfaces that can reversibly bind to weak hydrophobic sites on the stationary phase. This binding is promoted in a culture medium with a relatively high salt concentration. Typically, in HIC, the sample to be purified is combined with the stationary phase in a high salt environment. Elution is then achieved by applying a gradient of decreasing salt concentration (continuously, or as a series of steps). A commonly used salt is ammonium sulfate. Species with different hydrophobicity levels will tend to elute at different salt concentrations, so the target species can be purified from impurities. Other factors (such as pH, temperature, and additives to the elution medium (such as detergents, chaotropic salts, and organic matter)) also affect the binding strength of the species to the HIC stationary phase. One or more of these factors can be adjusted or utilized to optimize the elution and purification of the product.

[0208] Viral vectors have hydrophobic moieties (such as proteins) on their surface and thus can be purified using HIC.

[0209] In some embodiments, HIC is performed after clarification of the harvest. d. Reverse Phase Chromatography (RPC)

[0210] In some embodiments, chromatography includes reverse phase chromatography. Similar to HIC, RPC separates species according to differences in the hydrophobicity levels of the species. Use a stationary phase that is more hydrophobic than the stationary phase used in HIC. The stationary phase is usually composed of a material (usually silica) that combines hydrophobic parts, such as alkyl or phenyl. Alternatively, the stationary phase can be an organic polymer without an attachment group. A sample containing a mixture of species to be dissociated is applied to a relatively high polar aqueous medium of the stationary phase, which promotes binding. Then, elution is achieved by adding an organic solvent (such as isopropanol or acetonitrile) to reduce the polarity of the aqueous medium. A gradient of increasing organic solvent concentration (continuous or as a series of steps) is usually used, and the species are eluted according to their respective hydrophobicity levels.

[0211] Other factors (such as the pH of the elution medium and the use of additives) may also affect the binding strength of the species to the RPC stationary phase. One or more of these factors can be adjusted or utilized to optimize the elution and purification of the product. A commonly used additive is trifluoroacetic acid (TFA). This suppresses the ionization of acidic groups (such as carboxyl moieties) in the sample. It also reduces the pH in the elution medium, and this suppresses the ionization of free silanol groups that may exist on the stationary phase surface with a silica matrix. TFA is one of a class of additives called ion pairing agents. These additives interact with ionic groups with opposite charges present on the species in the sample. The interaction tends to mask the charge, thereby increasing the hydrophobicity of the species. Anionic ion pairing agents (such as TFA and pentafluoropropionic acid) interact with positively charged groups on the species. Cationic ion pairing agents (such as triethylamine) interact with negatively charged groups.

[0212] Viral vectors have hydrophobic moieties (eg, proteins) on their surface and thus could potentially be purified using RPC.

[0213] In some embodiments, RPC is performed after clarification of the harvest. e. Affinity chromatography

[0214] In some embodiments, chromatography includes affinity chromatography. Affinity chromatography exploits the fact that certain ligands that specifically bind to biomolecules (such as proteins or nucleotides) can be fixed to a stationary phase. A modified stationary phase can then be used to separate the related biomolecules from the mixture. Examples of highly specific ligands are antibodies for purifying target antigens and enzyme inhibitors for purifying enzymes. More general interactions can also be utilized, such as using protein A ligands to separate a wide variety of antibodies.

[0215] Usually, affinity chromatography is carried out by applying a mixture containing the target species to a stationary phase to which the relevant ligand is attached. Under appropriate conditions, this will result in the binding of the species to the stationary phase. The unbound components are then washed away and an elution medium is subsequently applied. The elution medium is selected to destroy the binding of the ligand to the target species. This is usually achieved by selecting appropriate ionic strength, pH or by using a substance that can compete with the target species for the ligand site. For some combined species, a chaotropic agent such as urea is used to achieve displacement from the ligand. However, this may result in irreversible denaturation of the species.

[0216] Viral vectors have moieties such as proteins on their surface that may be able to specifically bind to appropriate ligands. This means that affinity chromatography could potentially be used for their separation.

[0217] In some embodiments, affinity chromatography is performed after clarification of the harvest. f. Immobilized Metal Ion Affinity Chromatography (IMAC)

[0218] In some embodiments, the chromatogram includes an immobilized metal ion affinity chromatography. Biomolecules (such as proteins) can have an electron-donating portion that can form a coordination bond with a metal ion on its surface. This can promote them to be combined with a stationary phase carrying an immobilized metal ion (such as Ni2+, Cu2+, Zn2+ or Fe3+). The stationary phase used in IMAC has a chelating agent (usually nitrilotriacetic acid or iminodiacetic acid) covalently attached to their surface, and it is the chelating agent that keeps the metal ion. The chelated metal ion must have at least one coordination site left to form a coordination bond with the biomolecule. There are potentially several parts on the surface of the biomolecule that may be able to bond with the immobilized metal ion. These parts include histidine, tryptophan and cysteine ​​residues and phosphate groups. However, for proteins, the main donor seems to be the imidazole group of histidine residues. If a natural protein exhibits a suitable donor portion on its surface, IMAC can be used to separate the natural protein. IMAC can also be used to separate recombinant proteins that carry chains of several connected histidine residues.

[0219] Typically, IMAC is performed by applying a mixture containing the target species to a stationary phase. Under appropriate conditions, this results in a coordination bond between the species and the stationary phase. The unbound components are then washed away and an elution medium is subsequently applied. For elution, a gradient (continuous, or as a series of steps) of increasing salt concentration or decreasing pH can be used. In addition, a common procedure is to apply a gradient of increasing imidazole concentration. Biomolecules having different donor properties (e.g., having histidine residues in different environments) can be separated by using gradient elution.

[0220] Viral vectors have moieties such as proteins on their surface that may be able to bind to an IMAC stationary phase. This means that IMAC could potentially be used for their separation.

[0221] In some embodiments, IMAC is performed after clarification of the harvest. 3. Ultrafiltration / diafiltration

[0222] In some embodiments, the method of the present disclosure includes concentrating the filtered viral particle preparation, wherein the concentration includes ultrafiltration. In some embodiments, the method of the present disclosure includes purifying the filtered viral particle preparation, wherein the purification includes ultrafiltration. In some embodiments, ultrafiltration is performed before chromatography. In some embodiments, ultrafiltration is performed after chromatography. In some embodiments, ultrafiltration also acts on removing impurities, such as HCP and HcDNA, from the filtered particle preparation. In some embodiments, ultrafiltration is used to further remove impurities, such as HCP and hcDNA, and concentrate the viral particles in the preparation from the filtered particle preparation and / or from the chromatography eluent. In some embodiments, the method of the present disclosure includes clarifying the suspended mixture by using a series of filters as described above, followed by a chromatography step, and then an ultrafiltration step.

[0223] According to an embodiment of the present disclosure, the filtered viral particle formulation is subjected to at least one ultrafiltration (also called diafiltration when used for buffer exchange) during the process, for example, for concentrating the vector and / or buffer exchange.

[0224] The process for concentrating viral particles according to the methods of the present disclosure may include any filtration process (e.g., ultrafiltration (UF)) in which the concentration of viral particles is increased by forcing a diluent through a filter in such a manner that the diluent is removed from the viral particle preparation while the viral particles are unable to pass through the filter and are thus retained in a concentrated form in the viral particle preparation. UF is described in detail in, for example, Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, NY, 1996); and Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9).

[0225] In some embodiments, tangential flow filtration ("TFF") is used, as described, for example, in the MILLIPORE catalog entitled "Pharmaceutical Process Filtration Catalogue" on pages 177-202 (Bedford, Massachusetts, 1995 / 96). TFF is widely used in the bioprocessing industry for cell harvesting, clarification, purification, and concentration of products including viruses. The system consists of three different process streams: a feed solution, a permeate, and a retentate. Depending on the application, filters with different pore sizes can be used in TFF. In some embodiments, the retentate contains the product (e.g., lentiviral particles).

[0226] In some embodiments, TFF is used to purify and concentrate viral particles using ultrafiltration and diafiltration (UF / DF). In embodiments using TFF, viral vector particles are retained in the system, while smaller components and impurities penetrate the membrane and are removed from the system.

[0227] In some embodiments, the pore size of the selected specific ultrafiltration membrane will be small enough to retain viral particles, but large enough to effectively remove impurities. According to the manufacturer and membrane type, the nominal molecular weight cut-off (NMWC) between 100kDa and 1000kDa may be appropriate, for example, a membrane with 300kDa or 500kDa NMWC. The composition of the membrane can be but is not limited to regenerated cellulose, polyethersulfone, polysulfone or its derivatives. The membrane can be a flat plate (also referred to as a flat screen) or a hollow fiber. UF generally involves the filtration performed using a filter having a pore size less than 0.1 μm. The product is usually retained while the volume can be reduced by penetration (or in the diafiltration process by adding buffer at the same speed as the speed of removing the permeate containing buffer and impurities on the permeate side to remain unchanged).

[0228] In the biopharmaceutical industry, the two most commonly used geometries for TFF are plate and frame (flat sieve) and hollow fiber modules. Hollow fiber modules consist of an array of self-supporting fibers with a dense skin layer. The fiber diameter ranges from 0.5 mm to 3 mm. The advantage of hollow fiber modules is the availability of filters from small membrane areas (about 16 cm2) to very large membrane areas (about 20 m2), allowing linear and simple scale-up.

[0229] In some embodiments, hollow fibers are used for TFF. These hollow fibers are reported to give less shear and better viral particles / infectious units (VP / IU) ratios than flat sieve membranes. In addition, the transmembrane pressure in hollow fibers is generally lower than that in flat sieves.

[0230] In certain embodiments, hollow fibers with a pore size of 500 kDa (0.05 μm) are used. Ultrafiltration can include diafiltration (DF) using an ultrafilter and is an ideal approach for: removal and exchange of salts, sugars, non-aqueous solvents; separation of free species from bound species; removal of low molecular weight materials; or rapid changes in ionic and / or pH environments. Microsolutes are most effectively removed by adding solvent to the solution being ultrafiltered at a rate equal to the UF rate. This washes out microspecies from the solution at a constant volume, thereby purifying the retained viral particles.

[0231] UF / DF can be used at different stages of the concentration process to concentrate and / or buffer exchange the viral particle suspension according to the present disclosure. In some embodiments, the method of the present disclosure utilizes a DF step to exchange the buffer of the supernatant after the chromatography or other purification steps. In some embodiments, the eluate of the chromatography step according to the present disclosure is concentrated and further purified by ultrafiltration-diafiltration. During this process, the viral particles are exchanged into the formulation buffer.

[0232] In some embodiments, ultrafiltration / diafiltration can be tangential flow diafiltration, stirred cell diafiltration and dialysis.In some embodiments, ultrafiltration / diafiltration may include multiple tangential flow diafiltrations (TFF).In some embodiments, the multiple may be 1,2,3,4 or more individual tangential flow diafiltrations.In some embodiments, the process includes two TFF steps and systems.In some embodiments, the first TFF step can be used to concentrate the chromatographic eluent (e.g., AEX collection) and buffer exchange to diafiltration buffer, and concentrate.In some embodiments, the second TFF can be used to further concentrate the collection from the first TFF, such as to achieve the final target titer of the bulk drug.In some embodiments, the first TFF and the second TFF are identical.In some embodiments, the first TFF and the second TFF are different.In some embodiments, the first TFF and the second TFF have the same NMWC, but the first TFF has a larger hold-up volume or surface area.

[0233] In some embodiments, TFF is performed with a hollow fiber filter (e.g., each of the TFFs is performed separately). In some embodiments, the nominal molecular weight cutoff (NMWC) of the hollow fiber filter is between 100 kDa and 1000 kDa. In some embodiments, the NMWC is between 100 kDa and 500 kDa. In some embodiments, the NMWC is between 300 kDa and 750 kDa. In some embodiments, the NMWC is between 500 kDa and 1000 kDa.

[0234] In some embodiments, the NMWC is 500 kDa. In some embodiments, the NMWC is 300 kDa. In some embodiments, the NMWC is 100 kDa.

[0235] In some embodiments, the pore size of the hollow fiber filter is between 0.1 μm and 0.65 μm. In some embodiments, the pore size is 0.1 μm. In some embodiments, the pore size is 0.2 μm. In some embodiments, the pore size is 0.45 μm. In some embodiments, the pore size is 0.65 μm.

[0236] In some embodiments, the length of the hollow fiber filter is 20 cm, 41.5 cm, 65 cm, 50 cm, 68 cm or 108 cm. In some embodiments, the lumen of the hollow fiber filter is 0.50 mm, 0.63 mm, 0.75 mm, 1 mm, 2 mm or 3 mm.

[0237] In some embodiments, the surface area of ​​the hollow fiber filter is 235 cm 2 With 1600cm 2 In some embodiments, the surface area is 235 cm 2 In some embodiments, the surface area is 790 cm 2 In some embodiments, the surface area is 1600 cm 2 .

[0238] In some embodiments, the hollow fiber filter holds a volume of 300 mL.

[0239] In some embodiments, the hollow fiber filter is a commercially available hollow fiber filter. Illustrative examples of commercially available hollow fiber filters include, but are not limited to Hollow fiber filter (Repligen).

[0240] In some embodiments, each TFF is performed with a hollow fiber filter comprising different boxes or modules that differ in one or more aspects of batch volume, pore size, membrane cutoff, lumen diameter, or length. In some embodiments, the hollow fiber filters of each TFF can have the same nominal molecular weight cutoff (NMWC) or pore size, but are different in surface area or volume. In some embodiments, the hollow fiber filters of each TFF can have different NMWC or pore size and are different in surface area or volume.

[0241] In some embodiments, HCP is measured after the UF / DF step. HCP can be measured by ELISA and normalized relative to the amount of viral particles. In some embodiments, HCP is measured in units / dose, where a dose is defined using a transduction unit (TU) of the virus. In some embodiments, HCP is measured in μg / dose, where a dose is 1E9 TU.

[0242] In some embodiments, after UF / DF, HCP is less than about 5000 μg / 1E9 TU, less than about 4500 μg / 1E9 TU, less than about 4000 μg / 1E9 TU, less than about 3500 μg / 1E9 TU, less than about 3000 μg / 1E9 TU, less than about 2500 μg / 1E9 TU, less than about 2000 μg / 1E9 TU, less than about 1500 μg / 1E9 TU, or less than about 1000 μg / 1E9 TU. In some embodiments, after the first UF / DF step, HCP is less than about 1500 μg / 1E9 TU. In some embodiments, after the second UF / DF step, HCP is undetectable. 4. Filter Sterilization

[0243] In some embodiments, the method of the present disclosure comprises aseptic filtration of a filtered and concentrated viral particle formulation as described herein. Filter sterilization is commonly used in processes for pharmaceutical grade materials and is known to those skilled in the art. Filter sterilization renders the resulting formulation substantially free of contaminants. After filter sterilization, the level of contaminants renders the formulation suitable for clinical use. Suitable sterilizing filters for use according to the present disclosure are well known to those skilled in the art.

[0244] In some embodiments, the concentrated mixture after UF / DF is filter sterilized to produce a sterilized formulation. In some embodiments, the sterilizing filter has a maximum pore size of 0.22 μm. In some embodiments, the sterilizing filter has a retention threshold of 0.2 μm.

[0245] In some embodiments, the sterilized formulation is formulated in a buffer to produce a bulk drug. In some embodiments, the amount of contaminants in the bulk drug is at an acceptable level for in vivo administration to a subject. In some embodiments, the contaminants include host cells. In some embodiments, the contaminants include host cell DNA. In some embodiments, the contaminants include host cell proteins. In some embodiments, the contaminants include host cell DNA and host cell proteins.

[0246] In some embodiments, clarification, chromatography, UF / DF and filtration sterilization steps are carried out in a time period of about 5-10 hours. In some embodiments, clarification, chromatography, UF / DF and filtration sterilization steps are carried out in a time period of about 5-8 hours. In some embodiments, clarification, chromatography, UF / DF and filtration sterilization steps are carried out in a time period of about 5-6 hours. In some embodiments, clarification, chromatography, UF / DF and filtration sterilization steps are carried out in a time period of about 6-7 hours. In some embodiments, clarification, chromatography, UF / DF and filtration sterilization steps are carried out in a time period of about 6 hours.

[0247] In some embodiments, each of the clarification, chromatography, UF / DF, and filtration sterilization steps is performed at a pH of 6-8. In some embodiments, each of the clarification, chromatography, UF / DF, and filtration sterilization steps is performed at a pH equal to: 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, each of the clarification, chromatography, UF / DF, and filtration sterilization steps is performed at a pH equal to: 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0248] In some embodiments, the viral vector preparations prepared by the above method do not contain impurities. In some embodiments, impurities include host cell DNA (hcDNA). In some embodiments, impurities include host cell proteins (HCP). In some embodiments, the viral vector preparations are substantially pure. In some embodiments, the viral vector preparations provided herein contain less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.5% hcDNA. In some embodiments, the viral vector preparations provided herein contain less than 1% hcDNA. In some embodiments, the method for preparing the viral vector preparation realizes that hcDNA is greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% reduction, such as relative to the suspension mixture. In some embodiments, the method for preparing the viral vector preparation realizes 99% reduction of hcDNA, such as compared with the suspension mixture.

[0249] In some embodiments, the viral vector preparations provided herein contain less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.5% HCP. In some embodiments, the viral vector preparations described herein contain less than 1% HCP. In some embodiments, the method for preparing the viral vector preparation realizes that HCP is greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% reduction, such as relative to the suspension mixture. In some embodiments, the method for preparing the viral vector preparation realizes 99% reduction of HCP, such as relative to the suspension mixture.

[0250] In some embodiments, the yield of the viral vector achieved by the above method of preparing a viral vector formulation is at least or at least about 10%, at least or at least about 12%, at least or at least about 15%, at least or at least about 18%, at least or at least about 20%, at least or at least about 22%, at least or at least about 25%, at least or at least about 28%, or at least or at least about 30%. In some embodiments, the yield of the viral vector achieved by the above method of preparing a viral vector formulation is 10% to 30%, such as or about 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28% or 30%, or any value between any of the foregoing values. In some embodiments, the yield of the viral vector achieved by the above method of preparing a viral vector formulation is at least or at least about 15% or about 15%.

[0251] In some embodiments, the downstream process produces a viral vector titer of 0.5 x 10 7 TU / mL to 5x10 8 TU / mL of viral vector formulation (which may be referred to as drug substance). In some embodiments, the viral vector titer is 1x10 7 TU / mL to 3x10 7 TU / mL, 2x10 7 TU / mL up to 4x10 7 TU / mL, 3x10 7 TU / mL to 5x10 7 TU / mL, 4x10 7 TU / mL up to 6x10 7 TU / mL, 5x10 7 TU / mL up to 7x10 7 TU / mL, 6x10 7 TU / mL up to 8x10 7 TU / mL, 7x10 7 TU / mL up to 9x107 TU / mL, 8x10 7 TU / mL to 1x10 8 TU / mL, 9x10 7 TU / mL to 2x10 8 TU / mL, 1x10 8 TU / mL to 3x10 8 TU / mL, 2x10 8 TU / mL up to 4x10 8 TU / mL, 3x10 8 TU / mL to 5x10 8 TU / mL. In some embodiments, the viral vector titer is 2.5x10 8 TU / mL to 4.7x10 8 TU / mL. In some embodiments, the downstream process produces a viral vector titer of 2.5x10 8 TU / mL to 4.7x10 8 TU / mL of a viral vector formulation (or drug substance). In some embodiments, the viral vector titer comprises at least about 2.5x10 8 TU / mL. In some embodiments, the viral vector titer comprises at least about 4.7 x 10 8 TU / mL. In some embodiments, the viral vector titer is 2.5x10 8 TU / mL. In some embodiments, the viral vector titer is 4.7x10 8 TU / mL.

[0252] In some embodiments, the volume of the viral vector preparation is between about 1 and 500 mL. In some embodiments, the viral vector preparation comprises the following volume: between about 1 and 10 mL, 5 to 15 mL, 10 to 20 mL, 15 to 25 mL, 20 to 30 mL, 25 to 35 mL, 30 to 40 mL, 35 to 45 mL, 40 to 50 mL, 50 to 100 mL, 75 to 125 mL, 100 to 300 mL, 200 to 400 mL, or 300 to 500. In some embodiments, the viral vector preparation comprises at least 10 mL, 50 mL, 100 mL, 200 mL, or 300 mL. In some embodiments, the viral vector preparation is 10 mL. In some embodiments, the viral vector preparation is 100 mL. In some embodiments, the viral vector preparation is 200 mL. In some embodiments, the viral vector preparation is 300 mL. In some embodiments, the viral vector preparation is 500 mL. In some embodiments, the viral vector is a retrovirus (eg, a lentivirus).

[0253] In some embodiments, the downstream process produces a viral vector titer of 5x10 10 TU to 5x10 12 TU viral vector formulation or API. In some embodiments, the viral vector titer is 1x10 11 TU to 3x10 11 TU, 2x10 11 TU to 4x10 11 TU, 3x10 11 TU to 5x10 11 TU, 4x10 11 TU to 6x10 11 TU, 5x10 11 TU to 7x10 11 TU, 6x10 11 TU to 8x10 11 TU, 7x10 11 TU to 9x10 11 TU, 8x10 11 TU to 1x10 12 TU, 9x10 11 TU to 2x10 12 TU, 1x10 12 TU to 3x10 12 TU, 2x10 12 TU to 4x10 12 TU, 3x10 12 TU to 5x10 12 TU. In some embodiments, the viral vector titer is 2.5x10 12 TU to 4.7x10 12 TU. In some embodiments, the viral vector titer is at least about 2.5 x 10 12 TU. In some embodiments, the viral vector titer is at least about 4.7 x 10 12 TU. In some embodiments, the viral vector titer is 2.5x10 12 TU. In some embodiments, the viral vector titer is 4.7x10 12 TU.

[0254] In some embodiments, the downstream process produces a viral vector titer of 5x10 8 TU to 5x10 10 TU viral vector formulation or API. In some embodiments, the viral vector titer is 1x10 9 TU to 3x10 9 TU, 2x10 9 TU to 4x10 9 TU, 3x10 9TU to 5x10 9 TU, 4x10 9 TU to 6x10 9 TU, 5x10 9 TU to 7x10 9 TU, 6x10 9 TU to 8x10 9 TU, 7x10 9 TU to 9x10 9 TU, 8x10 9 TU to 1x10 10 TU, 9x10 9 TU to 2x10 10 TU, 1x10 10 TU to 3x10 10 TU, 2x10 10 TU to 4x10 10 TU, 3x10 10 TU to 5x10 10 TU. In some embodiments, the viral vector titer is 2.5x10 10 TU to 4.7x10 10 TU. In some embodiments, the viral vector titer comprises at least about 2.5 x 10 10 TU. In some embodiments, the viral vector titer comprises at least about 4.7 x 10 10 TU. In some embodiments, the viral vector titer is 2.5x10 10 TU. In some embodiments, the viral vector titer is 4.7x10 10 TU. In some embodiments, the viral vector titer is 6x10 10 TU.

[0255] In some embodiments, the downstream process produces a viral vector titer of 1x10 9 TU to 1x10 11 TU of viral vector formulation or API. In some embodiments, the viral vector titer is 2x10 9 TU to 6x10 9 TU, 4x10 9 TU to 8x10 9 TU, 6x10 9 TU to 1x10 10 TU, 8x10 9 TU to 1.2x10 10 TU, 1x10 10 TU to 1.4x10 10 TU, 1.2x10 10 TU to 1.6x10 10TU, 1.4x10 10 TU to 1.8x10 10 TU, 1.6x10 10 TU to 2x10 10 TU, 1.8x10 10 TU to 4x10 10 TU, 2x10 10 TU to 6x10 10 TU, 4x10 10 TU to 8x10 10 TU, 6x10 10 TU to 1x10 11 TU. In some embodiments, the viral vector titer is 5x10 10 TU to 9.4x10 10 TU. In some embodiments, the viral vector titer comprises at least about 5x10 10 TU. In some embodiments, the viral vector titer comprises at least about 9.4 x 10 10 TU. In some embodiments, the viral vector titer is 5x10 10 TU. In some embodiments, the viral vector titer is 9.4x10 10 TU. In some embodiments, the viral vector titer is 6x10 10 TU.

[0256] In some embodiments, the downstream process produces a viral vector titer of 1.5 x 10 9 TU to 1.5x10 11 TU of viral vector formulation or API. In some embodiments, the viral vector titer is 3x10 9 TU to 9x10 9 TU, 6x10 9 TU to 1.2x10 10 TU, 9x10 9 TU to 1.5x10 10 TU, 1.2x10 10 TU to 1.8x10 10 TU, 1.5x10 10 TU to 2.1x10 10 TU, 1.8x10 10 TU to 2.4x10 10 TU, 2.1x10 10 TU to 2.7x10 10 TU, 2.4x10 10 TU to 3x10 10 TU, 2.7x10 10 TU to 6x10 10TU, 3x10 10 TU to 9x10 10 TU, 6x10 10 TU to 1.2x10 11 TU, 9x10 10 TU to 1.5x10 11 TU. In some embodiments, the viral vector titer is 7.5x10 10 TU to 1.4x10 11 TU. In some embodiments, the viral vector titer comprises at least about 7.5 x 10 10 TU. In some embodiments, the viral vector titer comprises at least about 1.4 x 10 11 TU. In some embodiments, the viral vector titer is 7.5x10 10 TU. In some embodiments, the viral vector titer is 1.4x10 11 TU. In some embodiments, the viral vector titer is 6x10 10 TU. C. Process Control and Characterization

[0257] In some embodiments, a method for preparing a viral particle formulation is provided herein. In some embodiments, a method for preparing a viral particle formulation includes a manufacturing process. In some embodiments, an exemplary manufacturing process is shown in Figure 1C In some embodiments, the manufacturing process provided herein includes the following: Figure 1C Evaluation of one or more manufacturing process controls shown. In some embodiments, manufacturing process controls include measurements of one or more features of the manufacturing process with the goal of ensuring consistency between different manufacturing runs. For example, determining the cell count at the start of the manufacturing process can ensure that similar cell yields will be consistent between different manufacturing runs, which will ultimately lead to consistent, predictable, and stable virus production.

[0258] In some embodiments, the manufacturing processes provided herein include Figure 1C An evaluation of one or more manufacturing process characterizations shown. In some embodiments, a manufacturing process characterization is a feature measured in order to provide information about the performance of a manufacturing process in retrospect. For example, measuring process impurities (i.e., impurities introduced due to reagents or other materials used in the manufacturing process) indicates how well certain steps of the manufacturing process work. Measuring process impurities for a harvest clarification step or an anion exchange chromatography step indicates how well a material (e.g., a column or membrane) removes process impurities.

[0259] In some embodiments, manufacturing process control in the upstream process includes measuring: cell count or cell viability during cell expansion (e.g., seed culture); or cell count, cell viability, pH, dissolved oxygen (DO) or temperature in a production bioreactor. In some embodiments, cell counting is used to determine whether the cells are growing, and cells are also allowed to be passaged at a non-restrictive density during seed culture expansion. In some cases, higher cell densities may induce stress on cells. In some embodiments, cell viability is used to confirm whether the cell count mainly contains live non-dead cells. In some embodiments, pH is used to ensure that cells remain viable. In some cases, pH<6.8 has a negative impact on viral production cell growth and transfection. In some embodiments, DO represents the total amount of oxygen available for viral production cells.

[0260] In some embodiments, manufacturing process control in the downstream process includes measuring: pressure or turbidity of the viral particle product in the harvest clarification step; pH, conductivity or flow rate of the viral particle product in the AEX chromatography step; viral particle count or p24 of the viral particle product in the UF / DF step; pressure or load factor of the viral particle product during the bulk drug (DS) filtration step; or the filling weight of the frozen drug product. In some embodiments, turbidity is measured to indicate the presence of cells or other contaminants. In some embodiments, pressure is measured to determine whether larger impurities need to be removed. In some cases, high pressure may cause problems in the membrane chromatography step. P24 is a capsid protein expressed by the virus. Therefore, in some embodiments, measuring p24 indicates the presence of cells that are producing viral particles. In some embodiments, the filling weight is the amount of drug product in a given container (e.g., a vial). In some embodiments, the drug product is assessed to see if it meets the acceptable weight range, otherwise the drug product can be rejected during clinical use.

[0261] In some embodiments, manufacturing process characterization in the upstream process includes measuring metabolites, p24, titer, turbidity, hcDNA during cell expansion (e.g., seed culture) and production bioreactor steps. In some embodiments, metabolites are used to determine whether the cells are healthy.

[0262] In some embodiments, manufacturing process characterization in the downstream process includes measuring: p24, titer, turbidity, or process impurities during the harvest clarification step; p24, titer, or process impurities during the AEX and UF / DF steps; or complete release, characterization, or stability during the DS filtration step.

[0263] In addition to step-by-step manufacturing process control and characterization, an analytical platform can also be used to support rapid scale-up of lentiviral particle formulations. In some embodiments, the analytical platform includes methods for assessing: (i) the identity of the viral particle formulation; (ii) the purity of the viral particle formulation; (iii) the potency of the viral particle formulation; and (iv) the safety of the viral particle formulation.

[0264] In some embodiments, methods employed in identifying viral particle formulations include sequencing to detect the presence of a virally expressed transgene, or performing a Western blot / ELISA to detect the presence of a virally expressed protein.

[0265] In some embodiments, the purity of the viral particle formulation is assessed using methods that include detecting impurities such as endonucleases (process-related impurities), host cell proteins, host cell DNA, and polyethyleneimine (PEI; process-related impurities). In some embodiments, viral particle formulation components that indicate purity include the presence of plasmid DNA, E1A DNA, or SV40 DNA.

[0266] In some embodiments, the method used to evaluate the effectiveness of a viral particle formulation includes physical titer, which is determined by measuring the number of p24, RNA genomes, and physical particles. In some embodiments, another method for evaluating effectiveness includes generating the transduction titer required for a viral particle product. In some embodiments, the transduction titer or infection titer is any infection titer provided herein. In some embodiments, another method for evaluating effectiveness includes measuring the expression of cell surface markers on viral particles or measuring cell surface marker functions (e.g., CAR functions).

[0267] In some embodiments, methods for assessing the safety of viral particle products include measuring endotoxin, bioburden (i.e., the number of contaminating organisms found in the viral particle formulation prior to sterilization), sterility, invisible particles (i.e., particles that are too large (e.g., about >0.1 μm) for analysis by size exclusion chromatography (SEC), but too small to be visible to the naked eye (e.g., <100 μm)), mycoplasma, adventitious viruses, and replication-competent lentiviruses. D. Exemplary Methods

[0268] In some embodiments, the present disclosure provides a method for preparing a viral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and viral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a double-layer filter assembly comprising a second filter and a third filter to produce a filtered viral particle preparation, wherein the second filter has a smaller retention threshold than the first filter, and the third filter has a smaller retention threshold than the second filter; (ii) purifying the filtered viral particle preparation; and (iii) concentrating the filtered viral particle preparation, wherein the concentration comprises chromatography and ultrafiltration.

[0269] In some embodiments, the present disclosure provides a method for preparing a retroviral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and retroviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a double-layer filter assembly comprising a second filter and a third filter to produce a filtered retroviral particle preparation, wherein the second filter has a smaller retention threshold than the first filter, and the third filter has a smaller retention threshold than the second filter; (ii) purifying the filtered retroviral particle preparation; and (iii) concentrating the filtered retroviral particle preparation, wherein the concentration comprises chromatography and ultrafiltration.

[0270] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and lentiviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a double-layer filter assembly comprising a second filter and a third filter to produce a filtered lentiviral particle formulation, wherein the second filter has a smaller retention threshold than the first filter, and the third filter has a smaller retention threshold than the second filter; (ii) purifying the filtered lentiviral particle formulation; and (iii) concentrating the filtered lentiviral particle formulation, wherein the concentration comprises chromatography and ultrafiltration.

[0271] In some embodiments, the present disclosure provides a method for preparing a viral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and viral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, (c) filtering the second filtrate with a double-layer filter assembly comprising the second filter and a third filter to produce a filtered viral particle preparation, wherein the third filter has a smaller retention threshold than the second filter; (ii) purifying the filtered viral particle preparation; and (iii) concentrating the filtered viral particle preparation, wherein the concentration comprises chromatography and ultrafiltration.

[0272] In some embodiments, the present disclosure provides a method for preparing a retroviral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and retroviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, (c) filtering the second filtrate with a double-layer filter assembly comprising the second filter and a third filter to produce a filtered viral particle preparation, wherein the third filter has a smaller retention threshold than the second filter; (ii) purifying the filtered retroviral particle preparation; and (iii) concentrating the filtered retroviral particle preparation, wherein the concentration comprises chromatography and ultrafiltration.

[0273] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and lentiviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, (c) filtering the second filtrate with a double-layer filter assembly comprising the second filter and a third filter to produce a filtered viral particle formulation, wherein the third filter has a smaller retention threshold than the second filter; (ii) purifying the filtered lentiviral particle formulation; and (iii) concentrating the filtered lentiviral particle formulation, wherein the concentration comprises chromatography and ultrafiltration.

[0274] In some embodiments, the present disclosure provides a method for preparing a viral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and viral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter, wherein the third filter is a double-layer filter comprising a first filter and a second filter, wherein the retention threshold of the first filter is the same as that of the second filter, and wherein the second filter has a smaller retention threshold than the first filter; (ii) purifying the filtered viral particle preparation; and (iii) concentrating the filtered viral particle preparation, wherein the concentration comprises chromatography and ultrafiltration.

[0275] In some embodiments, the present disclosure provides a method for preparing a retroviral particle preparation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and retroviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter, wherein the third filter is a double-layer filter comprising a first filter and a second filter, wherein the retention threshold of the first filter is the same as that of the second filter, and wherein the second filter has a smaller retention threshold than the first filter; (ii) purifying the filtered retroviral particle preparation; and (iii) concentrating the filtered retroviral particle preparation, wherein the concentration comprises chromatography and ultrafiltration.

[0276] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and lentiviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate with a third filter to produce a filtered lentiviral particle formulation, wherein the third filter is a double-layer filter comprising a first filter and a second filter, wherein the retention threshold of the first filter is the same as that of the second filter, and wherein the second filter has a smaller retention threshold than the first filter; (ii) purifying the filtered lentiviral particle formulation; and (iii) concentrating the filtered lentiviral particle formulation, wherein the concentration comprises chromatography and ultrafiltration.

[0277] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising (i) contacting a host cell population in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the host cell population of step (i) for a period of time sufficient to produce a suspension mixture comprising the host cell population and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, the filtration comprising: (a) contacting the mixture with an endonuclease, (b) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter having a retention threshold of at least 8 μm, (c) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a retention threshold of 0.45 μm, and (d) filtering the second filtrate with a third filter to produce a filtered lentiviral particle preparation, wherein the third filter has a retention threshold of 0.2 μm; (iv) purifying the filtered lentiviral particle preparation; and (v) concentrating the filtered lentiviral particle preparation, wherein the concentration comprises chromatography and ultrafiltration.

[0278] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising (i) contacting a host cell population in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the host cell population of step (i) for a period of time sufficient to produce a suspension mixture comprising the host cell population and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, the filtration comprising: (a) contacting the mixture with an endonuclease, (b) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter with a retention threshold of at least 8 μm, (c) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a retention threshold of 0.45 μm, and (d) filtering the second filtrate with a third filter to produce a filtered lentiviral particle preparation, wherein the third filter has a retention threshold of 0.2 μm; (iv) purifying the filtered lentiviral particle preparation; and (iv) concentrating the filtered lentiviral particle preparation, wherein the concentration comprises two steps of ion exchange chromatography and ultrafiltration.

[0279] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising (i) contacting a host cell population in a suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the host cell population of step (i) for a period of time sufficient to produce a suspension mixture comprising the host cell population and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, the filtration comprising: (a) contacting the mixture with an endonuclease, (b) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a preservative The invention relates to a method for preparing a lentiviral particle formulation comprising: (i) filtering the first filtrate through a depth filter having a retention threshold of at least 8 μm, (c) filtering the first filtrate through a second filter to obtain a second filtrate, wherein the second filter has a retention threshold of 0.45 μm, and (d) filtering the second filtrate through a third filter to produce a filtered lentiviral particle formulation, wherein the third filter has a retention threshold of 0.2 μm; (iv) purifying the filtered lentiviral particle formulation; (v) concentrating the filtered lentiviral particle formulation, wherein the concentration comprises two steps of ion exchange chromatography and ultrafiltration; and (vi) filter sterilizing the concentrated formulation to produce a sterile drug substance.

[0280] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and lentiviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter having a retention threshold of at least 8 μm, (b) filtering the first filtrate with a double-layer filter assembly comprising a second filter and a third filter to produce a filtered lentiviral particle formulation, wherein the second filter has a retention threshold of 0.45 μm and the third filter has a retention threshold of 0.2 μm; (ii) purifying the filtered lentiviral particle formulation; and (iii) concentrating the filtered lentiviral particle formulation, wherein the concentration comprises chromatography and ultrafiltration.

[0281] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and lentiviral particles to remove contaminants, wherein the filtration comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter having a retention threshold of at least 8 μm, (b) filtering the first filtrate with a double-layer filter assembly comprising a second filter and a third filter to produce a filtered lentiviral particle formulation, wherein the second filter has a retention threshold of 0.45 μm and the third filter has a retention threshold of 0.2 μm; (ii) purifying the filtered lentiviral particle formulation; and (iii) concentrating the filtered lentiviral particle formulation, wherein the concentration comprises two steps of ion exchange chromatography and ultrafiltration.

[0282] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising: (i) filtering a suspended mixture comprising a host cell population and lentiviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter having a retention threshold of at least 8 μm, (b) filtering the first filtrate with a double-layer filter assembly comprising a second filter and a third filter to produce a filtered lentiviral particle formulation, wherein the second filter has a retention threshold of 0.45 μm and the third filter has a retention threshold of 0.2 μm; (ii) purifying the filtered lentiviral particle formulation; and (iii) concentrating the filtered lentiviral particle formulation, wherein the concentration comprises two steps of ion exchange chromatography and ultrafiltration; and (iii) filter sterilizing the concentrated formulation to produce a sterile drug substance.

[0283] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising (i) contacting a host cell population in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the host cell population of step (i) for a period of time sufficient to produce a suspension mixture comprising the host cell population and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, the filtration comprising: (a) contacting the mixture with an endonuclease, (b) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter having a retention threshold of at least 8 μm, (c) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a retention threshold of 0.45 μm, and (d) filtering the second filtrate with a third filter to produce a filtered lentiviral particle formulation, wherein the third filter has a first filter with a retention threshold of 0.45 μm and a second filter with a retention threshold of 0.2 μm; (iv) purifying the filtered lentiviral particle formulation; and (v) concentrating the filtered lentiviral particle formulation, wherein the concentration comprises chromatography and ultrafiltration.

[0284] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising (i) contacting a host cell population in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the host cell population of step (i) for a period of time sufficient to produce a suspension mixture comprising a host cell population and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, the filtration comprising: (a) contacting the mixture with an endonuclease, (b) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a depth filter with a retention threshold of at least 8 μm, (c) filtering the first filtrate with a second filter to obtain a second filtrate, wherein the second filter has a retention threshold of 0.45 μm, and (d) filtering the second filtrate with a third filter, wherein the third filter has a first filter with a retention threshold of 0.45 μm and a second filter with a retention threshold of 0.2 μm; (iv) purifying the filtered lentiviral particle formulation; and (iv) concentrating the filtered lentiviral particle formulation, wherein the concentration comprises two steps of ion exchange chromatography and ultrafiltration.

[0285] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising (i) contacting a host cell population in a suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the host cell population of step (i) for a period of time sufficient to produce a suspension mixture comprising the host cell population and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, the filtration comprising: (a) contacting the mixture with an endonuclease, (b) filtering the mixture with a first filter to obtain a first filtrate, wherein the first filter is a filter having a retention threshold of at least (c) filtering the first filtrate through a second filter to obtain a second filtrate, wherein the second filter has a retention threshold of 0.45 μm, and (d) filtering the second filtrate through a third filter, wherein the third filter has a first filter with a retention threshold of 0.45 μm and a second filter with a retention threshold of 0.2 μm; (iv) purifying the filtered lentiviral particle formulation; (v) concentrating the filtered lentiviral particle formulation, wherein the concentration comprises two steps of ion exchange chromatography and ultrafiltration; and (vi) filter sterilizing the concentrated formulation to produce a sterile drug substance.

[0286] In some embodiments, the present disclosure provides a method for preparing a viral particle formulation, the method comprising: (i) expanding a host cell population in one or more seed cultures in a shake flask; (ii) inoculating a bioreactor with the host cell population; (iii) transfecting the host cell population in the bioreactor; (iv) adding a nuclease (e.g., a omnipotent nuclease); (v) harvesting a cell culture medium containing the host cell population from the bioreactor; (vi) clarifying the cell culture medium one or more times; (vii) purifying and concentrating the cell culture medium using AEX chromatography including an elution step; (viii) filtering the cell culture medium (e.g., tangential flow filtration); (ix) performing a drug substance filtration; and (x) storing the drug substance, i.e., the viral particle formulation.

[0287] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, the method comprising: (i) expanding a host cell population in one or more seed cultures in a shake flask; (ii) inoculating a bioreactor with the host cell population; (iii) transfecting the host cell population in the bioreactor; (iv) adding an endonuclease (e.g., a omnipotent nuclease); (v) harvesting a cell culture medium containing the host cell population from the bioreactor; (vi) clarifying the cell culture medium one or more times; (vii) purifying and concentrating the cell culture medium using AEX chromatography including an elution step; (viii) filtering the cell culture medium (e.g., tangential flow filtration); (ix) performing drug substance filtration; and (x) storing the drug substance, i.e., the lentiviral formulation.

[0288] In some embodiments, the present disclosure provides a method for preparing a retroviral formulation, the method comprising: (i) expanding a host cell population in one or more seed cultures in a shake flask; (ii) inoculating a bioreactor with the host cell population; (iii) transfecting the host cell population in the bioreactor; (iv) adding a nuclease (e.g., a omnipotent nuclease); (v) harvesting a cell culture medium containing the host cell population from the bioreactor; (vi) clarifying the cell culture medium one or more times; (vii) purifying and concentrating the cell culture medium using AEX chromatography including an elution step; (viii) filtering the cell culture medium (e.g., tangential flow filtration); (ix) performing drug substance filtration; and (x) storing the drug substance, i.e., the retroviral formulation. II. Virus Particles

[0289] As is well known in the art, a viral particle is a tool that allows or facilitates the transfer of an entity from one environment to another. According to the present disclosure, and by way of example, some viral particles used in recombinant DNA techniques allow an entity (such as a segment of DNA) to be transferred into a host cell. Examples of vectors used in recombinant DNA techniques include, but are not limited to, plasmids, chromosomes, artificial chromosomes, or viruses. The term "expression vector" means a construct capable of in vivo or in vitro / ex vivo expression. A. Retroviral particles

[0290] In some embodiments, the disclosure provides a method for preparing a viral formulation. In some embodiments, the virus is a retrovirus. A large number of different retroviruses have been identified. Examples of retroviruses include, but are not limited to: murine leukemia virus (MLV), human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29) and avian erythroblastosis virus (AEV). A detailed list of retroviruses can be found in Coffin et al., 1997, "Retroviruses", Cold Spring Harbor Laboratory Press editor: JM Coffin, SM Hughes, HE Varmus 758-763 pages.

[0291] Retroviruses include slow viruses, gamma retroviruses and alpha retroviruses, each of which can be used to deliver polynucleotides to cells using methods known in the art. Slow viruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to common retroviral genes gag, pol and env. The higher complexity enables the virus to regulate its life cycle, such as during latent infection. Some examples of slow viruses include human immunodeficiency virus (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV). Retroviral vectors have been produced by multiple weakening of HIV virulence genes, such as deletion of genes env, vif, vpr, vpu and nef, so that the vector is biologically safe.

[0292] The lentiviral vector of the present disclosure may be derived from any suitable lentivirus or may be derived from any suitable lentivirus. The recombinant retroviral vector particles are capable of transducing recipient cells with a target nucleotide (NOI). Once inside the cell, the RNA genome from the vector particles is reverse transcribed into DNA and integrated into the DNA of the recipient cell. In some embodiments of the present disclosure, at least a portion of one or more protein coding regions necessary for replication can be removed from the virus. This makes the viral vector replication-defective. It is also possible to replace a portion of the viral genome with an NOI to generate a vector comprising an NOI that is capable of transducing a target non-dividing host cell and / or integrating its genome into the host genome.

[0293] Illustrative lentiviral vectors include those described in Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873-9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Pat. No. 6,013,516; and U.S. Pat. No. 5,994,136, each of which is incorporated herein by reference in its entirety. Typically, these vectors are configured to carry sequences necessary for selecting cells containing the vector, incorporating foreign nucleic acids into lentiviral particles, and transferring nucleic acids into target cells.

[0294] Commonly used lentiviral vector systems are so-called third generation systems. The third generation lentiviral vector system includes four plasmids. "Transfer plasmid" encodes a polynucleotide sequence delivered to the target cell by the lentiviral vector system. The transfer plasmid usually has one or more target transgenic sequences, which are flanked by long terminal repeat (LTR) sequences, which promote the integration of the transfer plasmid sequence into the host genome. For safety reasons, the transfer plasmid is usually designed to make the resulting vector unable to replicate. For example, the transfer plasmid lacks the genetic elements necessary for producing infectious particles in the host cell. In addition, the transfer plasmid can be designed to lack 3'LTR, making the virus "self-inactivating" (SIN). See Dull et al. (1998) J.Virol.72:8463-71; Miyoshi et al. (1998) J.Virol.72:8150-57. The virus particle can also include a 3' untranslated region (UTR) and a 5'UTR. The UTR contains retroviral regulatory elements that support packaging, reverse transcription, and integration of the proviral genome into cells following contact of the cells with retroviral particles.

[0295] The third generation system also typically contains two "packaging plasmids" and one "envelope plasmid". The "envelope plasmid" typically encodes an Env gene operably linked to a promoter. In an illustrative third generation system, the Env gene is VSV-G, and the promoter is a CMV promoter. The third generation system uses two packaging plasmids, one encoding gag and pol, and the other encoding rev as another safety feature; this is an improvement over the single packaging plasmid of the so-called second generation system. Although safer, the third generation system may be more cumbersome to use and result in lower viral titers due to the addition of additional plasmids. Illustrative packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0296] Many retroviral vector systems rely on the use of a "packaging cell line". Typically, a packaging cell line is a cell line whose cells are capable of producing infectious retroviral particles when a transfer plasmid, one or more packaging plasmids, and an envelope plasmid are introduced into the cells. Various methods for introducing plasmids into cells can be used, including transfection or electroporation. In some cases, a packaging cell line is suitable for efficiently packaging a retroviral vector system into retroviral particles.

[0297] As used herein, the term "retroviral vector" or "lentiviral vector" is intended to mean a nucleic acid encoding a retroviral or lentiviral cis nucleic acid sequence required for genome packaging and one or more polynucleotide sequences to be delivered to a target cell. Retroviral particles and lentiviral particles typically contain an RNA genome (derived from a transfer plasmid), a lipid bilayer envelope embedded with Env protein, and other auxiliary proteins including integrase, protease, and matrix protein. As used herein, the terms "retroviral particles" and "lentiviral particles" refer to viral particles that include an envelope, have one or more characteristics of a lentivirus, and are capable of invading a target host cell. Such features include, for example, infection of non-dividing host cells, transduction of non-dividing host cells, infection or transduction of host immune cells, containing retroviral or lentiviral particles (which include one or more of the gag structural polypeptides), containing retroviral or lentiviral envelopes (which include one or more of the glycoproteins encoded by env), containing genomes (which include one or more retroviral or lentiviral cis-acting sequences that function in replication, proviral integration or transcription), containing genomes encoding retroviral or lentiviral proteases, reverse transcriptases or integrases, or containing genomes encoding regulatory activities (such as Tat or Rev). The transfer plasmid may contain a cPPT sequence, as described in U.S. Pat. No. 8,093,042.

[0298] The efficiency of the system is an important issue in vector engineering. The efficiency of retroviral or lentiviral vector systems can be evaluated in a variety of ways known in the art, including measuring vector copy number (VCN) or vector genome (vg) (such as by quantitative polymerase chain reaction (qPCR)), or viral titer in infection units / ml (IU / mL). For example, titer can be evaluated using a functional assay performed on a cultured tumor cell line HT1080, as described in the following literature: Humbert et al. Development of third-generation Cocal Envelope Producer Cell Lines for Robust Retroviral Gene Transfer into Hematopoietic Stem Cells and T-cells. Molecular Therapy 24: 1237–1246 (2016). When titer is assessed for a cell line cultured for continuous division, stimulation is not required, so the titer measured is not affected by the surface engineering of retroviral particles. Other methods for assessing the efficiency of retroviral vector systems are provided in Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006).

[0299] In some embodiments, the retroviral particles and / or lentiviral particles of the present disclosure comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a gated adapter. In some embodiments, the sequence encoding a receptor that specifically binds to a gated adapter is operably linked to a promoter. Illustrative promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, a SV40 promoter, a SV40 / CD43 promoter, and a MND promoter.

[0300] In some embodiments, the retroviral particle comprises a transduction enhancer. In some embodiments, the retroviral particle comprises a marker protein.

[0301] In some embodiments, each retroviral particle comprises a polynucleotide comprising, in 5' to 3' order: (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds to a ligand, and (iv) a 3' LTR or UTR.

[0302] In some embodiments, the retroviral particles comprise cell surface receptors that bind to surface markers on the target host cells, thereby allowing host cell transduction. The viral vector may comprise a heterologous viral envelope glycoprotein, thereby obtaining a pseudotyped viral vector. For example, the viral envelope glycoprotein may be derived from RD114 or one of its variants, VSV-G, gibbon ape leukemia virus (GALV), or an amphoteric envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the cell surface receptor is a VSV G protein from the Cocal strain or a functional variant thereof.

[0303] In some embodiments, the viral envelope comprises a viral envelope protein. In some embodiments, the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a Kokar virus G protein. In some embodiments, the viral particles comprise a modified VSV G protein lacking LDLR binding affinity. In some embodiments, these mutations include mutations at positions 47 (e.g., K47Q) and / or 354 (e.g., R354A).

[0304] In some embodiments, the viral envelope protein is a VSV G protein (Cocal glycoprotein) from a Cocal strain. In some embodiments, the VSV G protein is a Cocal envelope protein containing a mutation (R354) at position 354. In some embodiments, the VSV G protein is a Cocal envelope protein containing a mutation (K47) at position 47. In some embodiments, the VSV G protein is a Cocal envelope variant containing an R354Q mutation. In some embodiments, the VSV G protein is a Cocal envelope variant containing a K47Q mutation. In some embodiments, this variant may be referred to as a "blinded" Cocal envelope. Illustrative Cocal envelope variants are provided in, for example, US2020 / 0216502 A1, which is incorporated herein by reference in its entirety.

[0305] Various fusion glycoproteins can be used to pseudotype lentiviral vectors. Although the most commonly used example is the envelope glycoprotein (VSVG) from vesicular stomatitis virus, many other viral proteins have also been used for pseudotyping of lentiviral vectors. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present disclosure contemplates the replacement of various fusion glycoproteins. Notably, some fusion glycoproteins result in higher vector efficiency.

[0306] In some embodiments, pseudotyping of the fusion glycoprotein or its functional variant facilitates targeted transduction of specific cell types including, but not limited to, innate lymphocytes or NK cells. In some embodiments, the fusion glycoprotein or its functional variant is one or more full-length polypeptides, one or more functional fragments, one or more homologs, or one or more functional variants of the following: human immunodeficiency virus (HIV) gp160, murine leukemia virus (MLV) gp70, gibbon ape leukemia virus (GALV) gp70, feline leukemia virus (RD114) gp70, amphotropic retrovirus (Ampho) gp70, 10A1 MLV (10A1) gp70, ecotropic retrovirus (Eco) gp70, baboon simian leukemia virus (BaEV) gp70, measles virus (MV) H and F, Nipah virus (NiV) H and F, rabies virus (RabV) G, Mokola virus (MOKV) G, Ebola virus (EboZ) G, lymphocytic choriomeningitis virus (LCMV) GP1 and GP2, baculovirus GP64, chikungunya virus (CHIKV) E1 and E2, Ross River virus (RRV) E1 and E2, Semliki Forest virus (SFV) E1 and E2, Sindbis virus (SV) E1 and E2, Venezuelan equine encephalitis virus (VEEV) E1 and E2, Western equine encephalitis virus (WEEV) E1 and E2, influenza A, B, C, or D HA, fowl plague virus (FPV) HA, vesicular stomatitis virus VSV-G, or Chandipura virus and Persian virus CNV-G and PRV-G.

[0307] In some embodiments, the fusion glycoprotein or its functional variant is a full-length polypeptide, functional fragment, homologue or functional variant of the G protein of the following viruses: Vesicular Stomatitis Alagoas Virus (VSAV), Vesicular Stomatitis Caracas Virus (CJSV), Chandipura Vesicular Stomatitis Virus (CHPV), Cocal Vesicular Stomatitis Virus (COCV), Vesicular Stomatitis Indiana Virus (VSIV), Isfahan Vesicular Stomatitis Virus (ISFV), Maraba Vesicular Stomatitis Virus (MARAV), Vesicular Stomatitis New Jersey Virus (VSNJV), Bas-Congo Virus (BASV). In some embodiments, the fusion glycoprotein or its functional variant is Cocal virus G protein.

[0308] In some embodiments, the fusion glycoprotein or its functional variant is a full-length polypeptide, functional fragment, homologue or functional variant of the G protein of the following viruses: Vesicular Stomatitis Alagoas virus (VSAV), Carajas Vesicular Stomatitis Virus (CJSV), Chandipura Vesicular Stomatitis Virus (CHPV), Cocal Vesicular Stomatitis Virus (COCV), Vesicular Stomatitis Indiana Virus (VSIV), Isfahan Vesicular Stomatitis Virus (ISFV), Maraba Vesicular Stomatitis Virus (MARAV), Vesicular Stomatitis New Jersey Virus (VSNJV), Lower Congo Fever Virus (BASV). In some embodiments, the fusion glycoprotein or its functional variant is Cocal virus G protein.

[0309] The present disclosure further provides various retroviral vectors, including but not limited to γ-retroviral vectors, α-retroviral vectors and lentiviral vectors. In some embodiments, the vector can be a viral vector, a retroviral vector, a lentiviral vector, a γ-retroviral vector. In some embodiments, the viral vector comprises VSV G protein or a functional variant thereof. In some embodiments, the viral vector comprises Cokar G protein or a functional variant thereof. 1. Engineered Viral Envelope

[0310] In some embodiments, the viral envelope protein is engineered to express surface engineered proteins. In some embodiments, the surface engineered proteins are exposed on the surface of the lentiviral particles. In some embodiments, the surface engineered proteins are embedded in a lipid bilayer. In some embodiments, the surface engineered proteins are composed of single binding domain proteins that bind to target molecules on target cells. In some embodiments, the surface engineered proteins are composed of multi-binding domain proteins, wherein each binding domain binds to target molecules on target cells. In some embodiments, each binding domain of the multi-binding protein binds to different target molecules. In some embodiments, the binding domains of the multi-binding protein are connected by a joint. In some embodiments, the target cell is an immune cell, such as a T cell. In some embodiments, the surface engineered protein is a transduction enhancer. In some embodiments, the surface engineered protein is a part of the viral envelope as a fusion protein with the viral envelope protein.

[0311] In some embodiments, the viral envelope comprises a transduction enhancer. In some embodiments, the viral envelope comprises an immune cell activation protein. In some embodiments, the viral envelope comprises a co-stimulatory molecule. In some embodiments, the viral envelope comprises an immune cell activation protein and a co-stimulatory molecule. In some embodiments, the viral envelope comprises an adhesion molecule.

[0312] In some embodiments, the transduction enhancer is a single domain binding protein or a multi-domain binding protein. In some embodiments, the transduction enhancer comprises at least one binding domain that binds to a target molecule selected from an immune cell activation receptor, a T cell co-stimulatory receptor, or an adhesion molecule. In some embodiments, the immune cell activation receptor is a T cell activation receptor or a NK cell activation receptor.

[0313] In some embodiments, the transduction enhancer comprises a single binding domain, and the single binding domain binds to a target molecule selected from an immune cell activation receptor (e.g., a T cell activation receptor), a T cell co-stimulatory receptor, or an adhesion molecule. In some embodiments, the single domain fusion protein is encoded by a nucleic acid sequence of 600-900 nucleotides. In some embodiments, the single domain fusion protein is encoded by a nucleic acid sequence of about 720 nucleotides. In some embodiments, the length of a single fusion protein is 200-300 amino acids. In some embodiments, the length of a single fusion protein is about 240 amino acids.

[0314] In some embodiments, the transduction enhancer comprises multiple binding domains, and the multiple binding domains bind to two or more target molecules selected from immune cell activation receptors (e.g., T cell activation receptors), T cell co-stimulatory receptors or adhesion molecules. In some embodiments, the transduction enhancer comprises multiple binding domains, and the multiple binding domains each bind to different target molecules, and the target molecules are immune cell activation receptors (e.g., T cell activation receptors), T cell co-stimulatory receptors and adhesion molecules. In some embodiments, the viral envelope comprises immune cell activation proteins, co-stimulatory molecules and adhesion molecules. In some embodiments, the multi-domain fusion protein is encoded by a nucleic acid sequence of 1,000 to 3,000 nucleotides. In some embodiments, the multi-domain fusion protein is encoded by a protein of at least 2,000 nucleotides. In some embodiments, the length of the multi-domain fusion protein is at least 700 amino acids.

[0315] In some embodiments, the viral envelope comprises one or more transduction enhancers. In some embodiments, the transduction enhancer comprises a T cell activating receptor, a NK cell activating receptor and / or a co-stimulatory molecule. In some embodiments, one or more transduction enhancers comprise one or more of anti-CD3 scFv, CD86, CD80 and / or CD58. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv and CD58. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv and CD80. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv and CD86. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv, CD80 and CD58. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv, CD86 and CD58.

[0316] In some embodiments, the viral particles are surface engineered with proteins that bind to the target molecule on the target cell. In some embodiments, the surface engineered protein is a fusion of one or more binding domains of the target molecule on the target cell and a viral envelope protein. In some embodiments, the viral envelope protein is a heterologous viral envelope protein. In some embodiments, the viral particles include cell surface receptors that bind to the ligand on the target host cell, thereby allowing host cell transduction. In some embodiments, the viral particles include heterologous viral envelope glycoproteins, thereby producing pseudotyped viral particles. For example, the viral envelope glycoprotein can be derived from RD114 or one of its variants, VSV-G, gibbon ape leukemia virus (GALV), or a dual tropic envelope, measles envelope or baboon retrovirus envelope glycoprotein. In some embodiments, the viral envelope glycoprotein is a VSV G protein (Cokar glycoprotein) or a functional variant thereof from the Cokar strain.

[0317] In some embodiments, the viral envelope comprises more than one polypeptide on the surface. In some embodiments, the more than one polypeptide binds to the target immune cell and replicates the immune synapse. In some embodiments, the viral envelope comprises immune cell activation proteins, costimulatory molecules and adhesion molecules, wherein the immune cell activation proteins, costimulatory molecules and adhesion molecules each bind to the target immune cell. a. Immune cell activators

[0318] In some embodiments, the transduction enhancer comprises a mitogenic stimulator that is incorporated into a retroviral or lentiviral capsid so that the virus both activates and transduces T cells. This eliminates the need to add vectors and mitogens. In some embodiments, the transduction enhancer comprises a mitogenic transmembrane protein and / or one or more co-stimulatory molecules that are incorporated into the retrovirus when the retrovirus buds from the production / packaging cell membrane. In some embodiments, the transduction enhancer is expressed as a separate cell surface molecule on the production cell rather than as part of a viral envelope glycoprotein.

[0319] In some embodiments, the viral vectors described herein comprise a mitogenic transduction enhancer in the viral envelope. In some embodiments, the mitogenic transduction enhancer is derived from a host cell during the production of a retroviral vector. In some embodiments, the mitogenic transduction enhancer is produced by a packaging cell and expressed on the cell surface. When the nascent retroviral vector buds from the host cell membrane, the mitogenic transduction enhancer can be incorporated into the viral envelope as part of a lipid bilayer derived from a packaging cell. In some embodiments, the mitogenic enhancer is an antibody or a fragment thereof. In some embodiments, the mitogenic enhancer is a single domain antibody, such as a camelid antibody. In some embodiments, the mitogenic enhancer is a scFv. In some embodiments, the mitogenic enhancer is a nanobody.

[0320] In some embodiments, the transduction enhancer is host cell derived. The term "host cell derived" indicates that the mitogenic transduction enhancer originates from a host cell as described above and is not produced as a fusion or chimera from one of the viral genes, such as gag encoding a major structural protein; or env encoding an envelope protein.

[0321] The envelope protein is formed by two subunits, a transmembrane subunit (TM) that anchors the protein to the lipid membrane and a surface subunit (SU) that binds to cell receptors. In some embodiments, the packaging cell-derived mitogenic transduction enhancer of the present invention does not contain a surface envelope subunit (SU).

[0322] In some embodiments, the mitogenic transduction enhancer has the following structure: MS-TM, wherein M is a mitogenic domain; S is an optional spacer domain, and TM is a transmembrane domain.

[0323] The mitogenic domain is the portion of the mitogenic transduction enhancer that causes T cell activation. It can bind directly or indirectly to or otherwise interact with T cells, leading to T cell activation. In some embodiments, the mitogenic domain binds to T cell surface antigens such as CD3, CD28, CD134, and CD137.

[0324] CD3 is a T cell co-receptor. It is a protein complex composed of four different chains. In mammals, the complex contains a CD3y chain, a CD35 chain and two CD3e chains. These chains associate with the T cell receptor (TCR) and the z chain to generate activation signals in T lymphocytes. TCR, z chain and CD3 molecules together constitute the TCR complex. In some embodiments, the mitogenic domain is bound to the CD3 e chain.

[0325] In some embodiments, the mitogenic domain comprises all or part of an antibody or other molecule that specifically binds to a T cell surface antigen. In some embodiments, the antibody activates TCR or CD28. In some embodiments, the antibody binds TCR, CD3, or CD28. Examples of such antibodies include: OKT3, 15E8, and TGN1412. Other suitable antibodies include:

[0326] Anti-CD28: CD28.2, 10F3

[0327] Anti-CD3 / TCR: UCHT1, YTH12.5, TR66

[0328] In some embodiments, the mitogenic domain comprises a binding domain from OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5, or TR66.

[0329] In some embodiments, the mitogenic domain comprises all or part of a co-stimulatory molecule such as OX40L and 41BBL. For example, the mitogenic domain can comprise a binding domain from OX40L or 41BBL.

[0330] OKT3, also known as muromonab-CD3, is a monoclonal antibody that targets the CD3e chain. It is used clinically to reduce acute rejection in organ transplant patients. It is the first monoclonal antibody approved for human clinical use.

[0331] In some embodiments, the viral envelope comprises an immune cell activation protein. In some embodiments, the immune cell activation protein specifically binds to a receptor on an immune cell. In some embodiments, the immune cell activation protein provides a first signal for T cell activation.

[0332] In some embodiments, the immune cell activation protein specifically binds to CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80. In some embodiments, the immune cell activation protein specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immune cell activation protein specifically binds to CD3γ, CD3δ, CD3ε, CD9, CD5, CD22, CD33, CD37, CD64, CD45, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, CD16, CD56, NKG2D, NKp46, NKp44, NKp30, CD244, NKp80, TCRα chain, TCRβ chain, TCRγ chain or TCRδ chain. In some embodiments, the immune cell activation protein specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immune cell activation protein specifically binds to CD3.

[0333] In some embodiments, the immune cell activation protein is an antibody or antigen binding fragment thereof that specifically binds to a receptor on an immune cell. In some embodiments, the immune cell activation protein is an antibody or antigen binding fragment thereof that specifically binds to the following: CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80. In some embodiments, the immune cell activation protein is an antibody or antigen binding fragment thereof that specifically binds to the following: CD28, CD2, CD3γ, CD3δ, CD3ε, CD4, CD8, CD9, CD5, CD22, CD33, CD37, CD64, CD45, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, CD16, CD56, NKG2D, NKp46, NKp44, NKp30, CD244, NKp80, TCRα chain, TCRβ chain, TCRγ chain or TCRδ chain. In some embodiments, the immune cell activation protein is an antibody or antigen binding fragment thereof that specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immune cell activation protein is an antibody or antigen binding fragment thereof that specifically binds to CD3.

[0334] Antibodies targeting the polypeptides described herein are known to those of skill in the art. Methods for generating antibodies are known to those of skill in the art.

[0335] In some embodiments, the viral envelope comprises an anti-CD3ε antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CD3ε antibody or an antigen-binding fragment thereof is coupled to a transmembrane domain. An illustrative anti-CD3ε antibody is OKT3. OKT3, also known as muromonab-CD3, is a monoclonal antibody targeting the CD3ε chain.

[0336] In some embodiments, the viral envelope comprises a single-chain Fv fragment (scFv) of an anti-CD3 antibody.

[0337] In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of SEQ ID NO: 120. In some embodiments, the anti-CD3 scFv consists of the amino acid sequence of SEQ ID NO: 120.

[0338] In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-CD3 scFv consists of the amino acid sequence of SEQ ID NO: 122.

[0339] In some embodiments, the anti-CD3 scFV is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 121.

[0340] In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the anti-CD3 scFv is encoded by a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 123. b.Co-stimulatory molecules

[0341] In some embodiments, the viral envelope comprises at least one co-stimulatory molecule. In some embodiments, the co-stimulatory molecule specifically binds to a receptor on an immune cell. In some embodiments, the co-stimulatory molecule provides a signal for cell activation.

[0342] As used herein, the term "costimulatory molecule" refers to a molecule capable of producing costimulatory signals to T cells. Lymphocytes (such as T cells and natural killer (NK) cells) generally require several signals and interactions with antigen presenting cells (APC) for optimal initiation to obtain complete effector functions. For T cells, these include signal transduction via T cell receptor (TCR), costimulatory molecules (such as CD28 and CD2), cytokines and various adhesion molecules, and the signal transduction is necessary to allow sufficient time for appropriate synapse formation and signal transduction. NK cells require similar types of stimulation, but may depend on different activation receptors, such as NKG2D, NKp46 and DNAM-1. For T cells, in addition to TCR stimulation, appropriate costimulation is particularly important for effective initiation, and many studies have shown that single TCR stimulation may cause functional anergy and unresponsiveness. Costimulatory signals increase the function of T cells and NK cells by enhancing cell metabolism, cytokine production, differentiation and long-term persistence. Costimulation is an important factor in cell proliferation, differentiation and survival. In some embodiments, costimulatory molecules include but are not limited to CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In some embodiments, costimulatory molecules include but are not limited to binding agents that bind to any costimulatory molecule described herein, such as scFv, antibodies, single domain antibodies, antibody fragments, nanobodies. In some embodiments, these binding agents can include anti-CD28, anti-CD2, anti-CD45, anti-CD4, anti-CD5, anti-CD8, anti-CD9, anti-CD16, anti-CD22, anti-CD33, anti-CD37, anti-CD64, anti-CD80, anti-CD86, anti-CD137, anti-CD154, anti-CD28H, anti-LFA-1, anti-OX40, anti-4-1BB, anti-CD40L, anti-DNAM-1, anti-CD27, anti-ICOS, anti-LIGHT, anti-GITR, anti-CD30, anti-SLAM, anti-Ly-9, anti-CD84, anti-Ly108, anti-NKG2D, anti-NKp46, anti-NKp44, anti-NKp30, anti-CD244, anti-NKp80, anti-TCR alpha chain, anti-TCR beta chain, anti-TCR gamma chain, and anti-TCR delta chain agents.

[0343] In some embodiments, the costimulatory molecule is a ligand of CD28. CD28 is one of the proteins expressed on T cells, which provides the costimulatory signals required for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation via CD28 can provide effective signals for producing various interleukins (particularly IL-6). In some embodiments, the costimulatory molecule is an antibody or fragment thereof that binds CD28. Examples of such antibodies include: 15E8 and TGN1412. Other suitable antibodies include: CD28.2 and 10F3.

[0344] In some embodiments, the co-stimulatory molecule is CD86. CD86 (also known as B7-2) is a ligand of CD28. In some embodiments, the ligand of CD28 is CD86. In some embodiments, the co-stimulatory molecule is CD80. CD80 is another ligand of CD28. In some embodiments, the ligand of CD28 is CD80. In some embodiments, the ligand of CD28 is anti-CD28 antibody or anti-CD28 scFv. In some embodiments, the anti-CD28 antibody or anti-CD28 scFv is coupled to a transmembrane domain for display on the surface of the viral envelope.

[0345] In some embodiments, the costimulatory molecule is a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the costimulatory molecule is a CD86 polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5.

[0346] In some embodiments, the CD86 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the CD86 polypeptide is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6.

[0347] In some embodiments, the costimulatory molecule is a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the costimulatory molecule is a CD80 polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3.

[0348] In some embodiments, the CD80 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the CD80 polypeptide is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4.

[0349] In some embodiments, the costimulatory molecule is a CD80 extracellular domain polypeptide comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the costimulatory molecule is a CD80 extracellular domain polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7. In some embodiments, the costimulatory molecule is a CD86 extracellular domain polypeptide comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the costimulatory molecule is a CD86 extracellular domain polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8.

[0350] CD134 (also known as OX40) is a member of the TNFR receptor superfamily expressed on activated T cells. OX40 can promote cell division and survival. OX40 is a secondary co-stimulatory molecule expressed 24 to 72 hours after activation; its ligand OX40L is also not expressed on resting antigen-presenting cells, but is expressed after activation of the antigen-presenting cells. In some embodiments, the viral particle comprises a ligand of OX40 or a functional fragment thereof coupled to the native transmembrane domain or heterologous transmembrane domain of OX40.

[0351] CD137 (also known as 4-1BB) is a member of the tumor necrosis factor (TNF) receptor family. CD137 is expressed on activated T cells. In addition, CD137 expression is seen on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and vascular wall cells at inflammatory sites. The best characterized activity of CD137 is its co-stimulatory activity on activated T cells. The cross-linking of CD137 enhances T cell proliferation, IL-2 secretion survival and cytolytic activity. In some embodiments, the viral particle comprises a ligand or a functional fragment of 4-1BB coupled to a natural transmembrane domain or a heterologous transmembrane domain of 4-1BB. 4-1BBL is a cytokine belonging to the tumor necrosis factor (TNF) ligand family. This transmembrane cytokine is a bidirectional signal transduction protein acting as a 4-1BB ligand, and 4-1BB is a co-stimulatory receptor molecule in T lymphocytes. It has been shown that 4-1BBL, in addition to promoting T lymphocyte proliferation, also reactivates anergic T lymphocytes.

[0352] Viral particles containing one or more activation or co-stimulatory molecules can be prepared by engineering a packaging cell line by the methods provided by WO 2016 / 139463; or expressing one or more T cell activation or co-stimulatory molecules from a polycistronic helper vector, as described in International Patent Publication No. WO 2020 / 106992 A1, both of which are incorporated herein by reference in their entirety. c. Adhesion molecules

[0353] In some embodiments, the viral particles include adhesion molecules. As used herein, the term "adhesion molecules" refers to a subset of cell surface molecules that participate in the binding of cells to other cells. Adhesion molecules can help form more stable interactions between immune cells, such as immune synapses. Immune synapses are stable adhesion connections between polarized immune effector cells and cells carrying antigens. In some embodiments, adhesion molecules can provide costimulatory signals to target cells. In some embodiments, adhesion molecules include but are not limited to CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6 and B7-H6.

[0354] In some embodiments, the adhesion molecule is a fragment or extracellular domain of CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6 and B7-H6. In some embodiments, the adhesion molecule includes but is not limited to SLAMF2, B7-H2, B7-H5, B7-H3, B7x and TMIGD2. In some embodiments, the adhesion molecule is a fragment or extracellular domain of SLAMF2, B7-H2, B7-H5, B7-H3, B7x and TMIGD2.

[0355] In some embodiments, adhesion molecules include but are not limited to binding agents that bind to any adhesion molecules or costimulatory molecules described herein, such as scFv, antibodies, single domain antibodies, antibody fragments and nano antibodies. In some embodiments, these binding agents may include anti-CD28, anti-CD2, anti-CD28H, anti-LFA-1, anti-OX40, anti-4-1BB, anti-CD40L, anti-DNAM-1, anti-CD27, anti-ICOS, anti-LIGHT, anti-GITR, anti-CD30, anti-SLAM, anti-Ly-9, anti-CD84, anti-Ly108, anti-NKG2D, anti-NKp46, anti-NKp44, anti-NKp30, anti-CD244, anti-NKp80, anti-TCR alpha chain, anti-TCR beta chain, anti-TCR gamma chain and anti-TCR delta chain reagents.

[0356] In some embodiments, the adhesion molecule binds CD2. CD2 is also known as T11, LFA-2, and E rosette receptors and is a surface protein expressed on T lymphocytes and NK cells. CD2 is a natural ligand for CD58. In addition to performing adhesion functions, the engagement of CD2 provides co-stimulatory signals that can enhance activation and effector functions. In some embodiments, the lentiviral particles comprise molecules that bind CD2. In some embodiments, the lentiviral particles comprise antibodies, single domain antibodies, antibody fragments, and / or nanobodies that are specific for CD2. In some embodiments, the lentiviral particles comprise CD58 or a functional portion thereof that binds CD2.

[0357] In some embodiments, the adhesion molecule is CD58. In some embodiments, the adhesion molecule is a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the adhesion molecule is a CD58 polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.

[0358] In some embodiments, the CD58 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the CD58 polypeptide is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. In some embodiments, the adhesion molecule is a CD58 extracellular domain polypeptide comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the adhesion molecule is a CD58 extracellular domain polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9. d. Additional non-viral proteins

[0359] In some embodiments, the viral particle comprises at least one non-viral protein. In some embodiments, the viral particle comprises at least one non-viral protein in addition to those proteins described above.

[0360] In some embodiments, the viral particle comprises a targeting ligand. In some embodiments, the viral particle comprises CD19 or a functional fragment thereof coupled to a native transmembrane domain or a heterologous transmembrane domain of CD19. In some embodiments, CD19 acts as a ligand for blinatumomab, thereby providing an adaptor for coupling the particle to a T cell via the anti-CD3 portion of blinatumomab. In some embodiments, another type of particle surface ligand can be used to couple appropriately surface engineered lentiviral particles to T cells using a multispecific antibody comprising a binding portion of a particle surface ligand. In some embodiments, the multispecific antibody is a bispecific antibody, such as a bispecific T cell adaptor (BiTE).

[0361] In some embodiments, the non-viral protein is a cytokine. In some embodiments, the cytokine can be selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 and any combination thereof. In some embodiments, the cytokine is IL-12α. In some embodiments, the cytokine is IL-12β. When the non-viral protein used is a soluble protein (such as scFv or cytokine), it can be bound to the surface of the viral particle by fusion with a transmembrane domain (such as the transmembrane domain of CD8). Alternatively, it can be indirectly bound to the lentiviral particle by using a transmembrane protein engineered to bind to a soluble protein. Further comprising one or more cytoplasmic residues can increase the stability of the fusion protein.

[0362] Mitogenic transduction enhancers and / or cytokine-based transduction enhancers can include a "spacer sequence" to connect the antigen binding domain to the transmembrane domain. Flexible spacers allow the antigen binding domain to be oriented in different directions to facilitate binding. As used herein, the term "coupling" refers to the chemical connection of two proteins, i.e., direct C-terminal to N-terminal fusion; chemical connection with a non-peptide spacer; chemical connection with a polypeptide spacer; and two proteins are fused to the C-terminus of a polypeptide spacer (e.g., a spacer sequence) via a peptide bond to the N-terminus.

[0363] The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge, or a human CD8 stem or a mouse CD8 stem. The spacer may alternatively comprise an alternative linker sequence having similar length and / or domain spacing properties to an IgG1 Fc region, an IgG1 hinge, or a CD8 stem. The human IgG1 spacer may be altered to remove an Fc binding motif. In some embodiments, the spacer sequence may be derived from a human protein.

[0364] In some embodiments, the spacer sequence comprises a CD8-derived hinge.

[0365] In some embodiments, the spacer sequence comprises a "short" hinge. A short hinge is described as a hinge region that comprises fewer nucleotides relative to the CAR hinge regions known in the art.

[0366] The transmembrane domain is a transmembrane mitogenic transduction enhancer and / or a cytokine-based transduction enhancer sequence. The transmembrane domain may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from CD28. In some embodiments, the transmembrane domain is derived from a human protein.

[0367] The viral particles of the present invention may include a cytokine-based transduction enhancer in the viral envelope. In some embodiments, the cytokine-based transduction enhancer is derived from host cells during viral particle production. In some embodiments, the cytokine-based transduction enhancer is prepared by host cells and expressed on the cell surface. When the nascent viral particles bud from the host cell membrane, the cytokine-based transduction enhancer can be incorporated into the viral envelope as part of the lipid bilayer derived from the packaging cell.

[0368] Cytokine-based transduction enhancers can include a cytokine domain and a transmembrane domain. It can have a structure CS-TM, where C is a cytokine domain, S is an optional spacer domain (e.g., a spacer sequence), and TM is a transmembrane domain. The spacer domain and the transmembrane domain are as defined above.

[0369] The cytokine domain may comprise a T cell activating cytokine (e.g., from IL2, IL7, and IL15) or a functional fragment thereof. As used herein, a "functional fragment" of a cytokine is a fragment of a polypeptide that retains the ability to bind to a specific receptor of the polypeptide and activate T cells.

[0370] IL2 is one of the factors secreted by T cells to regulate the growth and differentiation of T cells and some B cells. IL2 is a lymphokine that induces the proliferation of responsive T cells. It is secreted as a single glycosylated polypeptide, and its activity requires the cleavage of a signal sequence. Solution NMR shows that the structure of IL2 contains a bundle of 4 helices (called AD), flanked by 2 shorter helices and several poorly defined loops. The residues in helix A and the residues in the loop region between helix A and B are important for receptor binding. 2. Payload

[0371] In some embodiments, the viral particle comprises a payload. In some embodiments, the payload is conjugated to the surface of the particle. In some embodiments, the payload is encapsulated by the particle. In some embodiments, the viral particle delivers the payload to a target cell.

[0372] In some embodiments, the payload is a nucleic acid. In some embodiments, the nucleic acid is a coding nucleic acid. In some embodiments, the nucleic acid encodes a polypeptide of interest. In some embodiments, the polypeptide of interest is a therapeutic polypeptide. In some embodiments, the polypeptide of interest is a chimeric antigen receptor. In some embodiments, the nucleic acid is transduced into a target cell and the polypeptide of interest is expressed in the target cell. In some embodiments, the nucleic acid is a non-coding nucleic acid. In some embodiments, the nucleic acid is a therapeutic non-coding nucleic acid. Non-coding nucleic acids are known to those skilled in the art and include, but are not limited to, siRNA, miRNA, and shRNA.

[0373] In some embodiments, the expression of the payload is driven by a promoter. In some embodiments, the promoter is a MND promoter (myeloproliferative sarcoma virus enhancer, negative regulatory region deleted, dl587rev primer binding site replaced), which is a synthetic promoter of viral origin, containing modified U3 region and myeloproliferative sarcoma virus enhancer 13 of Moloney murine leukemia virus (MoMuLV) LTR, and has high expression in human CD34+ stem cells, lymphocytes and other tissues. In some embodiments, a single protein is expressed, which is separated by a 2A peptide sequence, which induces ribosome jumping and cutting during translation. In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter is an EF1a promoter. In other embodiments, the promoter is an HTLV promoter. a. Synthetic cytokine receptor complex

[0374] In some embodiments, the retroviral vector comprises a nucleotide sequence encoding a synthetic cytokine receptor complex. The synthetic cytokine receptor of the present disclosure comprises a synthetic γ chain and a synthetic β chain each containing a dimerization domain. The dimerization domain controllably dimerizes in the presence of a non-physiological ligand, thereby activating the signal transduction of the synthetic cytokine receptor. In a preferred embodiment, the non-physiological ligand is rapamycin or rapalog, and this synthetic cytokine receptor is called a rapamycin-activated cytokine receptor (RACR).

[0375] The synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an intracellular domain of interleukin-2 receptor subunit gamma (IL-2RG). The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N or C-terminal to the intracellular domain of IL-2G).

[0376] The synthetic β chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the following: an interleukin-2 receptor subunit β (IL-2RB) intracellular domain, an interleukin-7 receptor subunit β (IL-7RB) intracellular domain, or an interleukin-21 receptor subunit β (IL-21RB) intracellular domain. The synthetic γ chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain. The dimerization domain can be extracellular (N-terminal of the transmembrane domain) or intracellular (C-terminal of the transmembrane domain and N or C-terminal of the IL-2RB or IL-7RB intracellular domain). i) Intracellular domain

[0377] In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises an interleukin-2 receptor subunit gamma (IL2Rg) domain.

[0378] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein (comprising an IL-2RG intracellular domain, a first dimerization domain), a second transmembrane receptor protein (comprising an IL-2RB intracellular domain and a second dimerization domain).

[0379] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein (comprising an IL-2RG intracellular domain and a first dimerization domain), a second transmembrane receptor protein (comprising an IL-7RB intracellular domain and a second dimerization domain).

[0380] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein (comprising an IL-2RG intracellular domain and a first dimerization domain), a second transmembrane receptor protein (comprising an IL-21RB intracellular domain and a second dimerization domain). ii) Dimerization domain

[0381] The dimerization domain may be a heterodimerization domain, including but not limited to the 12 kD FK506 binding protein (FKBP) and the FKBP12-rapamycin binding (FRB) domain, both of which are known in the art to dimerize in the presence of rapamycin or rapalog.

[0382] Alternatively, the first dimerization domain and the second dimerization domain may be a 12 kD FK506 binding protein (FKBP) and a calcineurin domain, both of which are known in the art to dimerize in the presence of FK506 or an analog thereof.

[0383] In some embodiments, the dimerization domain is a homodimerization domain selected from: i) FK506 binding protein (FKBP) with a size of 12 kD; ii) Cyclophilin A (CypA); or iii) gyrase B (CyrB); The corresponding non-physiological ligands are i) FK1012, AP1510, AP1903 or AP20187; ii) Cyclosporine-A (CsA); or iii) coumermycin or its analogs.

[0384] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a cyclophilin domain.

[0385] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

[0386] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a calcineurin domain and a cyclophilin domain.

[0387] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are PYR1-like 1 (PYL1) and abscisic acid-insensitive protein 1 (ABI1). iii) Transmembrane domain

[0388] The transmembrane domain is a sequence that spans the membrane in a synthetic cytokine receptor. The transmembrane domain may comprise a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein. iv) Cytoplasmic FRB

[0389] The FRB domain is a domain of approximately 100 amino acids derived from the mTOR protein kinase. It can be expressed in the cytosol as a freely diffusible soluble protein. Advantageously, the FRB domain reduces the inhibitory effect of rapamycin on mTOR in transduced cells and promotes consistent activation of transduced cells, thereby giving the cells a proliferative advantage over native cells.

[0390] In some embodiments, the synthetic cytokine receptor complex comprises a cytoplasmic polypeptide bound to a ligand or a complex comprising a ligand.

[0391] In some embodiments, the cytoplasmic polypeptide comprises a FRB domain. In some embodiments, the cytoplasmic polypeptide comprises a FRB domain and the ligand is rapamycin. Advantageously, the cytoplasmic FRB confers resistance to the immunosuppressive effects of non-physiological ligands (e.g., rapamycin or rapalog). b. Chimeric Antigen Receptor

[0392] In some embodiments, the viral particles described herein are used to transduce nucleic acid sequences (polynucleotides) encoding one or more chimeric antigen receptors (CAR) into cells (e.g., T lymphocytes). In some embodiments, the transduction of viral particles causes one or more CARs to be expressed in transduced cells.

[0393] Conventionally, CAR is produced by fusing a polynucleotide encoding VL, VH or scFv to the 5' end of a polynucleotide encoding a transmembrane and intracellular domain, and, if desired, transducing cells with the polynucleotide and with the corresponding VH or VL. Many variations of CAR are well known in the art, and the present disclosure contemplates the use of any known variation. In addition, VL / VH pairs and scFvs of numerous haptens are known in the art, or can be conventionally produced by conventional methods. Therefore, the present disclosure contemplates the use of any known hapten binding domain.

[0394] In some embodiments, the binding portion of the CAR can be, for example, a single chain fragment variable region (scFv), Fab, Fv, Fc or (Fab')2 fragment of an antibody, etc. The use of unmodified (ie, full-size) antibodies (such as IgG, IgM, IgA, IgD or IgE) in or as a CAR is excluded from the scope of the present invention.

[0395] In some embodiments, the costimulatory domain is used to enhance the proliferation and survival of lymphocytes after CAR is combined with the targeted portion. The identity of the costimulatory domain is limited only in that it has the ability to enhance cell proliferation and survival activation after CAR is combined with the targeted portion. Suitable costimulatory domains include, but are not limited to: CD28 (see, for example, Alvarez-Vallina, L. et al., Eur J Immunol. 1996. 26 (10): 2304-9); CD137 (4-1BB), which is a member of the tumor necrosis factor (TNF) receptor family (see, for example, Imai, C. et al., Leukemia. 2004. 18: 676-84); and CD134 (OX40), which is a member of the TNFR receptor superfamily (see, for example, Latza, U. et al., Eur. J. Immunol. 1994. 24: 677). It will be appreciated by a skilled person that sequence variants of these costimulatory domains can be used, wherein the variant has the same or similar activity to the domain modeled therewith. In various embodiments, such variants have at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the domain from which they are derived.

[0396] In some embodiments of the present invention, the CAR construct comprises two costimulatory domains. Although a specific combination includes all possible variations of the four described domains, specific examples include: 1) CD28+CD137 (4-1BB) and 2) CD28+CD134 (OX40).

[0397] In some embodiments, the activation signaling domain is used to activate cells after CAR binds to the targeted portion. The identity of the activation signaling domain is limited only in that it has the ability to induce activation of selected cells after CAR binds to the targeted portion. Suitable activation signaling domains include CD3 ζ chain and Fc receptor γ. Skilled technicians will understand that sequence variants of these activation signaling domains can be used without adversely affecting the present invention, wherein the variants have the same or similar activity as the domains modeled by them. Such variants may have at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with the amino acid sequence of the domain from which they are derived.

[0398] In some embodiments, the CAR may include additional elements, such as a signal peptide to ensure proper export of the fusion protein to the cell surface, a transmembrane domain to ensure that the fusion protein is maintained as an integral membrane protein, and a hinge domain to confer flexibility to the recognition region and allow strong binding to the targeted moiety.

[0399] In some embodiments, the payload may include a CAR-like construct. For example, the payload may include nucleic acids encoding both a receptor and a reporter. In some embodiments, this construct may include a structure S-ETD-MBD-IRES-R, wherein S is a signal sequence, ETD includes an extracellular targeting domain, MBD includes a membrane-bound domain, IRES includes an internal ribosome entry site, and R encodes a reporter. In some embodiments, the reporter may be a fluorescent protein or an antibiotic resistance marker. In some embodiments, the extracellular targeting domain may include an antibody, an antibody fragment, a small molecule ligand, or a peptide. In some embodiments, the peptide may include some or all of a cytokine or an interleukin.

[0400] Illustrative CAR constructs suitable for CAR-NK cells are provided below: (1) scFv-CD8 TM -4-1BB IC -CD3ζ (see, e.g., Liu E, Tong Y, Dotti G, et al., Leukemia. 2018; 32: 520-531); (2) scFv-CD28 TM+IC -CD3ζ (see, e.g., Han J, Chu J, Keung CW et al., Sci Rep. 2015; 5: 11483; Kruschinski A, Moosmann A, Poschke I et al., Proc Natl Acad Sci US A. 2008; 105: 17481-17486; and Chu J, Deng Y, Benson DM et al., Leukemia. 2014; 28: 917-927); (3) scFv-DAP12 TM+IC (See, e.g., Muller N, Michen S, Tietze S et al., J Immunother. 2015; 38: 197-210); (4) scFv-CD8 TM -2B4 IC -CD3ζ (see, e.g., Xu Y, Liu Q, Zhong M et al., J Hematol Oncol. 2019; 12:49); (5) scFv-2B4 TM+IC -CD3ζ (see, e.g., Altvater B, Landmeier S, Pscherer S et al., Clin Cancer Res. 2009; 15: 4857-4866); (6) scFv-CD28 TM+IC -4-1BB IC -CD3ζ (see, e.g., Kloss S, Oberschmidt O, Morgan M et al., Hum Gene Ther. 2017; 28: 897-913); (7) scFv-CD16 TM -2B4 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (8)scFv-NKp44 TM -DAP10 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (9)scFv-NKp46 TM -2B4 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (10)scFv-NKG2D TM -2B4 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (11)scFv-NKG2D TM -4-1BB IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (12)scFv-NKG2D TM -2B4 IC -DAP12 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (13)scFv-NKG2D TM -2B4 IC -DAP10 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (14)scFv-NKG2D TM -4-1BB IC -2B4 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); and (15)scFv-NKG2D TM -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192).

[0401] Although the affinity of a CAR expressed by a lymphocyte to bind to a targeted moiety can vary, and in some cases low affinity binding may be preferred (e.g., about 50 nM), the binding affinity of the CAR to the targeted ligand will generally be at least about 100 nM, 1 pM, or 10 pM, preferably at least about 100 pM, 1 fM, or 10 fM, and even more preferably at least about 100 fM. i) CAR extracellular domain

[0402] In some embodiments, CAR comprises an extracellular domain that binds to an antigen of interest. In some embodiments, the extracellular domain comprises a receptor or a portion of a receptor that binds to the antigen. In some embodiments, the extracellular domain comprises or is an antibody or an antigen-binding portion thereof. In some embodiments, the extracellular domain comprises or is a single-chain Fv domain. The single-chain Fv domain may comprise, for example, a VL connected to a VH by a flexible linker, wherein the VL and VH are from an antibody that binds to the antigen.

[0403] In some embodiments, the extracellular domain of CAR may contain any polypeptide that binds to a desired antigen (e.g., a prostate neoantigen). The extracellular domain may include a portion of an scFv, an antibody, or an alternative scaffold. CAR may also be engineered to bind to two or more desired antigens, which may be arranged in series and separated by a linker sequence. For example, one or more domain antibodies, scFv, llama VHH antibodies, or other VH-only antibody fragments may be organized in series via a linker to provide bispecific or multispecific CAR.

[0404] The antigen bound by the extracellular domain of the polypeptide can be any target antigen, for example, it can be an antigen on a tumor cell. The tumor cell can be, for example, a cell in a solid tumor or a cell of a blood cancer. The antigen can be any antigen expressed on a cell of any tumor or cancer type, for example, a cell of the following tumor or cancer: lymphoma, lung cancer, breast cancer, prostate cancer, adrenocortical carcinoma, thyroid cancer, nasopharyngeal carcinoma, melanoma (such as malignant melanoma), skin cancer, colorectal cancer, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, Ewing's sarcoma, peripheral primitive neuroectodermal tumor, solid germ cell tumor, hepatoblastoma, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms' tumor, glioblastoma, myxoma, fibroma, lipoma, etc. In some embodiments, the lymphoma can be chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, intranodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, T-lymphocytic prolymphoma, In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), and the B cells of CLL have a normal karyotype. In some embodiments, wherein the cancer is chronic lymphocytic leukemia (CLL), the B cells of the CLL carry a 17p deletion, a 11q deletion, a 12q trisomy, a 13q deletion, or a p53 deletion.

[0405] In some embodiments, the antigen is expressed on the following cells: B cell malignancy cells, relapsed / refractory CD19-expressing malignancy cells, diffuse large B cell lymphoma (DLBCL) cells, Burkitt's large B cell lymphoma (B-LBL) cells, follicular lymphoma (FL) cells, chronic lymphocytic leukemia (CLL) cells, acute lymphocytic leukemia (ALL) cells, mantle cell lymphoma (MCL) cells, hematological malignancy cells, colon cancer cells, lung cancer cells, liver cancer cells, breast cancer cells, renal cancer cells, prostate cancer cells, ovarian cancer cells, skin cancer cells, melanoma cells, bone cancer cells, brain cancer cells, squamous cell cancer cells, leukemia cells, myeloma cells, B cell lymphoma cells, kidney cancer cells, uterine cancer cells, adenocarcinoma cells, pancreatic cancer cells, chronic myeloid leukemia cells, glioblastoma cells, neuroblastoma cells, medulloblastoma cells, or sarcoma cells.

[0406] In some embodiments, the antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In some embodiments, without limitation, the tumor-associated antigen or tumor-specific antigen is B cell maturation antigen (BCMA), B cell activating factor (BAFF), GPRC5D, FCRL5, ROR1, L1-CAM, CD22, folate receptor, carboxyanhydrase IX (CAIX), claudin 18.2, FAP, mesothelin, IL13Ra2, Lewis Y, CCNA1, WT-1, TACI, CD38, SLAMF7, CD138, DLL3, transmembrane 4L six family member 1 (TM4SF1), epithelial cell adhesion molecule (EpCAM), PD-1, PD-L1, CTLA-4, AXL, ROR2, phosphatidylinositol proteoglycan-3 (GPC3), CD133, CD147, EGFR, MUC1, GD2, Her2, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), EGFRvIII, cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-related Antigen (MAGE), CD19, CD20, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), giant cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melanin-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilaments, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, vascular endothelial growth factor receptor (VEGFR), dimeric form of pyruvate kinase isozyme M2 (tumor M2-PK), abnormal ras protein, or abnormal p53 protein.

[0407] In other embodiments, the CAR is a universal CAR and does not specifically target a tumor antigen itself. For example, the CAR may comprise a tag-specific scFv such that an exogenous agent comprising a tag and a tumor targeting domain can direct the universal CAR T cell to the target tumor.

[0408] In some embodiments, the CAR is a second generation CAR composed of an anti-fluorescein scFv linked to a 4-1BB co-stimulatory domain and a CD3ζ intracellular signaling domain.

[0409] In some embodiments, the antigen is CD 19. CARs targeting CD19 are described in, for example, U.S. Publication No. 20160152723, U.S. Patent No. 10,736,918, U.S. Patent No. 10,357,514, and U.S. Patent No. 7,446,190, each of which is incorporated by reference.

[0410] In some embodiments, CAR comprises an extracellular domain containing an FMC63scFv binding domain for CD19 binding. In some embodiments, CAR is a second generation CAR consisting of an FMC63 mouse anti-human CD19 scFv connected to a 4-1BB co-stimulatory domain and a CD3ζ intracellular signaling domain. In some embodiments, CAR comprises a binding domain of CD19, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, CAR comprises a binding domain of CD19, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, CAR comprises a binding domain of CD19, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, CAR comprises an extracellular domain containing an FMC63 scFv binding domain for CD19 binding, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 ζ signaling domain. In some embodiments, CAR comprises an extracellular domain containing an FMC63 scFv binding domain for CD19 binding, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 ζ signaling domain. In some embodiments, CAR comprises an extracellular domain containing an FMC63 scFv binding domain for CD19 binding, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 ζ signaling domain.

[0411] In some embodiments, CAR is a second generation CAR, which is composed of FMC63 mouse anti-human CD19 scFv connected to a CD28 costimulatory domain and a CD3 ζ intracellular signaling domain. In some embodiments, CAR is a second generation CAR, which is composed of FMC63 mouse anti-human CD19 scFv connected to a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 ζ intracellular signaling domain.

[0412] In some embodiments, the antigen is BCMA. CAR T therapy targeting BCMA has been approved by the FDA and includes Abecma and Carvykti. CAR targeting BCMA is described in, for example, U.S. Publication No. 2020 / 0246381, U.S. Patent No. 10,918,665, and U.S. Publication No. 2019 / 0161553, each of which is incorporated herein by reference. In some embodiments, CAR comprises a binding domain for BCMA, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, CAR comprises a binding domain for BCMA, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, CAR comprises a binding domain for BCMA, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ signaling domain.

[0413] In some embodiments, the antigen is G protein coupled receptor C class 5 family member D (GPRC5D). CAR targeting GRC5D is described in, for example, U.S. Publication Nos. 2018 / 0118803 and 2021 / 10393689, each of which is incorporated herein by reference. In some embodiments, CAR comprises a binding domain of GRC5D, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. In some embodiments, CAR comprises a binding domain of GRC5D, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. In some embodiments, CAR comprises a binding domain of GRC5D, a CD28 hinge, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3ζ signaling domain.

[0414] In some embodiments, the antigen is Fc receptor-like 5 (FcRL5). CARs targeting FcRL5 are described in, for example, U.S. Publication No. US2017 / 0275362, which is incorporated herein by reference. In some embodiments, CAR comprises a binding domain of FcRL5, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, CAR comprises a binding domain of FcRL5, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, CAR comprises a binding domain of FcRL5, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ signaling domain.

[0415] In some embodiments, the antigen is a receptor tyrosine kinase-like orphan receptor 1 (ROR1). CARs targeting ROR1 are described in, for example, U.S. Publication No. 2022 / 0096651, which is incorporated herein by reference. In some embodiments, CAR comprises a binding domain of ROR1, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, CAR comprises a binding domain of ROR1, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, CAR comprises a binding domain of ROR1, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ signaling domain.

[0416] In some embodiments, CAR is a second generation CAR, which is composed of an anti-BCMA scFv connected to a 4-1BB costimulatory domain and a CD3 ζ intracellular signaling domain. In some embodiments, CAR is a second generation CAR, which is composed of an anti-GPRC5D scFv connected to a 4-1BB costimulatory domain and a CD3 ζ intracellular signaling domain. In some embodiments, CAR is a second generation CAR, which is composed of an anti-ROR1 scFv connected to a 4-1BB costimulatory domain and a CD3 ζ intracellular signaling domain.

[0417] In some embodiments, the TAA or TSA is a cancer / testis (CT) antigen, such as BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE.

[0418] In some embodiments, the TAA or TSA is a carbohydrate or ganglioside, such as fuc-GM1, GM2 (carcinoembryonic antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, etc.

[0419] In some embodiments, the TAA or TSA is alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein-Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-All, hsp70-2, KIAAO205, Mart2, Mum-1, 2 and 3, neo-PAP, class I myosin, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Gage3, Gage 4, Gage 5, Gage6, Gage 7. GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, TRP2-Int2, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA19-9, CA72-4, CAM 17.1. NuMa, K-ras, 13-catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, CD27, CD30, CD70, CD123, CD133, B cell maturation antigen, CS1, GPCR5, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternative reading frame protein), Trp-p8, STEAP1 (prostatic six-transmembrane epidermal antigen 1), abnormal ras protein or abnormal p53 protein. In some embodiments, the tumor-associated antigen or tumor-specific antigen is integrin αVβ3 (CD61), prolactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene) or Ral-B. Other tumor-associated antigens and tumor-specific antigens are known to those skilled in the art. .

[0420] Antibodies and scFvs that bind to TSA and TAA include those known in the art, as do nucleotide sequences encoding them.

[0421] In some embodiments, the antigen is an antigen that is not considered to be a TSA or TAA, but is still associated with tumor cells or damage caused by a tumor. In some embodiments, for example, the antigen is, for example, a growth factor, a cytokine, or an interleukin, for example, a growth factor, a cytokine, or an interleukin associated with angiogenesis or vasculogenesis. Such growth factors, cytokines, or interleukins may include, for example, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8). Tumors may also produce a hypoxic environment locally in the tumor. Therefore, in some embodiments, the antigen is a hypoxia-related factor, such as HIF-1α, HIF-1β, HIF-2a, HIF-2β, HIF-3α, or HIF-3β. Tumors may also cause local damage to normal tissues, thereby causing the release of molecules known as damage-associated molecular pattern molecules (DAMPs; also known as alarmins). Thus, in some embodiments, the antigen is a DAMP, such as a heat shock protein, chromatin-associated protein high mobility group box 1 (HMGB1), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), serum amyloid A (SAA), or may be deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.

[0422] In some embodiments of the polypeptides described herein, the extracellular domain is directly joined to the transmembrane domain, or is connected via a linker, spacer, or hinge polypeptide sequence (e.g., a sequence from CD28 or a sequence from CTLA4).

[0423] In some embodiments, the extracellular domain that binds a desired antigen can be derived from an antibody or antigen-binding fragment thereof generated using the techniques described herein.

[0424] Exemplary anti-CD19 CARs are shown in Table 1, wherein the different portions thereof include the extracellular domain.

[0425] In some embodiments, CAR is anti-CD20 CAR, and the extracellular binding domain of CD20 CAR is specific to CD20 (for example, human CD20). In some embodiments, the extracellular binding domain of CD20 CAR is derived from an antibody with specificity to CD20, and the antibody includes, for example, Leu16, IF5, 1.5.3, rituximab, obitutuzumab, ibritumomab tiuxetan, ofatumumab, tositumomab, onituzumab, veltuzumab, utoximab and auretuzumab. In any of these embodiments, the extracellular binding domain of CD20 CAR may include VH, VL and / or one or more CDR of any of the antibodies or be composed thereof. Exemplary anti-CD20 CAR is shown in Tables 2 and 3, wherein different parts thereof include its extracellular domain. Universal CAR

[0426] In some embodiments, CAR targeting is not produced or expressed by the cells of the treated subject. Therefore, this CAR allows cells to be concentrated on the target cells (such as cancer cells). By administering small conjugate molecules, CAR cell responses can be made to target only those cells expressing tumor receptors, thereby reducing off-target toxicity, and due to the rapid removal of small conjugate molecules, the activation of CAR cells can be more easily controlled. As an additional advantage, cells expressing CAR can be used as "universal" cytotoxic cells to target a variety of tumors without the need to prepare separate CAR constructs. The targeted portion recognized by CAR can also remain unchanged. Only the ligand portion of the small conjugate molecule needs to be changed to allow the system to target cancer cells of different identities.

[0427] Various methods for targeting CAR and cells expressing CAR have been described in the art, including, for example, in US2020 / 0123224, the disclosure of which is incorporated herein by reference. For example, fluorescein or fluorescein isothiocyanate (FITC) moieties can be conjugated to reagents that bind to desired target cells (such as cancer cells), so that CAR cells expressing anti-fluorescein / FITC chimeric antigen receptors can be selectively targeted to target cells labeled by the conjugate. In a modification, other haptens recognized by CAR can be used instead of fluorescein / FITC. CAR can be produced using various scFv sequences known in the art or scFv sequences produced by conventional and routine methods. Other illustrative scFv sequences for fluorescein / FITC and for other haptens are provided in, for example, WO 2021 / 076788, and their disclosures are incorporated herein by reference.

[0428] In some embodiments, the CAR is an anti-FITC CAR, and the ligand is composed of a fluorescein or fluorescein isothiocyanate (FITC) portion conjugated to an agent that is bound to a desired target cell (such as a cancer cell). Exemplary ligands are shown below. In some embodiments, the ligand is FITC-folic acid.

[0429] Exemplary anti-FITC CARs and their various portions are shown in Table 4.

[0430] In some embodiments, CAR comprises a scFv domain. In some embodiments, the scFv domain comprises anti-fluorescein isothiocyanate (FITC) E2. In some embodiments, the scFv domain comprises a light chain variable domain (VL), a joint and a heavy chain variable domain (VH).

[0431] In some embodiments, the E2 scFv VL comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 45. In some embodiments, the E2 scFv VL comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 45. In some embodiments, the E2 scFv VL comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 45. In some embodiments, the E2 scFv VL comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 45. In some embodiments, the E2 scFv VL comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 45. In some embodiments, the E2 scFv VL comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 45. In some embodiments, the E2 scFv VL comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 45. In some embodiments, the E2 scFv VL comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 45. In some embodiments, the E2 scFv VL comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 45. In some embodiments, the E2 scFv VL comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 45. In some embodiments, the E2 scFv VL comprises the nucleotide sequence of SEQ ID NO: 45. In some embodiments, the E2 scFv VL consists of the nucleotide sequence of SEQ ID NO: 45.

[0432] In some embodiments, the E2 scFv VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the E2 scFv VL comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the E2 scFv VL comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the E2 scFv VL comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the E2 scFv VL comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the E2 scFv VL comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the E2 scFv VL comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the E2 scFv VL comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the E2 scFv VL comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the E2 scFv VL comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the E2 scFv VL comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, the E2 scFv VL consists of the amino acid sequence of SEQ ID NO: 47.

[0433] In some embodiments, the E2 scFv VH comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the E2 scFv VH comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the E2 scFv VH comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the E2 scFv VH comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the E2 scFv VH comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the E2 scFv VH comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the E2 scFv VH comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the E2 scFv VH comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the E2 scFv VH comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the E2 scFv VH comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the E2 scFv VH comprises the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the E2 scFv VH consists of the nucleotide sequence of SEQ ID NO: 48.

[0434] In some embodiments, the E2 scFv VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the E2 scFv VH comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the E2 scFv VH comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the E2 scFv VH comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the E2 scFv VH comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the E2 scFv VH comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the E2 scFv VH comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the E2 scFv VH comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the E2 scFv VH comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the E2 scFv VH comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the E2 scFv VH comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the E2 scFv VH consists of the amino acid sequence of SEQ ID NO: 50.

[0435] In some embodiments, the E2 scFv linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the E2 scFv linker comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the E2 scFv linker comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the E2 scFv linker comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the E2 scFv linker comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the E2 scFv linker comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the E2 scFv linker comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the E2 scFv linker comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the E2 scFv linker comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the E2 scFv linker comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the E2 scFv linker comprises the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the E2 scFv linker consists of the nucleotide sequence of SEQ ID NO: 51.

[0436] In some embodiments, the E2 scFv linker comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 scFv linker comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 scFv linker comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 scFv linker comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 scFv linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 scFv linker comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 scFv linker comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 scFv linker comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 scFv linker comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 scFv linker comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 scFv linker comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 scFv linker consists of the amino acid sequence of SEQ ID NO: 53.

[0437] In some embodiments, the E2 scFv comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, the E2 scFv comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, the E2 scFv comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, the E2 scFv comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, the E2 scFv comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, the E2 scFv comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, the E2 scFv comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, the E2 scFv comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, the E2 scFv comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, the E2 scFv comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, the E2 scFv comprises the nucleotide sequence of SEQ ID NO: 54. In some embodiments, the E2 scFv consists of the nucleotide sequence of SEQ ID NO: 54.

[0438] In some embodiments, the E2 scFv comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the E2 scFv comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the E2 scFv comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the E2 scFv comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the E2 scFv comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the E2 scFv comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the E2 scFv comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the E2 scFv comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the E2 scFv comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the E2 scFv comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the E2 scFv comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, the E2 scFv consists of the amino acid sequence of SEQ ID NO: 56.

[0439] In some embodiments, the CAR system uses a conjugate molecule as a bridge between cells expressing CAR and targeted cancer cells. The conjugate molecule is a conjugate comprising a hapten and a cell targeting portion (such as any suitable tumor cell specific ligand). Exemplary haptens that can be recognized and combined by CAR include small molecular weight organic molecules, such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin and digoxin, and fluorescein and its derivatives (including FITC (fluorescein isothiocyanate), NHS-fluorescein and pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives), knotting element, centyrin and DARPin. Suitable cell targeting portions that can serve as haptens of CAR themselves include knotting element (see Kolmar H. et al., The FEBS Journal. 2008. 275 (11): 26684-90), centyrin and DARPin (see Reichert, JMMAbs 2009. 1 (3): 190–209).

[0440] In some embodiments, the cell targeting moiety is DUPA (DUPA-(99m)Tc), a molecule that binds to PSMA-positive human prostate cancer cells with nanomolar affinity (K D =14 nM; see Kularatne, SA et al., Mol Pharm. 2009. 6(3):780-9) bound ligands. In one embodiment, the DUPA derivative can be a ligand of a small molecule ligand linked to a targeting moiety, and the DUPA derivatives are described in WO 2015 / 057852, which is incorporated herein by reference.

[0441] In some embodiments, the cell targeting moiety is a CCK2R ligand, a ligand bound by CCK2R-positive cancer cells (e.g., cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stromal, and colon; see Wayua. C. et al., Molecular Pharmaceutics. 2013. ePublication).

[0442] In some embodiments, the cell targeting moiety is folate, folic acid, or an analog thereof, a ligand bound by a folate receptor on cancer cells, including cancers of the ovary, cervix, endometrium, lung, kidney, brain, breast, colon, and head and neck; see Sega, EI et al., Cancer Metastasis Rev. 2008. 27(4):655-64).

[0443] In some embodiments, the cell targeting moiety is a NK-1R ligand. Receptors for NK-1R ligands are found on cancers of, for example, the colon and pancreas. In some embodiments, the NK-1R ligand can be synthesized according to the methods disclosed in International Patent Application No. PCT / US2015 / 044229, which is incorporated herein by reference.

[0444] In some embodiments, the cell targeting moiety can be a peptide ligand, for example, the ligand can be a peptide ligand that is an endogenous ligand of the NK1 receptor. In some embodiments, the small conjugate molecule ligand can be a regulatory peptide belonging to the tachykinin family that targets the tachykinin receptor. Such regulatory peptides include substance P (SP), neurokinin A (K substance) and neurokinin B (neuromediatin K) (see Hennig et al., International Journal of Cancer: 61, 786-792).

[0445] In some embodiments, the cell targeting moiety is a CAIX ligand. Receptors for the CAIX ligand are found, for example, on renal cancer, ovarian cancer, vulvar cancer, and breast cancer. The CAIX ligand may also be referred to herein as CA9.

[0446] In some embodiments, the cell targeting moiety is a ligand for gamma-glutamyl transpeptidase, which is overexpressed in, for example, ovarian cancer, colon cancer, liver cancer, astrocytic glioma, melanoma, and leukemia.

[0447] In some embodiments, the cell targeting moiety is a CCK2R ligand. Receptors for the CCK2R ligand are found, inter alia, on cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon.

[0448] In one embodiment, the cell targeting moiety can have a mass of less than about 10,000 Daltons, less than about 9000 Daltons, less than about 8,000 Daltons, less than about 7000 Daltons, less than about 6000 Daltons, less than about 5000 Daltons, less than about 4500 Daltons, less than about 4000 Daltons, less than about 3500 Daltons, less than about 3000 Daltons, less than about 2500 Daltons, less than about 2000 Daltons, less than about 1500 Daltons, less than about 1000 Daltons, or less than about 500 Daltons. In another embodiment, the small molecule ligand can have a mass of about 1 to about 10,000 Daltons, about 1 to about 9000 Daltons, about 1 to about 8,000 Daltons, about 1 to about 7000 Daltons, about 1 to about 6000 Daltons, about 1 to about 5000 Daltons, about 1 to about 4500 Daltons, about 1 to about 4000 Daltons, about 1 to about 3500 Daltons, about 1 to about 3000 Daltons, about 1 to about 2500 Daltons, about 1 to about 2000 Daltons, about 1 to about 1500 Daltons, about 1 to about 1000 Daltons, or about 1 to about 500 Daltons.

[0449] In an illustrative embodiment, the connection in the conjugate described herein can be a direct connection (e.g., a reaction between the isothiocyanate group of FITC and the free amine group of the small molecule ligand), or the connection can be achieved through an intermediate linker. In one embodiment, if present, the intermediate linker can be any biocompatible linker known in the art, such as a divalent linker. In an illustrative embodiment, the divalent linker can include about 1 to about 30 carbon atoms. In another illustrative embodiment, the divalent linker can include about 2 to about 20 carbon atoms. In other embodiments, a divalent linker with a lower molecular weight (i.e., those with an approximate molecular weight of about 30 to about 300 Da) is used. In another embodiment, suitable linker lengths include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 or more atoms.

[0450] In some embodiments, the hapten and the cell targeting moiety can be directly conjugated by means such as the reaction between the isothiocyanate group of FITC and the free amine group of a small ligand (e.g., folic acid, DUPA, and CCK2R ligand). However, the use of a linker domain to connect the two molecules can be helpful because it can provide flexibility and stability. Examples of suitable linker domains include: 1) polyethylene glycol (PEG); 2) polyproline; 3) hydrophilic amino acids; 4) sugars; 5) non-natural peptidoglycans; 6) polyvinyl pyrrolidone; 7) Pluronic F-127. Suitable linker lengths include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 or more atoms.

[0451] In some embodiments, the linker can be a bivalent linker, which can include one or more spacers.

[0452] Illustrative conjugates of the present disclosure include the following molecules: FITC-(PEG) 12 -Folic acid, FITC-(PEG) 20 -Folic acid, FITC-(PEG) 108 -Folic acid, FITC-DUPA, FITC-(PEG) 12 -DUPA, FITC-CCK2R ligand, FITC-(PEG) 12 -CCK2R ligand, FITC-(PEG) 11 -NK1R ligand and FITC-(PEG)2-CA9.

[0453] Although the affinity with which a ligand binds to a cancer cell receptor can vary, and in some cases low affinity binding may be preferred (e.g., about 1 μM), the binding affinity of a ligand to a cancer cell receptor will typically be at least about 100 μM, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM or 10 pM, and even more preferably at least about 100 pM.

[0454] Examples of conjugates and methods for making them are provided in U.S. patent applications US 2017 / 0290900, US 2019 / 0091308, and US 2020 / 0023009, all of which are incorporated herein by reference.

[0455] In some embodiments, the viral particles described herein include nucleotide sequences encoding universal modular anti-tag chimeric antigen receptors (UniCAR). This system allows immune cells transplanted with UniCAR to be retargeted against multiple antigens (see, e.g., U.S. Patent Publication US20170240612 A1, which is incorporated herein by reference in its entirety; Cartellieri et al., (2016) Blood Cancer Journal 6, e458, which is incorporated herein by reference in its entirety).

[0456] In some embodiments, the viral particles described herein include nucleotide sequences encoding switchable CAR and / or CAR effector cell (CAR-EC) switches. In this system, the CAR-EC switch has a first region bound by a CAR on the CAR-EC and a second region that binds to a cell surface molecule on a target cell, thereby stimulating an immune response from the CAR-EC that is cytotoxic to the bound target cell. In some embodiments, the CAR-EC switch can act as an "on-switch" for CAR-EC activity. Activity can be "turned off" by reducing or stopping the application of the switch. These CAR-EC switches can be used with CAR-EC disclosed herein and existing CAR T cells for the treatment of diseases or disorders, such as cancer, in which the target cells are malignant cells. This treatment may be referred to herein as switchable immunotherapy (U.S. Patent Publication US9624276B2, which is incorporated herein by reference in its entirety).

[0457] In some embodiments, the viral particle comprises a nucleotide sequence encoding a universal immune receptor (e.g., switchable CAR, sCAR) binding to a peptide neo-epitope (PNE). In some embodiments, the peptide neo-epitope (PNE) has been incorporated into different positions determined in an antibody (antibody switch) targeting an antigen. Therefore, sCAR-T cell specificity is redirected only to PNE (not occurring in the human proteome), thereby allowing orthogonal interactions between sCAR-T cells and the antibody switch. In this way, sCAR-T cells strictly rely on the presence of an antibody switch to be fully activated, thereby excluding the off-target recognition of CAR T cells to endogenous tissues or antigens in the absence of the antibody switch (Arcangeli et al., (2016) Transl Cancer Res 5 (Suppl 2): ​​S174-S177, which is incorporated herein by reference in its entirety). Other examples of switchable CARs are provided in U.S. Patent Application US20160272718A1, which is incorporated herein by reference in its entirety.

[0458] As used herein, the term "tag" encompasses universal immune receptors, tags, switches or Fc regions of i...

Claims

1. A method for preparing a lentiviral formulation, the method comprising (i) filtering the suspension mixture comprising the host cell population and the lentiviral particles to remove contaminants, wherein the filtering comprises: (a) filtering the mixture through a first filter to obtain a first filtrate, wherein the first filter is a depth filter, (b) filtering the first filtrate through a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (c) filtering the second filtrate through a third filter, thereby producing a filtered lentiviral particle preparation, wherein the third filter has a lower retention threshold than the second filter; and (ii) concentrating the filtered lentiviral particle preparation, wherein concentrating comprises chromatography and ultrafiltration.

2. A method for preparing a lentiviral formulation, the method comprising (i) contacting the host cell population in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the host cell population of step (i) for a period of time sufficient to produce a suspension mixture comprising the host cell population and lentiviral particles; (iii) filtering the suspended mixture to remove contaminants, the filtering comprising: (a) contacting the mixture with an endonuclease, (b) filtering the mixture through a first filter to obtain a first filtrate, wherein the first filter is a depth filter (c) filtering the first filtrate through a second filter to obtain a second filtrate, wherein the second filter has a smaller retention threshold than the first filter, and (d) filtering the second filtrate through a third filter, thereby producing a filtered lentiviral particle preparation, wherein the third filter has a lower retention threshold than the second filter; and (iv) concentrating the filtered lentiviral particle preparation, wherein concentrating comprises chromatography and ultrafiltration.

3. The method of claim 1 or 2, wherein the host cell comprises a human cell.

4. The method according to claim 3, wherein the human cells comprise HEK293 cells, HEK293T cells, HEK293F cells, HEK293FT cells, Te671 cells, HT1080 cells or CEM cells.

5. The method of claim 3 or 4, wherein the cells comprise HEK293 cells.

6. The method of claim 3 or 4, wherein the cells comprise HEK293T cells.

7. The method according to any one of claims 1 to 6, wherein the first filter has a retention threshold of 1-60 μm. The method of claim 7 , wherein the first filter has a retention threshold of 60 μm.

9. The method of any one of claims 1-8, wherein the second filter has a retention threshold of 0.4-4 μm.

10. The method of claim 9, wherein the second filter has a retention threshold of 0.45 μm.

11. The method according to any one of claims 1 to 10, wherein the third filter has a retention threshold of 0.45 μm ± 0.2 μm.

12. The method according to any one of claims 1-10, wherein the third filter has a retention threshold of 0.2-0.3 μm. The method of claim 12 , wherein the third filter has a retention threshold of 0.2 μm.

14. The method of any one of claims 1-10, 12 and 13, wherein the first filter has a retention threshold of 60 μm, the second filter has a retention threshold of 0.45 μm, and the third filter has a retention threshold of 0.2 μm.

15. The method of any one of claims 1 and 3-14, wherein an endonuclease is present in each of steps (i)(a)-(i)(c).

16. The method according to any one of claims 2 to 15, wherein the endonuclease is present in all steps (iii)(b) to (iii)(d).

17. The method of any one of claims 1-16, wherein the second filter and the third filter are two layers within a double layer filter assembly.

18. The method according to any one of claims 1-16, wherein the third filter is a double-layer filter including a first layer filter and a second layer filter, wherein the second layer filter has a smaller retention threshold than the first layer filter.

19. The method of claim 18, wherein the retention threshold of the first filter is 60 μm, the retention threshold of the second filter is 0.45 μm, the retention threshold of the first layer of filters is 0.45 μm, and the retention threshold of the second layer of filters is 0.2 μm.

20. The method of any one of claims 1-19, wherein the chromatography is anion exchange chromatography (AEX).

21. The method of claim 20, wherein the AEX chromatography comprises eluting the lentiviral particles with a saline buffer.

22. The method of claim 21, wherein the salt buffer comprises NaCl.

23. The method of claim 22, wherein the concentration of NaCl is about 0.5M to 3M.

24. The method of claim 22, wherein the concentration of NaCl is about 0.5M to 1M.

25. The method of any one of claims 22-24, wherein the NaCl is or is about 0.75M.

26. The method of claim 22, wherein the concentration of NaCl is about 1 M to 3 M.

27. The method of claim 22, wherein the concentration of NaCl is about 1.5M to 2.5M.

28. The method of any one of claims 22, 26 or 27, wherein the NaCl is about 2M.

29. The method of any one of claims 1-28, wherein the chromatography is performed prior to ultrafiltration.

30. The method of any one of claims 1-29, wherein the ultrafiltration is ultrafiltration / diafiltration (UF / DF).

31. The method of claim 30, wherein the UF / DF is performed by one or more tangential flow filtration (TFF) steps.

32. The method of claim 31 , wherein the one or more TFF filters are hollow fiber filters.

33. The method of claim 32, wherein the hollow fiber filter has a nominal molecular weight cutoff (NMWC) of at or about 500 kDa.

34. The method of any one of claims 31-33, wherein the TFF comprises a first tangential flow filtration (TFF) step and a second tangential flow filtration (TFF) step.

35. The method of claim 33, wherein the first TFF is performed with a first hollow fiber filter and the second TFF is performed with a second hollow fiber filter.

36. The method of claim 35, wherein the first hollow fiber filter and the second hollow fiber filter have the same nominal molecular weight cutoff (NMWC).

37. The method of claim 36, wherein the NMWC is 500 kDa.

38. The method of claim 35, wherein the first hollow fiber filter has a greater nominal molecular weight cutoff (NMWC) than the second hollow fiber filter.

39. The method of claim 38, wherein the first hollow fiber filter has a NMWC of 500 kDa.

40. The method of any one of claims 35-39, wherein the first hollow fiber filter has a greater surface area than the second hollow fiber filter.

41. The method of claim 40, wherein the surface area of ​​the first hollow fiber filter is 790 cm 2 Up to 1600cm 2 between.

42. The method of any one of claims 35-41, wherein the first hollow fiber filter accommodates a larger volume than the second hollow fiber filter.

43. The method of claim 42, wherein the first hollow fiber filter has a capacity of 300 mL.

44. The method of any one of claims 1-43, comprising sterile filtering the filtered formulation after concentration, thereby producing a sterilized formulation.

45. The method of claim 44, wherein sterile filtering comprises filtering the filtered formulation with a fourth filter.

46. ​​The method of claim 45, wherein the fourth filter has a retention threshold of 0.2 μm.

47. A method according to any one of claims 44-46, comprising formulating the sterilized formulation in a buffer to produce a drug substance.

48. The method of any one of claims 1-47, wherein the method is performed at a pH of 6-8.

49. The method of any one of claims 1-48, wherein the lentiviral formulation is for in vivo administration to a subject.

50. The method of any one of claims 1-49, wherein the amount of contaminants in the filtered preparation is reduced compared to the amount of contaminants in the second filtrate.

51. The method of any one of claims 44-50, wherein the amount of contaminants in the sterilized formulation is at an acceptable level for in vivo administration to a subject.

52. The method of any one of claims 1-51, wherein the contaminants comprise host cells, host cell DNA (hcDNA) and / or host cell proteins (HCP).

53. The method of claim 52, wherein the amount of hcDNA is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.5% of the sterilized formulation.

54. The method of claim 52, wherein the amount of hcDNA in the sterilized formulation is reduced by greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99%.

55. The method of claim 52, wherein the amount of HCP is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.5% of the sterilized formulation.

56. The method of claim 52, wherein the amount of HCP in the sterilized formulation is reduced by greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

57. The method of any one of claims 52-56, wherein the amount of hcDNA in the filtered preparation is less than about 2500 ng / 1E9 TU.

58. The method of any one of claims 52-57, wherein the amount of hcDNA in the filtered preparation is at least about 80 times lower than the amount of hcDNA in the suspension mixture.

59. The method of any one of claims 52-58, wherein the amount of hcDNA in the filtered preparation is at least about 5-fold lower than the amount of hcDNA in the second filtrate.

60. The method of any one of claims 52-59, wherein the amount of HCP after chromatography is less than about 3000 μg / 1E9TU.

61. The method of any one of claims 52-60, wherein the amount of HCP after chromatography is at least about 40 times lower than the amount of HCP before chromatography.

62. The method of any one of claims 52-61, wherein the amount of HCP after chromatography is at least about 99% lower than the amount of HCP before chromatography.

63. The method of any one of claims 52-62, wherein after the first UF / DF step, the amount of HCP is less than about 1500 μg / 1E9 TU.

64. The method of any one of claims 52-63, wherein after the second UF / DF step, the amount of HCP is undetectable.

65. The method of any one of claims 1-64, wherein the suspension mixture comprises a culture medium, wherein the culture medium is free of serum and / or animal by-products.

66. The method of any one of claims 2-65, wherein the culturing of step (ii) continues for 40-48 hours.

67. The method of any one of claims 1-66, wherein said filtering and said concentrating are performed over a period of 5-8 hours.

68. The method of any one of claims 1-67, wherein the volume of the suspension mixture is 3-50 liters.

69. The method of any one of claims 1-67, wherein the volume of the suspension mixture is from 5 L to 200 L.

70. The method of any one of claims 1-67, wherein the volume of the suspension mixture is 100L to 200L.

71. The method of any one of claims 1-67, wherein the volume of the suspension mixture is between at or about 180 L and at or about 200 L.

72. The method of any one of claims 1-71, wherein the lentiviral particle comprises at least one payload.

73. The method of claim 72, wherein the at least one payload comprises a non-coding nucleic acid, optionally wherein the non-coding nucleic acid is a siRNA, miRNA or shRNA.

74. The method of claim 72, wherein the at least one payload is a polynucleotide encoding a polypeptide of interest.

75. The method of any one of claims 2-74, wherein the at least one plasmid is a polynucleotide encoding a polypeptide of interest.

76. The method of claim 74 or 75, wherein the polypeptide of interest is a chimeric antigen receptor (CAR).

77. The method of claim 76, wherein the CAR is specific for a tumor-associated antigen.

78. The method of claim 77, wherein the tumor-associated antigen is CD19, BCMA, GPRC5D, ROR1, FcRL5, alpha-fetoprotein, or Her2.

79. The method of claim 76, wherein the CAR is a universal CAR.

80. The method of claim 79, wherein the universal CAR comprises an extracellular domain comprising a tag binding domain.

81. The method of claim 80, wherein the label is fluorescein.

82. The method of claim 76, wherein the CAR comprises an extracellular domain comprising a hapten binding domain.

83. The method of any one of claims 1-82, wherein the lentiviral particle comprises a surface engineered fusion protein exposed on the surface of the lentiviral particle, optionally wherein the surface engineered protein is embedded in a lipid bilayer.

84. The method of claim 83, wherein the surface engineered protein consists of a single binding domain protein that binds a target molecule on a target cell.

85. The method of claim 84, wherein the surface engineered protein is composed of a multi-binding domain protein, wherein each binding domain binds a target molecule on a target cell, optionally wherein each binding domain binds a different target molecule.

86. The method of claim 84 or claim 85, wherein the single binding domain protein or the multiple binding domain protein is a transduction enhancer protein.

87. The method of claims 83-86, wherein the surface engineered protein is a fusion protein comprising a transduction enhancer and a viral envelope protein.

88. The method of any one of claims 1-87, wherein the lentiviral particle comprises a viral envelope comprising a transduction enhancer protein and a viral envelope protein.

89. The method of any one of claims 2-88, wherein the at least one plasmid is a plasmid encoding a fusion protein comprising a transduction enhancer protein and a viral envelope protein, optionally wherein the transduction enhancer comprises an immune cell activation protein and / or a co-stimulatory molecule.

90. The method of any one of claims 2-89, wherein the at least one plasmid is a plasmid encoding a fusion protein comprising an immune cell activation protein and a viral envelope protein.

91. The method of claim 89 or claim 90, wherein the immune cell activation protein comprises at least one binding domain, and the at least one binding domain specifically binds to CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244, TCRα chain, TCRβ chain, TCRζ chain, TCRγ chain, TCRδ chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR subunit or NKp80 or a combination thereof.

92. The method of any one of claims 2-89, wherein the at least one plasmid is a plasmid encoding a fusion protein comprising at least one co-stimulatory molecule and a viral envelope protein.

93. The method of claim 89 and claim 92, wherein the co-stimulatory molecule is CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, OX40, 4-1BB, CD40L, or any combination thereof.

94. The method of any one of claims 86-90, wherein the transduction enhancer comprises at least one binding domain that binds to a target molecule selected from an immune cell activation receptor, a T cell co-stimulatory receptor, or an adhesion molecule.

95. The method of any one of claims 86-94, wherein: The transduction enhancer comprises a single binding domain that binds to a target molecule selected from an immune cell activation receptor, a T cell co-stimulatory receptor, or an adhesion molecule; The transduction enhancer comprises a plurality of binding domains that bind to two or more target molecules selected from immune cell activation receptors, T cell co-stimulatory receptors, and adhesion molecules; or The transduction enhancer comprises a plurality of binding domains, each of which binds to a different target molecule, wherein the target molecule is an immune cell activation receptor, a T cell co-stimulatory receptor and an adhesion molecule.

96. The method of any one of claims 94-95, wherein: The immune cell activation receptor is CD3; The co-stimulatory molecule is CD28, CD137 or CD134; and / or The adhesion molecule is CD58 or CD2.

97. The method of any one of claims 84-96, wherein each of the at least one binding domain is independently selected from an antibody or antigen binding fragment, or an extracellular domain of a natural ligand of the target molecule.

98. The method of any one of claims 87-97, wherein the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a Cocal virus G protein.

99. The method of any one of claims 2-98, wherein the at least one plasmid is a plasmid encoding a helper virus protein.

100. The method of claim 99, wherein the helper viral protein is rev and / or gagpol.

101. The method of any one of claims 2-100, wherein the host cell population is contacted with a plasmid mixture comprising (i) a plasmid encoding a gene of interest; (ii) a plasmid encoding a rev viral protein; (iii) a plasmid encoding a gagpol viral protein; and (iv) A plasmid encoding a viral envelope protein.

102. The method of claim 101, wherein the plasmid mixture comprises (v) a plasmid encoding an immune cell activating protein, (vi) a plasmid encoding a co-stimulatory molecule, or (vii) any combination of (v)-(vi).

103. A lentiviral formulation produced by the method of any one of claims 1-102.

104. The lentiviral formulation of claim 103, wherein the infectious titer of the formulation is 2.0 TU / mL to 6 x 10 8 TU / mL.

105. The lentiviral formulation of claim 103 or claim 104, wherein the formulation has an infectious titer of 2.5 TU / mL to 4.7 x 10 8 TU / mL.

106. The lentiviral formulation of claim 104 or claim 105, wherein the total number of infectious units in the formulation is 4 x 10 10 TU to 8x 10 10 TU.

107. The lentiviral formulation of any one of claims 104-106, wherein the total number of infectious units in the formulation is 5 x 10 10 TU to 7x 10 10 TU, optionally at or about 6 x 10 10 TU.

108. The lentiviral formulation of any one of claims 104-107, wherein the formulation comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% HCP.

109. The lentiviral formulation of any one of claims 104-108, wherein the formulation comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.5% hcDNA.

110. The lentiviral formulation of any one of claims 104-109, wherein the formulation comprises greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% reduction in HCPs.

111. The lentiviral formulation of any one of claims 104-110, wherein the formulation comprises greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% reduction in hcDNA, optionally compared to the filtered formulation prior to the concentration.

112. The lentiviral formulation of any one of claims 104-111, wherein the formulation comprises greater than 99% reduction in hcDNA and greater than 99% reduction in HCPs, optionally compared to the filtered formulation prior to the concentration.

113. The lentiviral formulation of any one of claims 104-112, wherein the formulation comprises less than 1% hcDNA and less than 1% HCP.

114. A lentiviral preparation comprising a titer of 2.5 to 4.7 x 10 8 TU / mL of a lentiviral vector, wherein the formulation comprises less than 1% hcDNA and less than 1% HCP.

115. The lentiviral formulation of any one of claims 103-114, wherein the volume of the formulation is 1 mL to 500 mL, optionally 10 mL to 100 mL.

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