Production of virus particles

Abstract

Methods for the large-scale production of virus particles, as well as compositions and methods for using such virus particles, are provided. TIFF2025518534000022.tif89159

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 342,975, filed May 17, 2022; U.S. Provisional Application No. 63 / 371,756, filed Aug. 17, 2022; U.S. Provisional Application No. 63 / 371,864, filed Aug. 18, 2022; and U.S. Provisional Application No. 63 / 440,093, filed Jan. 19, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0002] Incorporation by Reference of Sequence Listing This application has been filed with an electronic sequence listing. The sequence listing is provided as a file named 260132000440SeqList.xml, created on May 17, 2023, with a size of 207,707 bytes. The information in the electronic form of the sequence listing is hereby incorporated by reference in its entirety.

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

Background Art

[0004] Background Gene therapy has shown significant clinical success in the treatment of cancer and other diseases. However, the use of these life-saving therapies is limited due to important challenges in cost, supply chain, and manufacturing. Retroviruses are often used as delivery agents to introduce one or more nucleotides of interest into one or more sites of interest. Lentiviral vectors are of considerable interest among retroviruses because they can infect non-dividing cells. In addition, lentiviral vectors enable stable long-term expression of genes of interest. In the smallest applications, vectors can be concentrated and purified by relatively simple methods using centrifugation techniques. However, scaling up purification methods for large-scale production for clinical use is a major challenge.

[0005] In particular, when considering the production of viral vectors for human use, the vector purification process is directly linked to safety in terms of purity. There remains a need for large-scale retroviral vector purification methods that yield high-purity products. SUMMARY OF THE INVENTION

[0006] Summary The present disclosure is based, at least in part, on the discovery of methods for producing viral particles (e.g., lentiviral particles) for in vivo administration to a subject. Specifically, as demonstrated herein, subjecting a mixture of host cells and viral particles to at least two filtration steps reduces contaminants (e.g., host cell DNA and proteins) prior to concentrating the mixture via chromatography and ultrafiltration. Without wishing to be bound by theory, the filtration steps described herein reduce contaminants to an amount sufficient for in vivo administration. Thus, in some aspects, the present disclosure provides (i) a step of filtering a suspension mixture comprising a population of host cells and lentiviral particles to remove contaminants, comprising (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate, (b) Filtering the first filtrate with a second filter having a retention threshold smaller than that of the first filter, thereby resulting in a second filtrate, and (c) Filtering the second filtrate with a third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of lentiviral particles including; and (ii) A step of concentrating the filtered preparation of lentiviral particles, including chromatography and ultrafiltration A method for preparing a lentiviral preparation is provided.

[0007] In other aspects, the present disclosure (i) Contacting a population of host cells in suspension with at least one plasmid encoding a lentiviral protein; (ii) Culturing the population of host cells of step (i) for a period sufficient to produce a suspension mixture containing the population of host cells and lentiviral particles; (iii) A step of filtering the suspension mixture to remove contaminants, (a) Contacting the mixture with an endonuclease, (b) Filtering the mixture with a first filter which is a depth filter, thereby resulting in a first filtrate, (c) Filtering the first filtrate with a second filter having a retention threshold smaller than that of the first filter, thereby resulting in a second filtrate, and (d) Filtering the second filtrate with a third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of lentiviral particles including; and (iv) A step of concentrating the filtered preparation of lentiviral particles, including chromatography and ultrafiltration A method for preparing a lentiviral preparation is provided.

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

[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 two - layer filter component. In some aspects, the third filter is a two - layer filter including a first - layer filter and a second - layer filter, and 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, the endonuclease is present in steps (i)(a) - (i)(c). In some aspects, the endonuclease is present in steps (iii)(b) - (iii)(d).

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

[0013] In some scenarios, chromatography is performed prior to ultrafiltration. In some scenarios, the ultrafiltration is ultrafiltration / diafiltration (UF / DF). In some scenarios, the UF / DF is by one or more tangential flow filtration (TFF) steps. In some scenarios, the one or more TFF filters are hollow fiber filters. In some scenarios, the nominal molecular weight cut-off (NMWC) of the hollow fiber filter is 500 kDa or about 500 kDa. In some scenarios, the TFF includes a first tangential flow filtration (TFF) step and a second tangential flow filtration (TFF) step. In some scenarios, the first TFF is performed using a first hollow fiber filter and the second TFF is performed using a second hollow fiber filter. In some scenarios, the first and second hollow fiber filters have the same nominal molecular weight cut-off (NMWC). In some scenarios, the NMWC is 500 kDa. In some scenarios, the first hollow fiber filter has a larger nominal molecular weight cut-off (NMWC) than the second hollow fiber filter. In some scenarios, the first hollow fiber filter is 500 kDa. In some scenarios, the first hollow fiber filter has a larger surface area than the second hollow fiber filter. In some scenarios, the first hollow fiber filter is from 790 cm2 to 1600 cm2. In some scenarios, the first hollow fiber filter accommodates a larger volume than the second hollow fiber filter. In some scenarios, the volume of the first hollow fiber filter is 300 mL.

[0014] In some scenarios, the method includes sterile filtration of the filtered formulation after concentration, thereby producing a sterile formulation. In some scenarios, the sterile filtration includes filtering the filtered formulation through a fourth filter. In some scenarios, the fourth filter has a retention threshold of 0.2 μm. In some scenarios, the method includes formulating the sterile formulation in a buffer, thereby producing a drug substance.

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

[0016] In some aspects, the amount of contaminants in the filtered formulation is reduced as compared to the amount of contaminants in the second filtrate. In some aspects, the amount of contaminants in the sterilized formulation is at a level acceptable for in vivo administration to a subject. In some aspects, the contaminants include host cells, host cell DNA (hcDNA), and / or host cell protein (HCP). In some aspects, 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. In some aspects, the amount of hcDNA 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 the sterilized formulation. In some aspects, 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. In some aspects, the amount of HCP 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 the sterilized formulation.

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

[0018] In some scenarios, the suspension mixture contains a medium, provided that the medium does not contain serum and / or animal by-products. In some scenarios, the culturing step of step (ii) lasts for 40 to 48 hours. In some scenarios, the filtering and concentrating occur over 5 to 8 hours. In some scenarios, the suspension mixture has a volume of 3 to 50 liters. In some scenarios, the suspension mixture has a volume of 5 L to 200 L. In some scenarios, the suspension mixture has a volume of 100 L to 200 L. In some scenarios, the suspension mixture has a volume of 180 L or about 180 L to 200 L or about 200 L.

[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, and optionally, 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, 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 for a tumor-associated antigen. 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 particle comprises a surface-engineered fusion protein exposed on the surface of the lentiviral particle, and optionally, the surface-engineered protein is embedded in the lipid bilayer. In some aspects, the surface-engineered protein is composed of a single-binding domain protein that binds to a target molecule on the 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 the target cell, and optionally, 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 comprise a viral envelope that includes an immunocyte activation protein and a viral envelope protein. In some aspects, at least one plasmid is a plasmid encoding an immunocyte activation protein and a plasmid encoding a viral envelope protein. In some aspects, the immunocyte activation protein is a protein that 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, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, or NKp80.

[0022] In some aspects, the immunocyte activating protein comprises at least one binding domain that binds to a target molecule selected from the group consisting of a T cell activation receptor, a costimulatory molecule, or an adhesion molecule. In some aspects, the immunocyte activating protein comprises a single binding domain that binds to one target molecule selected from the group consisting of a T cell activation receptor, a costimulatory molecule, or an adhesion molecule. In some aspects, the immunocyte activating protein comprises multiple binding domains that bind to two or more target molecules selected from the group consisting of a T cell activation receptor, a costimulatory molecule, and an adhesion molecule. In some aspects, the immunocyte activating protein comprises multiple binding domains that each bind to a different target molecule that is a T cell activation receptor, a costimulatory molecule, and an adhesion molecule. In some aspects, the immunocyte activating protein comprises at least one binding domain that binds to 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 of a target molecule or an ectodomain of a native ligand. In some aspects, the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a nipha virus envelope protein, or a cocal virus G protein.

[0023] In some aspects, the viral envelope protein is the VSV-G envelope protein, the measles virus envelope protein, the Nipah virus envelope protein, or the coxsackievirus G protein. In some aspects, the viral envelope protein comprises at least one costimulatory molecule. In some aspects, at least one plasmid is a plasmid encoding a costimulatory molecule. In some aspects, at least one costimulatory 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, at least one plasmid is a plasmid encoding a helper viral protein. In some aspects, the helper viral protein is rev and / or gagpol.

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

[0025] In some aspects, the disclosure provides a lentiviral preparation produced by the methods described herein. In some aspects, the lentiviral preparation has an infectivity titer of 2.0 - 6×10 8 TU / mL. In some aspects, the preparation has an infectivity titer of 2.5 - 4.7×10 8It has an infectious titer of TU / mL. In some aspects, the total number of infectious units in the formulation is 4×10^10 TU to 8×10^10 TU. In some aspects, the total number of infectious units in the formulation is 5×10^10 TU to 7×10^10 TU, optionally 6×10^10 TU or about 6×10^10 TU. In some aspects, the formulation contains 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 HCP. In some aspects, the formulation contains 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 hcDNA. In some aspects, the formulation includes a reduction of 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% of HCP. In some aspects, the formulation includes a reduction of 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% of hcDNA, optionally this is a reduction compared to the filtered formulation before the concentration step. In some aspects, the formulation includes a reduction of more than 99% of hcDNA and a reduction of more than 99% in HCP, optionally this is a reduction compared to the filtered formulation before the concentration step. In some aspects, the formulation contains less than 1% of hcDNA and less than 1% of HCP. In some aspects, the lentiviral formulation contains a lentiviral vector with a titer of 2.5 to 4.7×10 8 TU / mL, wherein the formulation contains less than 1% of hcDNA and less than 1% of 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 and a more complete understanding will be apparent and more easily understood by referring to the following detailed description and the appended claims in conjunction with the accompanying drawings.

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[0027] Detailed Description In some aspects, the disclosure provides methods for preparing a formulation of infectious virus particles suitable for use in the manufacture of a formulation and / or for in vivo use. In some aspects, the disclosure provides methods for preparing a formulation of virus particles for direct in vivo administration. In some aspects, preparing a formulation of virus particles includes subjecting a mixture of host cells and virus particles to at least two filtration steps to clarify the mixture. In some aspects, preparing a formulation of virus particles includes subjecting a mixture of host cells and virus particles to at least two filtration steps to clarify the mixture and subsequently concentrating the clarified mixture via chromatography and diafiltration.

[0028] In some aspects, preparing a formulation of virus particles includes subjecting a mixture of host cells and virus particles to three filtration steps to clarify the mixture. In some aspects, preparing a formulation of virus particles includes subjecting a mixture of host cells and virus particles to three filtration steps to clarify the mixture and subsequently concentrating the clarified mixture via chromatography and diafiltration.

[0029] Aspects of the present disclosure may use conventional techniques in chemistry, molecular biology, microbiology, recombinant DNA, and immunology that are within the capabilities of those skilled in the art. Such techniques are described in the literature. For example, J. Sambrook, E. F. Fritsch, and T. Maniatis (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 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; J. M. Polak and James O'D. McGee (1990) In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and D. M. J. Lilley and J. E. Dahlberg (1992) Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA; Methods in Enzymology, Academic Press. Roe, Simon, ed. Protein Purification Techniques. 2nd ed. Oxford: Oxford University Press, 2001, Sofer, Gail and Hagel, Lars.See 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 ed. New York: John Wiley & Sons, Inc., 1998, Masters, John R.W., ed. Animal Cell Culture: A Practical Approach. 3rd ed. Oxford: Oxford University Press, 2000. Each of these general texts is incorporated herein by reference.

[0030] I. Method for Producing Virus Particles Scaling up virus particle production for large-scale manufacturing for clinical use is a challenge regarding purification and yield. The present disclosure aims to overcome this challenge by providing a scalable method for producing sufficiently pure virus particles for use in direct in vivo injection into human subjects.

[0031] In some embodiments, the present disclosure provides a scalable suspension cell culture-based manufacturing method. Some embodiments of the methods of the present disclosure provide consistent cell culture titers for scale-up of bioreactors from 3 L to 10 L and further to 40 L.

[0032] In some embodiments, upstream and downstream processes in producing and purifying viral vector particles for in vivo use are described. The present disclosure also provides methods for effectively removing host cell DNA and proteins, which are important quality characteristics directly correlated with the safety of the formulation. The present disclosure provides methods that can be translated to clinical and commercial scale manufacturing of viral particles for in vivo administration.

[0033] A. Upstream Process In some embodiments, the present disclosure provides a method for preparing a viral particle formulation.

[0034] In some embodiments, the method for preparing a viral particle formulation includes a seed train, growth in a bioreactor, transfection with a vector, and vector production, which are referred to herein as the upstream process. The goal of the upstream process is to increase cell yield. In some embodiments, the upstream process includes the upstream process steps demonstrated in FIG. 1A.

[0035] In some embodiments, the methods described herein include an upstream process for generating viral particles. In some embodiments, the upstream process includes (i) expansion of host cells; (ii) plasmid transfection; (iii) recovery of viral particles; or (iv) any combination of (i)-(iii). In some embodiments, the methods described herein generate retroviral particles. In some embodiments, the methods described herein generate lentiviral particles.

[0036] In some embodiments, the present disclosure provides a method for preparing a viral particle preparation, comprising: (i) contacting a population of host cells in suspension with at least one plasmid encoding a viral protein; (ii) culturing the population of host cells of step (i) for a period sufficient to produce a suspension mixture comprising the population of host cells and viral particles; (iii) filtering the suspension mixture to remove contaminants, the filtering comprising: (a) contacting the mixture with an endonuclease; (b) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (c) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (d) filtering the second filtrate through a third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of viral particles; and (iv) concentrating the filtered preparation of viral particles, the concentrating comprising chromatography and ultrafiltration.

[0037] In some embodiments, the present disclosure provides a method for preparing a retroviral particle preparation, comprising: (i) contacting a population of host cells in suspension with at least one plasmid encoding a retroviral protein; (ii) culturing the population of host cells of step (i) for a period sufficient to produce a suspension mixture comprising the population of host cells and retroviral particles; (iii) filtering the suspension mixture to remove contaminants, the filtering comprising: (a) contacting the mixture with an endonuclease; (b) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (c) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (d) filtering the second filtrate through a third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of retroviral particles; and (iv) concentrating the filtered preparation of viral particles, the concentrating comprising chromatography and ultrafiltration.

[0038] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, comprising: (i) contacting a population of host cells in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the population of host cells of step (i) for a period of time sufficient to produce a suspension mixture comprising the population of host cells and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, comprising: (a) contacting the mixture with an endonuclease; (b) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (c) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (d) filtering the second filtrate through a third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of lentiviral particles; and (iv) concentrating the filtered preparation of lentiviral particles, comprising chromatography and ultrafiltration.

[0039] 1. Expansion of host cells Viral vectors can be suitably propagated within cells (also referred to as "host cells"). The cells according to the present disclosure can be any cells in which the desired viral vector can be propagated. By using a stable producer / packaging cell line, it is possible to increase the amount of viral vector particles for subsequent purification (e.g., to prepare a viral vector with an appropriate titer).

[0040] As used herein, the terms "producer cell" or "host cell" refer to a cell that contains all the elements necessary for the production of lentiviral vector particles. As used herein, the term "packaging cell" refers to a cell that contains the elements necessary for the production of an infectious recombinant virus that is lacking in the RNA genome. Typically, such packaging cells contain one or more producer plasmids capable of expressing viral structural proteins (e.g., codon-optimized gag-pol and env), but they do not contain a packaging signal.

[0041] In some embodiments, the host cells / packaging cells of the present disclosure are derived from mammalian cells, and any type of cell capable of assisting viral replication would be acceptable for the practice of the methods of the present disclosure.

[0042] In some embodiments, the host cell can be selected from any cell that enables the production of an enveloped virus. In some embodiments, the host cell is selected from human cells (HEK293, HEK293T, HEK293F, HEK293FT, Te671, HT1080, CEM), murine cells (muridae) (NIH-3T3), mustelid cells (mustelidae) (Mpf), and canine cells (canid) (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, the host cell is readily available from commercial sources (e.g., ATCC, Rockville, Md.).

[0043] In some embodiments, the host cells / packaging cells of the present disclosure are derived from primate cells such as human embryonic kidney cells. In some embodiments, the cells may be derived from an existing cell line, for example, the HEK293 cell line. In some embodiments, the host cell is the HEK293 cell line. In some embodiments, the cells may be derived from an existing cell line, for example, the HEK293T cell line. In some embodiments, the host cell is the HEK293T cell line. In some embodiments, the cells are adapted to suspension culture.

[0044] In some embodiments, the host cells are cultured in a medium suitable for culturing mammalian cells and producing enveloped viruses. The medium may be supplemented with additives well known in the art, such as antibiotics and serum (especially fetal bovine serum, among others) added at appropriate concentrations. The medium used may or may not contain serum, in particular. Culture media for mammalian cells are well known in the art and include, but are not limited to, DMEM (Dulbecco's Modified Eagle Medium), RPMI 1640, or various mixtures of culture media such as DMEM / F12, or serum-free media such as optiMEM®, optiPRO®, optiPRO-SFM®, CD293®, Freestyle F17® (Life Technologies) or Ex-Cell® 293 (Sigma-Aldrich), and LV-MAX™ medium (ThermoFisher Scientific #A3583402).

[0045] In some embodiments, the host cells are cultured in a medium that does not contain serum. In some embodiments, the host cells are cultured in a medium that does not contain animal products.

[0046] In some embodiments, the host cells are first diluted in a suitable medium before centrifugation to remove the medium. In some embodiments, the seed train expansion of the host cells is performed following removal of the medium. In some embodiments, the seed train expansion is performed to achieve a target cell number. In some embodiments, a series of target cell numbers are achieved. In some embodiments, the different target cell numbers correspond to containers of different sizes.

[0047] In some embodiments, the seed expansion is first performed in multiple containers until a sufficient number of cells for bioreactor inoculation is achieved.

[0048] In some embodiments, the seed expansion includes a target inoculation cell density and a target passage cell density. In some embodiments, the seed train expansion of the host cells is performed to achieve the target inoculation cell density. In some embodiments, the seed train expansion of the host cells is performed to achieve the target passage cell density. In some embodiments, the seed expansion is initiated by inoculating the container with the target inoculation cell density. In some embodiments, the target passage cell density is achieved after culturing the host cells in the container for host cell expansion. In some embodiments, after the target passage cell density is achieved, the host cells are passaged one or more times (e.g., continuously) into another container. In some embodiments, each passage into a new container is at the target inoculation density and the culture is maintained under conditions to achieve the target passage density. In some embodiments, the seed expansion is performed to achieve the target number of cells for bioreactor inoculation.

[0049] In some embodiments, the cells are inoculated into one or more containers for culturing in a medium until a target passage cell density is achieved. In some embodiments, the containers include tissue culture flasks, dishes, or roller bottles. In some embodiments, the container is a tissue culture flask. In some embodiments, the tissue culture flask is a shaking flask. In some embodiments, the tissue culture flask is suitable for suspension cells. In some embodiments, the tissue culture flask is an untreated flask. In some embodiments, the container is a bioreactor. In some embodiments, the bioreactor is suitable for suspension cells.

[0050] In some embodiments, the seed train expansion is carried out continuously in a plurality of containers. In some embodiments, the plurality of containers includes different containers. In some embodiments, the different containers are of different sizes. In some embodiments, the size of the container can accommodate a volume of 0.125 L to 50 L, such as 0.125 L to 10 L or 0.125 L to 5 L, and each is larger in size compared to the previous container in a series of containers for expansion. In some embodiments, the size of the container can accommodate a volume of 0.125 L to 5 L, and each is larger in size compared to the previous container in a series of containers for expansion. In some embodiments, the container can accommodate a volume of 0.125 L, 0.25 L, 0.5 L, 1 L, 1.6 L, 2 L, or 5 L. In some embodiments, the plurality of containers includes two or more shaking flasks. In some embodiments, the plurality of containers is for continuously passaging the cells. In some embodiments, the cells are inoculated at a target inoculation density of 2×10 5 cells / mL to 5×10 5 cells / mL, such as 3×10 5 cells / mL to 5×10 5 cells / mL or about 3×10 5 cells / mL to 5×10 5 cells / mL, and the cells reach about 3.5 to 6×10 6 cells / mL, such as 4.0 to 6×10 6When the target passage density of cells / mL is reached, the cells are serially passaged for inoculation into larger vessels. In some embodiments, the method for seed train expansion prior to bioreactor inoculation involves two, three, four, five or more passages of the cells. In some embodiments, the method for seed train expansion involves passaging the cells at least five times. In some embodiments, the cells are passaged into vessels that can accommodate volumes of, for example, 0.125 L, 0.5 L, 1 L, 1.6 L, and 5 L to increase their size.

[0051] In some embodiments, seed train expansion is performed in multiple shake flasks. In some embodiments, seed train expansion is performed in two or more, three or more, or four or more shake flasks. In some embodiments, seed train expansion is performed in at least two, at least three, at least four, or at least five shake flasks.

[0052] In some embodiments, seed train expansion is initiated by inoculating a host cell that has reached the target inoculation cell density into a first shake flask. In some embodiments, the first shake flask accommodates a volume of 0.125 L. In some embodiments, the target inoculation cell density for the first shake flask is 5×10 5 cells / mL or more.

[0053] In some embodiments, after the inoculated cells reach the target passage cell density in the first shake flask, the host cells are passaged at the target inoculation cell density into a second shake flask. In some embodiments, the second shake flask accommodates a volume of 0.5 L. In some embodiments, the target inoculation cell density for the second shake flask is about 2×10 5 cells / mL to 5×10 5 cells / mL, for example, 3×10 5 cells / mL to 5×10 5 cells / mL or about 3×10 5 cells / mL to 5×10 5cells / mL. In some embodiments, the target seeding cell density for the second shake flask is 5×10 5 cells / mL or more. In some embodiments, the target seeding cell density for the second shake flask is about 5×10 5 cells / mL. In some embodiments, the target passage cell density of the first flask is about 3.5 - 6×10 6 cells / mL. In some embodiments, the target passage cell density for the first shake flask is 4×10 6 cells / mL. In some embodiments, the target passage cell density for the first shake flask is 5×10 6 cells / mL. In some embodiments, the target passage cell density for the first shake flask is 6×10 6 cells / mL.

[0054] In some embodiments, after the inoculated cells achieve the target passage cell density in the second shake flask, the host cells are passaged into the third shake flask at the target seeding cell density. In some embodiments, the third shake flask has a volume of 1 L. In some embodiments, the target seeding cell density for the third shake flask is about 2×10 5 cells / mL - 5×10 5 cells / mL, for example, 3×10 5 cells / mL - 5×10 5 cells / mL or about 3×10 5 cells / mL - 5×10 5 cells / mL. In some embodiments, the target seeding cell density for the third shake flask is 5×10 5 cells / mL or more. In some embodiments, the target seeding cell density for the third shake flask is about 5×10 5 cells / mL. In some embodiments, the target passage cell density of the second shake flask is about 3.5 - 6×10 6 cells / mL. In some embodiments, the target passage cell density for the second shake flask is 4×10 6 cells / mL. In some embodiments, the target passage cell density for the second shake flask is 5×10 6cells / mL. In some embodiments, the target passage cell density for the second shake flask is 6×10 6 cells / mL.

[0055] In some embodiments, after the inoculated cells achieve the target passage cell density in the third shake flask, the host cells are passaged into the fourth shake flask at the target inoculation cell density. In some embodiments, the fourth shake flask has a volume of 1.6 L. In some embodiments, the target inoculation cell density for the fourth shake flask is from about 2×10 5 cells / mL to 5×10 5 cells / mL, for example, from 3×10 5 cells / mL to 5×10 5 cells / mL or from about 3×10 5 cells / mL to 5×10 5 cells / mL. In some embodiments, the target inoculation cell density for the fourth shake flask is 5×10 5 cells / mL or more. In some embodiments, the target inoculation cell density for the fourth shake flask is about 5×10 5 cells / mL. In some embodiments, the target passage cell density of the third shake flask is about 3.5 - 6×10 6 cells / mL. In some embodiments, the target passage cell density for the third shake flask is 4×10 6 cells / mL. In some embodiments, the target passage cell density for the third shake flask is 5×10 6 cells / mL. In some embodiments, the target passage cell density for the third shake flask is 6×10 6 cells / mL.

[0056] In some embodiments, after the inoculated cells achieve the target passage cell density in the fourth shake flask, the host cells are passaged into the fifth shake flask at the target inoculation cell density. In some embodiments, the fifth shake flask has a volume of 5 L. In some embodiments, the target inoculation cell density for the fifth shake flask is from about 2×10 5 cells / mL to 5×10 5 cells / mL, for example, from 3×10 5 cells / mL to 5×105 cells / mL or about 3×10 5 cells / mL to 5×10 5 cells / mL. In some embodiments, the target seeding cell density for the fifth shake flask is 5×10 5 cells / mL or more. In some embodiments, the target seeding cell density for the fifth shake flask is about 5×10 5 cells / mL. In some embodiments, the target subculture cell density of the fourth shake flask is about 3.5 to 6×10 6 cells / mL. In some embodiments, the target subculture cell density for the fourth shake flask is 4×10 6 cells / mL. In some embodiments, the target subculture cell density for the fourth shake flask is 5×10 6 cells / mL. In some embodiments, the target subculture cell density for the fourth shake flask is 6×10 6 cells / mL.

[0057] In some embodiments, when the host cells in the fifth shake flask achieve the target subculture cell density, the seed train scale-up growth is completed. In some embodiments, the target subculture cell density of the fifth shake flask is about 3.5 to 6×10 6 cells / mL. In some embodiments, the target subculture cell density for the fifth shake flask is 4×10 6 cells / mL. In some embodiments, the target subculture cell density for the fifth shake flask is 5×10 6 cells / mL. In some embodiments, the target subculture cell density for the fifth shake flask is 6×10 6 cells / mL. In some embodiments, the target subculture cell density of the fifth shake flask is about 6 to 7×10 9 cells / mL. In some embodiments, when the seed train scale-up growth is completed, the host cells are subcultured into the bioreactor at the target seeding cell density. In some embodiments, the target seeding cell density for the bioreactor is 5×10 5 cells / mL or more. In some embodiments, the target seeding cell density for the bioreactor is about 2×10 5 cells / mL to 5×10 5cells / mL, for example, 3×10 5 cells / mL to 5×10 5 cells / mL or about 3×10 5 cells / mL to 5×10 5 cells / mL. In some embodiments, the target seeding cell density for the second shake flask is about 5×10 5 cells / mL.

[0058] In some embodiments, the bioreactor can accommodate a volume of 3L to 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 bioreactor has a volume of about 20mL to about 1500mL, for example, 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 bioreactor has a volume of about 40mL to about 200mL. In some embodiments, the bioreactor has a volume of 40mL or about 40mL. In some embodiments, the bioreactor has a volume of 50mL or about 50mL. In some embodiments, the bioreactor has a volume of 100mL or about 100mL. In some embodiments, the bioreactor has a volume of 200mL or about 200mL.

[0059] In some embodiments, the host cells are inoculated and grown under the control of dissolved oxygen and pH in each of one or more bioreactors. In some embodiments, the host cells are inoculated into the first bioreactor and grown under the control of dissolved oxygen and pH. In some embodiments, the host cells are transferred from the first bioreactor to the second bioreactor and grown under the control of dissolved oxygen and pH. In some embodiments, the bioreactor is suitable for transfection.

[0060] 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) each having an increasing volume until a sufficient number of cells for bioreactor inoculation is achieved. In some embodiments, a sufficient number of cells for bioreactor inoculation is 6 - 7×10 9 cells. In some embodiments, a sufficient cell density for bioreactor inoculation is 3.5 - 6×10 6 cells / mL.

[0061] In some embodiments, seed train expansion begins at 5×10 5 cells / mL or greater. In some embodiments, cells are seeded into a 125 mL flask. In some embodiments, cells are seeded into a 500 mL shake flask. In some embodiments, cells are seeded into a 1 L shake flask. In some embodiments, cells are seeded into a 1.6 L shake flask. In some embodiments, cells are seeded into a 5 L shake flask.

[0062] In some embodiments, after seed train expansion has been carried out up to a 5 L shake flask, the bioreactor is inoculated.

[0063] In some embodiments, a series of target cell numbers is achieved from culturing in each of a plurality of containers. In some embodiments, different target cell numbers correspond to containers of different sizes.

[0064] In some embodiments, a series of target cell densities is achieved from culturing in each of a plurality of containers. In some embodiments, different target cell densities correspond to containers of different sizes.

[0065] In some embodiments, in a bioreactor of a scale desirable for expansion, about 4×10 6 cells / mL - 6×10 6Expansion is continued in one or more bioreactors until a final target cell density of cells / mL is achieved. In some embodiments, the bioreactor has a size of 40 mL to 500 mL, such as 40 mL to 200 mL or about 40 mL to 200 mL. In some embodiments, the bioreactor is 40 mL or about 40 mL in size. In some embodiments, the bioreactor is 50 mL or about 50 mL in size. In some embodiments, the bioreactor is 100 mL or about 100 mL in size. In some embodiments, the bioreactor is 200 mL or about 200 mL in size.

[0066] In some embodiments, seed expansion begins at a first target cell density of 5×10 5 cells / mL or about 5×10 5 cells / mL. In some embodiments, the second target cell density is 4 - 6×10 6 cells / mL. In some embodiments, the second target cell density is achieved in a 125 - 500 mL vessel. In some embodiments, the third target cell number is 6 - 7×10 9 cells. In some embodiments, the third target cell number is achieved at a density of 5×10 5 cells / mL or about 5×10 5 cells / mL. In some embodiments, the third target cell number is achieved in a 1 - 5 L vessel. In some embodiments, the third target cell number is suitable for inoculation into a vessel of about 50 L, optionally where the vessel is a bioreactor. In some embodiments, the bioreactor is a first bioreactor and the cells are transferred to a second bioreactor for full-scale expansion until a final target cell density is achieved. In some embodiments, when the final target cell density is achieved, the host cells are transfected with the plasmid.

[0067] In some embodiments, the host cells are inoculated and grown in a first bioreactor under the control of dissolved oxygen and pH. In some embodiments, the first bioreactor is about 50 L. In some embodiments, the target cell density in the first bioreactor is 4 - 6×10 6 cells / mL.

[0068] In some embodiments, after reaching the target cell density 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 at subculture is 1 - 5×10 5 cells / mL. In some embodiments, the host cells grow in the second bioreactor until the final target cell density is reached. In some embodiments, the final target cell density in the second bioreactor is 4 - 6×10 6 cells / mL. In some embodiments, the final target cell density in the second bioreactor is about 5×10 6 cells / mL. In some embodiments, the final target cell density in the second bioreactor is suitable for transfection.

[0069] In some embodiments, the seed expansion is started at a first cell density of about 5×10 5 cells / mL in a first container. In some embodiments, the cells are cultured in the first container until a second target cell density of 4 - 6×10 6 cells / mL is achieved. In some embodiments, the cells from the first container are transferred to a second container, and the cells are 6 - 7×10 9Cells are cultured in a second container until expanded growth to a target number of cells for bioreactor inoculation at cells / mL. In some embodiments, each of the first and second containers is a container of increasing size. In some embodiments, the first and second containers are shake flasks. In some embodiments, the first container (e.g., shake flask) has a volume of 125 - 500 mL. In some embodiments, the second container (e.g., shake flask) has a volume of 1 - 5 L. In some embodiments, the first shake flask holds a volume of 125 mL. In some embodiments, the first shake flask holds a volume of 500 mL. In some embodiments, the second shake flask holds a volume of 1 L. In some embodiments, the second shake flask holds a volume of 1.6 L. In some embodiments, the second shake flask holds a volume of 5 L.

[0070] In some embodiments, the target number of cells expanded from seed train expansion in a shake flask is used to inoculate a bioreactor. In some embodiments, cells from the target number of cells from the second container are inoculated into a first bioreactor, and the cells are cultured in the bioreactor until a target cell density in the range of 4 - 6×10 6 cells / mL is achieved. In some embodiments, cells from the first bioreactor are inoculated at scale into a second bioreactor, and the cells are cultured in the second bioreactor until a final target cell density in the range of 4 - 7×10 6 cells / mL is achieved. In some embodiments, the final target cell density in the second bioreactor is about 5×10 6cells / mL. In some embodiments, the first bioreactor has a smaller volume than the second bioreactor. In some embodiments, the first bioreactor has a volume of 3 to 50 L. In some embodiments, the second bioreactor has a volume of 40 to 200 L. In some embodiments, the first bioreactor accommodates a volume of 3 L. In some embodiments, the first bioreactor accommodates a volume of 10 L. In some embodiments, the first bioreactor accommodates a volume of 50 L. In some embodiments, the first bioreactor accommodates a volume of 40 L. In some embodiments, in some embodiments, the second bioreactor accommodates a volume of 200 L.

[0071] In some embodiments, when the final target cell density is achieved, the host cell is transfected with the plasmid.

[0072] 2. Plasmid Transfection In some embodiments, the methods described herein include the step of transfecting a host cell with at least one plasmid to generate virus particles. In some embodiments, the methods described herein include the step of contacting a population of host cells with at least one plasmid encoding virus particles for a time sufficient to produce virus particles. In some embodiments, the step of contacting a population of host cells with at least one plasmid encoding a viral protein is performed by transfection.

[0073] In some embodiments, transfection is performed in a bioreactor after the target transfection density is reached. In some embodiments, the target transfection density is the final target density achieved by the seed expansion method for the expansion of the host cells described above. Optionally, fresh medium may 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 10×10 6cells / mL. In some embodiments, the target transfection density is about 1 to about 3×10 6 cells / mL. In some embodiments, the target transfection density is about 3 to 6×10 6 cells / mL. In some embodiments, the target transfection density is about 4 to about 7×10 6 cells / mL. In some embodiments, the target transfection density is about 1×10 6 cells / mL, about 2×10 6 cells / mL, about 3×10 6 cells / mL, about 4×10 6 cells / mL, about 5×10 6 cells / mL or about 6×10 6 cells / mL, or any value in between any of the foregoing. In some embodiments, the target transfection density is 4×10 6 cells / mL.

[0074] In some embodiments, the target transfection density is determined by or depends on the host cell. In some cases, a particular host cell has higher productivity. For example, HEK293T cells can exhibit higher productivity than HEK293 cells, and as a result, the target transfection density when using HEK293T cells can be more than 3-fold lower, for example 2- to 3-fold lower, than the target transfection density when using HEK293 cells. The ability to achieve the desired yield while using a lower cell density improves the process by providing a lower residual impurity load to downstream steps.

[0075] In some embodiments, the host cell is HEK293T cells. In some embodiments, the target transfection density is about 1 to about 3×10 6 cells / mL. In some embodiments, the target transfection density is about 1×10 6 cells / mL. In some embodiments, the target transfection density is about 2×10 6 cells / mL. In some embodiments, the target transfection density is about 3×10 6cells / mL.

[0076] In some embodiments, the host cell is a HEK293 cell. In some embodiments, the target transfection density is about 3 - 6×10 6 cells / mL. In some embodiments, the target transfection density is about 4×10 6 cells / mL. In some embodiments, the target transfection density is about 5×10 6 cells / mL. In some embodiments, the target transfection density is about 6×10 6 cells / mL.

[0077] In some embodiments, the contents of the bioreactor are diluted with fresh medium to reach the target transfection density. In some embodiments, the target transfection density is 4×10 6 cells / mL. In some embodiments, a bolus of glucose is added to the bioreactor with fresh medium to the target glucose level. In some embodiments, glucose is supplemented in the medium for transfection. In some embodiments, the target glucose level in the medium for transfection is 1 g / L - 10 g / L, for example, 2.5 g / L - 7.5 g / L. In some embodiments, the target glucose level is 5.5 g / L or about 5.5 g / L. In some embodiments, glucose at a concentration up to 5.5 g / L is supplemented.

[0078] In some embodiments, transfection is performed immediately after reaching the target transfection density. The transfection method may be performed using methods well known in the art. For example, the transfection process may be performed using commercially available formulations such as calcium phosphate or Lipofectamine™ 2000CD (Invitrogen, CA) or polyethyleneimine (PEI).

[0079] Commercially available equipment and reagents may be used for transfection. In some embodiments, the equipment and reagents used for transfection are specific to large-scale virus production. Illustrative examples of such commercially available reagents include PEIpro® (Polyplus-transfection®).

[0080] In some embodiments, at least one plasmid used for transfection comprises non-coding nucleic acid. In some embodiments, the non-coding nucleic acid is siRNA, miRNA, or shRNA.

[0081] Exemplary genes of interest include any payload described in Section II.A.2.b.v. In some embodiments, at least one plasmid used for transfection comprises a polynucleotide encoding a polypeptide or gene of interest (e.g., also referred to as a payload gene). In some embodiments, at least one plasmid comprising a polynucleotide encoding a gene or polypeptide of interest is an introduction plasmid. In some embodiments, the gene of interest is not expressed by the host cell and the introduction plasmid is incorporated into viral particles as nucleic acid. In some embodiments, the polypeptide of interest is a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is specific for a tumor-associated antigen. 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 fluorescein and its derivatives, including FITC (fluorescein isothiocyanate), NHS-fluorescein, and pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives, together with DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin, and digoxigenin, notchins, centyrins, and DARPins.

[0082] 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 coxsackievirus G protein. In some embodiments, the viral envelope protein is a coxsackievirus G protein.

[0083] In some embodiments, the coxsackievirus 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 coxsackievirus 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 coxsackievirus 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 coxsackievirus 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 coxsackievirus 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 coxsackievirus 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 coxsackievirus 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 coxsackievirus 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 coxsackievirus 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 coxsackievirus 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 coxsackievirus G envelope protein comprises the amino acid sequence of SEQ ID NO:124. In some embodiments, the coxsackievirus G envelope protein consists of the amino acid sequence of SEQ ID NO:124.

[0084] 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 a nucleotide consisting of the nucleotide sequence of SEQ ID NO: 125.

[0085] In some embodiments, the cocalvirus 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 cocalvirus 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 cocalvirus 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 cocalvirus 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 cocalvirus 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 cocalvirus 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 cocalvirus 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 cocalvirus 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 cocalvirus 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 cocalvirus 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 cocalvirus G envelope protein is encoded by a nucleotide that comprises 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.

[0086] The envelope expression cassette can include one of several envelopes such as VSV-G or various murine retroviral envelopes such as 4070A. In some embodiments, the viral envelope protein is a fusion protein with an immune cell activating protein. The immune activating 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 activating protein contains at least one binding domain that binds to a primary T cell receptor (e.g., CD3), a co-stimulatory molecule, or an adhesion molecule. In some embodiments, the viral envelope protein includes a binding domain that binds to at least one co-stimulatory molecule.

[0087] In some embodiments, at least one plasmid used for transfection encodes a protein that binds to 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 of the co-stimulatory molecules described in Section II.A.1.b.

[0088] In some embodiments, at least one plasmid used for transfection encodes an immune cell activating protein. In some embodiments, the immune cell activating 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, TCRα chain, TCRβ chain, TCRζ chain, TCRγ chain, TCRδ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, or NKp80. In some embodiments, the co-stimulatory molecule is any of the co-stimulatory molecules described in Section II.A.1.a.

[0089] In some embodiments, at least one plasmid used for transfection encodes a protein that binds to an 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, the adhesion molecule is any of the adhesion molecules described in Section II.A.1.c.

[0090] In some embodiments, the host cell is engineered to improve safety, in which case the 3' LTR of the provirus is deleted. In such cells, two recombination events would be required to produce wild-type virus. In some embodiments, further safety improvements involve introducing the gag-pol gene and the env gene on separate constructs. These constructs can be introduced sequentially to prevent recombination during transfection. In these split construct cell lines, further reduction in recombination can be achieved by exchanging codons. This technique, based on the redundancy of the genetic code, aims to reduce homology between separate constructs, for example, between overlapping regions in the gag-pol and env open reading frames.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] In some embodiments, the rev 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:128. In some embodiments, the rev protein is encoded by 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 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 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 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 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 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 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 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 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 that comprises 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.

[0097] In some embodiments, a mixture of plasmids is used for transfection. In some embodiments, the mixture of plasmids includes a plasmid encoding a gene of interest; a plasmid encoding a rev viral protein; and a plasmid encoding a gagpol viral protein.

[0098] In some embodiments, the mixture of plasmids includes 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.

[0099] In some embodiments, the mixture of plasmids includes 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 immunocyte activation protein.

[0100] In some embodiments, the mixture of plasmids includes 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.

[0101] In some embodiments, the mixture of plasmids includes 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 costimulatory molecule.

[0102] In some embodiments, the mixture of plasmids 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 activating protein.

[0103] In some embodiments, the mixture of plasmids 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 activating protein; and a plasmid encoding a costimulatory molecule.

[0104] In some embodiments, the mixture of plasmids 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 costimulatory molecule.

[0105] In some embodiments, the mixture of plasmids 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 activating protein; and a plasmid encoding a costimulatory molecule.

[0106] In some embodiments, the 5-plasmid system is used for transfection. In some embodiments, the 5-plasmid system comprises a plasmid expressing a gene of interest, a plasmid expressing the lentiviral gagpol gene, a plasmid expressing the lentiviral rev gene, a plasmid expressing a viral envelope protein, and a plasmid expressing an immune activating protein.

[0107] In some embodiments, the plasmid system is used for transfection with more than five plasmids. In some embodiments, the plasmid system includes a plasmid expressing a gene of interest, a plasmid expressing the lentiviral gagpol gene, a plasmid expressing the lentiviral rev gene, a plasmid expressing a viral envelope protein, a plasmid expressing an immune activating protein, and at least one additional plasmid expressing a protein for incorporation into the viral envelope (e.g., a costimulatory molecule or other viral particle surface molecule described herein).

[0108] In some embodiments, transient transfection is used to generate the viral production cells of the present disclosure. Transient transfection can be used when the vector genome or lentiviral packaging components are toxic to the cells, as it avoids the longer time required to generate a stable vector production cell line. In the process of using transiently transfected cells, any agent that enables plasmid transfection can be used. Other agents may be contemplated by those skilled in the art, but illustrative examples include calcium phosphate or polyethyleneimine (Ansorge et al. 2010). The conditions (e.g., number of plasmids, ratio between plasmids, ratio of plasmid to transfection agent, type of medium, etc.) and transfection time can be adapted by those skilled in the art depending on the characteristics of the virus produced and / or the transgenes introduced into the introduced plasmids.

[0109] 3. Recovery of Viral Particles In some embodiments, cells transfected with a viral vector (also referred to herein as vector-producing cells) are cultured to increase the number of cells and virus and / or the viral titer. 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 in an appropriate medium. The methods can include growth attached to a surface, growth in suspension, or a combination thereof.

[0110] In some embodiments, the culturing step is performed in a tissue culture flask, dish, roller bottle, or in a bioreactor using a batch, fed-batch, continuous system, hollow fiber, or the like to achieve large-scale production of the virus by cell culture. In some embodiments, the vector-producing cells can be grown in suspension. Conditions suitable for culturing the cells are known to those skilled in the art (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and R.I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9)).

[0111] In some embodiments, the vector-producing cells are cultured to a target density suitable for harvest. In some embodiments, harvest occurs 40 - 50 hours after transfection. In some embodiments, harvest occurs about 48 hours after transfection.

[0112] In some embodiments, the endonuclease is added to the vector-producing cells before harvest. In some embodiments, the endonuclease is added to the vector-producing cells 1 to 5 hours before harvest. In some embodiments, the endonuclease is added to the vector-producing cells approximately 2 hours before harvest. In some embodiments, the endonuclease is a nuclease. In some embodiments, the endonuclease is a salt-activated nuclease. Various endonucleases are known and can be used. In some embodiments, the endonuclease is benzonase. In some embodiments, the endonuclease is denarase.

[0113] In some embodiments, the endonuclease is added to the bioreactor to achieve a concentration of 1 U / mL to 100 U / mL, such as 1 U / mL to 50 U / mL, 1 U / mL to 20 U / mL, 1 U / mL to 10 U / mL, 10 U / mL to 100 U / mL, 10 U / mL to 50 U / mL, 10 U / mL to 20 U / mL, 20 U / mL to 100 U / mL, 20 U / mL to 50 U / mL, or 50 U / mL to 100 U / mL. In some embodiments, the concentration is 5 U / mL to 20 U / mL. In some embodiments, the concentration is 20 U / mL.

[0114] In some embodiments, the vector-producing cells are supplemented with MgCl2 before harvest. In some embodiments, the vector-producing cells are supplemented with MgCl2 1 to 5 hours before harvest. In some embodiments, the vector-producing cells are supplemented with MgCl2 approximately 2 hours before harvest. In some embodiments, MgCl2 is added to the bioreactor to achieve a concentration of 0.5 mM to 5 mM, such as 0.5 mM to 4 mM, 0.5 mM to 2 mM, 0.5 mM to 1 mM, 1 mM to 5 mM, 1 mM to 4 mM, 1 mM to 2 mM, 2 mM to 5 mM, 2 mM to 4 mM, or 4 mM to 5 mM. In some embodiments, the concentration is 0.5 mM to 4 mM. In some embodiments, the concentration is 2 mM.

[0115] In some embodiments, the recovered cell culture may also be referred to as a suspension mixture. In some embodiments, the suspension mixture has a volume of about 3 to 50 liters. In some embodiments, the suspension mixture has a volume of about 50 to 500 liters. In some embodiments, the suspension mixture has a volume of about 200 liters.

[0116] In some embodiments, the upstream process produces a suspension mixture having a recovered titer of infectious units that includes from 0.5×10 5 TU / mL to 5×10 6 TU / mL. In some embodiments, the titer of infectious units is from 1×10 5 TU / mL to 3×10 5 TU / mL, from 2×10 5 TU / mL to 4×10 5 TU / mL, from 3×10 5 TU / mL to 5×10 5 TU / mL, from 4×10 5 TU / mL to 6×10 5 TU / mL, from 5×10 5 TU / mL to 7×10 5 TU / mL, from 6×10 5 TU / mL to 8×10 5 TU / mL, from 7×10 5 TU / mL to 9×10 5 TU / mL, from 8×10 5 TU / mL to 1×10 6 TU / mL, from 9×10 5 TU / mL to 2×10 6 TU / mL, from 1×10 6 TU / mL to 3×10 6 TU / mL, from 2×10 6 TU / mL to 4×10 6 TU / mL, from 3×10 6 TU / mL to 5×10 6 TU / mL. In some embodiments, the titer of infectious units is 2×10 6 TU / mL to 4×10 6 TU / mL.

[0117] In some embodiments, the titer of infectious units is at least about 2×10 6It contains TU / mL. In some embodiments, the infectivity titer is at least about 3×10 6 It contains TU / mL. In some embodiments, the infectivity titer is at least about 4×10 6 It contains TU / mL. In some embodiments, the infectivity titer is 2×10 6 TU / mL. In some embodiments, the infectivity titer is 3×10 6 TU / mL. In some embodiments, the infectivity titer is 4×10 6 TU / mL.

[0118] In some embodiments, the suspension mixture is about 3L to 200L. In some embodiments, the suspension mixture contains a volume of about 1 - 10L, 5 - 15L, 10 - 20L, 15 - 25L, 20 - 30L, 25 - 35L, 30 - 40L, 35 - 45L, 40 - 50L, 45 - 55L, 50 - 60L, 55 - 65L, 60 - 70L, 65 - 75L, 70 - 80L, 75 - 85L, 80 - 90L, 85 - 95L, 90 - 100L, 95 - 105L or 100 - 200L. In some embodiments, the suspension mixture contains at least 3L, 10L, 40L, 50L, 100L or 200L, or any value between any of the foregoing. In some embodiments, the suspension mixture contains at least 3L, 10L, 40L, 50L, 100L or 200L. In some embodiments, the suspension mixture is about 40L. In some embodiments, the suspension mixture is about 200L. In some embodiments, the suspension mixture is about 160L - 200L, for example, 160L, 170L, 180L, 190L or 200L, or about 160L, 170L, 180L, 190L or 200L, or any value between any of the foregoing.

[0119] In some embodiments, the upstream process produces a suspension mixture having a recovered product with an infectivity titer of 2×10 9 TU to 2×10 11 TU. In some embodiments, 4×10 9 TU to 12×10 9 TU, 8×10 9 TU to 16×10 9 TU, 12×10 9 TU to 2×1010 TU, 16×10 9 TU ~ 2.4×10 10 TU, 2×10 10 TU ~ 2.8×10 10 TU, 2.4×10 10 TU ~ 3.2×10 10 TU, 2.8×10 10 TU ~ 3.6×10 10 TU, 3.2×10 10 TU ~ 4×10 10 TU, 3.6×10 10 TU ~ 8×10 10 TU, 4×10 10 TU ~ 12×10 10 TU, 8×10 10 TU ~ 16×10 10 TU, 12×10 10 TU ~ 2×10 11 The infectivity titer of TU. In some embodiments, the upstream process is 8×10 10 TU ~ 16×10 10 produces a suspension mixture having a recovered product of the infectivity titer of TU.

[0120] In some embodiments, the infectivity titer is at least about 8×10 10 TU. In some embodiments, the infectivity titer is at least about 12×10 10 TU. In some embodiments, the infectivity titer is at least about 16×10 10 TU. In some embodiments, the infectivity titer is 8×10 10 TU. In some embodiments, the infectivity titer is 12×10 10 TU. In some embodiments, the infectivity titer is 16×10 10 TU.

[0121] In some embodiments, the upstream process is 1×10 10 TU ~ 1×10 12 produces a suspension mixture having a recovered product of the infectivity titer of TU. In some embodiments, the infectivity titer is 2×10 10 TU ~ 6×10 10 TU, 4×10 10 TU ~ 8×10 10 TU, 6×1010 TU ~ 10×10 10 TU, 8×10 10 TU ~ 12×10 10 TU, 10×10 10 TU ~ 14×10 10 TU, 12×10 10 TU ~ 16×10 10 TU, 14×10 10 TU ~ 18×10 10 TU, 16×10 10 TU ~ 2×10 11 TU, 18×10 10 TU ~ 4×10 11 TU, 2×10 11 TU ~ 6×10 11 TU, 4×10 11 TU ~ 8×10 11 TU, 6×10 11 TU ~ 10×10 11 It is TU. In some embodiments, the upstream process is 4×10 11 TU ~ 8×10 11 to produce a suspension mixture having a recovered product of the infectivity titer of TU.

[0122] In some embodiments, the infectivity titer is at least about 4×10 11 including TU. In some embodiments, the infectivity titer is at least about 6×10 11 including TU. In some embodiments, the infectivity titer is at least about 8×10 11 including TU. In some embodiments, the infectivity titer is about 4×10 11 TU. In some embodiments, the infectivity titer is about 6×10 11 TU. In some embodiments, the infectivity titer is about 8×10 11 TU.

[0123] B. Downstream process In some embodiments, the present disclosure provides a method for preparing a viral particle formulation.

[0124] In some embodiments, the method for preparing a viral particle preparation includes the introduction of a nuclease, clarification of the harvest, concentration and purification of the harvest, and formulation and concentration of the harvest, which is referred to herein as the downstream process. The goal of the downstream process is to increase the yield of viral particles and improve the purity of the viral particles. In some embodiments, the downstream process includes the downstream process shown in FIG. 1B.

[0125] In some embodiments, the present disclosure provides a method for preparing a viral particle preparation from a suspension mixture of a population of host cells and viral particles. In some embodiments, the suspension mixture is prepared from the upstream process described in the foregoing section. In some embodiments, preparing the viral particle preparation comprises (i) clarifying a mixture of host cells and viral particles to produce a filtered preparation of viral particles, and (ii) concentrating the filtered preparation to produce a concentrated preparation of viral particles. In some embodiments, the method comprises (iii) sterile filtering the concentrated preparation of viral particles to produce the viral particle preparation. In some embodiments, the viral particle preparation is suitable for in vivo administration. In some embodiments, the process of preparing the viral particle preparation purifies the viral vector to remove impurities.

[0126] In some embodiments, the present disclosure provides a method for preparing a viral particle preparation comprising (i) a step of filtering a suspension mixture comprising a population of host cells and viral particles to remove contaminants, comprising (a) filtering the mixture through a first filter that is a depth filter to result in a first filtrate, (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter to result in a second filtrate, and (c) filtering the second filtrate through a third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of viral particles; and (ii) a step of concentrating the filtered preparation of viral particles, comprising chromatography and ultrafiltration.

[0127] In some embodiments, the present disclosure provides a method for preparing a retroviral particle formulation, comprising: (i) a step of filtering a suspension mixture comprising a population of host cells and retroviral particles to remove contaminants, the step comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (c) filtering the second filtrate through a third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered retroviral particle formulation; and (ii) a step of concentrating the filtered retroviral particle formulation, the step comprising chromatography and ultrafiltration.

[0128] In some embodiments, the present disclosure provides a method for preparing a retroviral (e.g., lentiviral) particle formulation, comprising: (i) a step of filtering a suspension mixture comprising a population of host cells and lentiviral particles to remove contaminants, the step comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (c) filtering the second filtrate through a third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered lentiviral particle formulation; and (ii) a step of concentrating the filtered lentiviral particle formulation, the step comprising chromatography and ultrafiltration.

[0129] 1. Clarification of Bioreactor Recoveries In some embodiments, the methods described herein include filtering a mixture of a population of host cells 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.

[0130] In some embodiments, clarifying a suspension mixture of host cells and viral particles is performed to remove host cells, cell debris, and precipitated impurities from the suspension mixture in the bioreactor harvest while retaining viral vector particles. In some embodiments, clarification can be performed using a filter or by centrifugation. In some embodiments, centrifugation can include density gradient centrifugation, ultracentrifugation, and fractionation centrifugation. In some embodiments, clarifying or clarification includes centrifugation and depth filtration. In some embodiments, clarification is performed using depth filtration or membrane filtration. In some embodiments, one or more of the filters are hollow fiber filters.

[0131] In some embodiments, the clarification process step is performed using a series of filters having a decreasing pore size. In some embodiments, at least one filter is a depth filter that is useful for capturing contaminants within its structure and filtering a turbid process pool at a high throughput. In some embodiments, at least one filter is a membrane filter that typically traps contaminants larger than the pore size of the addressed surface of the membrane. In some embodiments, the processing step includes a series of filters where the first filter is a depth filter, followed by filtration using a membrane filter. In some embodiments, using a depth filter to reduce the total number of large particles in the pool enables an improvement in throughput through a membrane filter having a smaller pore size.

[0132] In some embodiments, the clarification process uses primary, secondary, and tertiary filtration.

[0133] In some embodiments, clarifying or clarification includes centrifugation. In some embodiments, centrifugation can include density gradient centrifugation, ultracentrifugation, and fractionation centrifugation. In some embodiments, clarifying or clarification includes centrifugation and depth filtration.

[0134] In some embodiments, the step of filtering the mixture includes (a) filtering the mixture through a first filter that is a depth filter to result in a first filtrate, (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter to result in a second filtrate, and (c) filtering the second filtrate through a third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of viral particles.

[0135] In some embodiments, the method includes the step of filtering a mixture of a population of host cells and retroviral particles. In some embodiments, the step of filtering includes (a) filtering the mixture through a first filter that is a depth filter to result in a first filtrate, (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter to result in a second filtrate, and (c) filtering the second filtrate through a third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of retroviral particles.

[0136] In some embodiments, the method includes filtering a mixture of a population of host cells and lentiviral particles. In some embodiments, the filtering step comprises: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (c) filtering the second filtrate through a third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of lentiviral particles.

[0137] In some embodiments, the first filter has a retention threshold of about 1 to 60 μm, about 5 to 60 μm, about 10 to 60 μm, about 15 to 60 μm, about 20 to 60 μm, about 25 to 60 μm, about 30 to 60 μm, about 35 to 60 μm, about 40 to 60 μm, about 45 to 60 μm, about 50 to 60 μm, about 55 to 60 μm, or greater than 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.

[0138] In some embodiments, the first filter is a depth filter. In some embodiments, the first filter is a commercially available depth filter. Exemplary commercially available first filters include, but are not limited to, the Clarisolve® depth filter (Millipore®).

[0139] In some embodiments, the second filter has a retention threshold of about 0.2 to 4 μm, about 0.3 to 4 μm, about 0.4 to 4 μm, about 0.5 to 4 μm, about 0.6 to 4 μm, about 0.7 to 4 μm, about 0.8 to 4 μm, about 0.9 to 4 μm, about 1 to 4 μm, about 2 to 4 μm, or about 3 to 4 μm. In some embodiments, the second filter has a retention threshold of about 0.4 to 3 μm, about 0.4 to 2 μm, about 0.4 to 1 μm, about 0.4 to 0.9 μm, about 0.4 to 0.8 μm, about 0.4 to 0.7 μm, about 0.4 to 0.6 μm, or about 0.4 to 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.

[0140] In some embodiments, the second filter is a commercially available filter. Illustrative examples of commercially available second filters include, but are not limited to, the Sartopure® PP3 filter (Sartorius).

[0141] In some embodiments, the third filter has a retention threshold of about 0.2 to 1 μm, about 0.2 to 0.9 μm, about 0.2 to 0.8 μm, about 0.2 to 0.7 μm, about 0.2 to 0.6 μm, about 0.2 to 0.5 μm, about 0.2 to 0.4 μm, or about 0.2 to 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.

[0142] In some embodiments, the third filter is a commercially available filter. Illustrative examples of commercially available third filters include, but are not limited to, the Sartopure® 2HF filter (Sartorius).

[0143] 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.

[0144] In some embodiments, the second filter and the third filter are two layers within a two - layer filter component. In some embodiments, the third filter is a two - layer filter that includes a first - layer filter and a second - layer filter. In some embodiments, each layer of the two - layer filter component has a different retention threshold. In some embodiments, the first layer has a retention threshold of 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 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 first layer has a retention threshold of about 0.45 μm and the second layer has a retention threshold of about 0.2 μm.

[0145] In some embodiments, the two - layer filter component is commercially available. Illustrative examples of commercially available two - layer filter components include, but are not limited to, Sartopure® 2 (pore size: 0.45|0.2 μm), Sartopure® 2XLG (pore size: 0.8|0.2 μm), and Sartopure® 2XLI (pore size: 0.35|0.2 μm).

[0146] In some embodiments, the present disclosure provides a method for preparing a viral particle formulation, comprising the steps of: (i) filtering a suspension mixture comprising a population of host cells and viral particles to remove contaminants, comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a two-layer filter component comprising a second filter and a third filter, wherein the second filter has a retention threshold smaller than that of the first filter and the third filter has a retention threshold smaller than that of the second filter, thereby producing a filtered formulation of viral particles.

[0147] In some embodiments, the present disclosure provides a method for preparing a retroviral particle formulation, comprising the steps of: (i) filtering a suspension mixture comprising a population of host cells and retroviral particles to remove contaminants, comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a two-layer filter component comprising a second filter and a third filter, wherein the second filter has a retention threshold smaller than that of the first filter and the third filter has a retention threshold smaller than that of the second filter, thereby producing a filtered formulation of retroviral particles.

[0148] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, comprising the steps of: (i) filtering a suspension mixture comprising a population of host cells and lentiviral particles to remove contaminants, comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a two-layer filter component comprising a second filter and a third filter, wherein the second filter has a retention threshold smaller than that of the first filter and the third filter has a retention threshold smaller than that of the second filter, thereby producing a filtered formulation of lentiviral particles.

[0149] In some embodiments, the present disclosure provides a method for preparing a viral particle formulation, comprising the steps of: (i) filtering a suspension mixture comprising a population of host cells and viral particles to remove contaminants, comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; (c) filtering the second filtrate through a two-layer filter component comprising the second filter and a third filter, wherein the third filter has a retention threshold smaller than that of the second filter, thereby producing a filtered formulation of viral particles.

[0150] In some embodiments, the present disclosure provides a method for preparing a retroviral particle formulation, comprising the steps of: (i) filtering a suspension mixture comprising a population of host cells and retroviral particles to remove contaminants, comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; (c) filtering the second filtrate through a two-layer filter component comprising the second filter and a third filter, wherein the third filter has a retention threshold smaller than that of the second filter, thereby producing a filtered formulation of retroviral particles.

[0151] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, comprising the steps of: (i) filtering a suspension mixture comprising a population of host cells and lentiviral particles to remove contaminants, including (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (c) filtering the second filtrate through a two-layer filter component comprising the second filter and a third filter, wherein the third filter has a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of lentiviral particles.

[0152] In some embodiments, the present disclosure provides a method for preparing a viral particle formulation, comprising the steps of: (i) filtering a suspension mixture comprising a population of host cells and viral particles to remove contaminants, including (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (c) filtering the second filtrate through a third filter that is a two-layer filter component comprising a first layer filter and a second layer filter, wherein the second layer filter has a retention threshold smaller than that of the first layer filter, thereby producing a filtered preparation of viral particles.

[0153] In some embodiments, the present disclosure provides a method for preparing a retroviral particle preparation, comprising the step of filtering a suspension mixture comprising a population of host cells and retroviral particles to remove contaminants, the step comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (c) filtering the second filtrate through a third filter, wherein the third filter is a two-layer filter component comprising a first layer filter and a second layer filter, and the second layer filter has a retention threshold smaller than that of the first layer filter, thereby producing a filtered preparation of retroviral particles.

[0154] In some embodiments, the present disclosure provides a method for preparing a lentiviral preparation, comprising the step of filtering a suspension mixture comprising a population of host cells and lentiviral particles to remove contaminants, the step comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (c) filtering the second filtrate through a third filter, wherein the third filter is a two-layer filter component comprising a first layer filter and a second layer filter, and the second layer filter has a retention threshold smaller than that of the first layer filter, thereby producing a filtered preparation of lentiviral particles.

[0155] In some embodiments, the present disclosure provides a method for preparing a viral particle formulation, comprising the steps of: (i) filtering a suspension mixture comprising a population of host cells and viral particles to remove contaminants, comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (c) filtering the second filtrate through a third filter, wherein the third filter is a two-layer filter component comprising a first layer filter and a second layer filter, the first layer filter having the same retention threshold as the second filter, and the second layer filter having a retention threshold smaller than that of the first layer filter, thereby producing a filtered formulation of viral particles.

[0156] In some embodiments, the present disclosure provides a method for preparing a retroviral particle formulation, comprising the steps of: (i) filtering a suspension mixture comprising a population of host cells and retroviral particles to remove contaminants, comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (c) filtering the second filtrate through a third filter, wherein the third filter is a two-layer filter component comprising a first layer filter and a second layer filter, the first layer filter having the same retention threshold as the second filter, and the second layer filter having a retention threshold smaller than that of the first layer filter, thereby producing a filtered formulation of retroviral particles.

[0157] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, comprising a step of filtering a suspension mixture comprising a population of host cells and lentiviral particles to remove contaminants, the step comprising: (a) filtering the mixture through a first filter which is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (c) filtering the second filtrate through a third filter, wherein the third filter is a two-layer filter component comprising a first layer filter and a second layer filter, the first layer filter having the same retention threshold as the second filter, and the second layer filter having a retention threshold smaller than that of the first layer filter, thereby producing a filtered preparation of lentiviral particles.

[0158] In some embodiments, one or more of the filters are hollow fiber filters (also described below as hollow fiber modules). In some embodiments, the hollow fiber filter has a nominal molecular weight cut-off (NMWC) of 100 kDa to 1000 kDa. In some embodiments, the NMWC is 100 kDa to 500 kDa. In some embodiments, the NMWC is 300 kDa to 750 kDa. In some embodiments, the NMWC is 500 kDa to 1000 kDa.

[0159] In some embodiments, the NMWC is 500 kDa. In some embodiments, the NMWC is 300 kDa. In some embodiments, the NMWC is 100 kDa.

[0160] In some embodiments, the hollow fiber filter has a pore size of 0.1 μm to 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.

[0161] In some embodiments, the hollow fiber filter has a length of 20 cm, 41.5 cm, 65 cm, 50 cm, 68 cm, or 108 cm. In some embodiments, the hollow fiber filter has an inner lumen of 0.50 mm, 0.63 mm, 0.75 mm, 1 mm, 2 mm, or 3 mm.

[0162] In some embodiments, the hollow fiber filter is 235 cm 2 ~1600 cm 2 in surface area. 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 .

[0163] In some embodiments, the hollow fiber filter accommodates a volume of 300 mL.

[0164] 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, the Spectrum® hollow fiber filter (Repligen).

[0165] In some embodiments, the filtering step is performed under aseptic conditions. In some embodiments, each of the filter steps is performed in a closed system. In some embodiments, one or more of the filters can be functionally connected in-line in a closed system. In some embodiments, the first filter is connected in-line with a bioreactor. In some embodiments, the second or third filter is connected in-line with the first filter, where all the filters are functionally connected. In some embodiments, the filtering step is within a closed system and is functionally connected in-line with a bioreactor, where the recovery of the suspension from the bioreactor is sent directly through the first filter and optionally further through the second and third filters, all in a closed system directly. In some embodiments, the closed system does not transport any substances from the system, for example, it does not expose substances to the atmosphere or the external environment.

[0166] In some embodiments, the endonuclease is added before filtering the suspension 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 formulation.

[0167] In some embodiments, the endonuclease is present throughout the step of filtering the mixture with the third filter and the step of concentrating the filtered formulation. In some embodiments, the endonuclease is present throughout the step of filtering the mixture with the second filter and the step of concentrating the filtered formulation. In some embodiments, the endonuclease is present throughout the step of filtering the mixture with the first filter and the step of concentrating the filtered formulation.

[0168] In some embodiments, residual host cell DNA (hcDNA) is measured after the filtration step. hcDNA is measured by qPCR and can be normalized to the amount of viral particles. In some embodiments, hcDNA is measured as units / dose, where one dose is defined using viral transduction units (TU). In some embodiments, hcDNA is measured as ng / dose, where one dose is 1E9 TU. The unit of 1E9 is the same as 1×10 9 the same as

[0169] In some embodiments, hcDNA is less than about 5000 ng / 1E9 TU, less than about 4500 ng / 1E9 TU, less than about 4000 ng / 1E9 TU, less than about 3500 ng / 1E9 TU, less than about 3000 ng / 1E9 TU, less than about 2500 ng / 1E9 TU, less than about 2000 ng / 1E9 TU, less than about 1500 ng / 1E9 TU, or less than about 1000 ng / 1E9 TU after the third filter. In some embodiments, hcDNA is less than about 2500 ng / 1E9 TU after the third filter. In some embodiments, hcDNA is undetectable after the third filter.

[0170] In some embodiments, 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 compared to hcDNA at harvest (in the suspension mixture). In some embodiments, hcDNA after the third filter is at least 80-fold lower compared to hcDNA at harvest.

[0171] In some embodiments, the hcDNA after the third filter is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, or at least 10-fold lower compared to the hcDNA after the second filter. In some embodiments, the hcDNA after the third filter is at least 5-fold lower compared to the hcDNA after the second filter.

[0172] 2. Chromatography In some embodiments, the methods of the disclosure include the step of concentrating a filtered preparation of viral particles. In some embodiments, the concentrating step can be by chromatography. In some embodiments, chromatography also acts to remove impurities, such as HCP and HcDNA, from the filtered preparation of particles. In some embodiments, the disclosed methods include a chromatography step following clarification of the suspension mixture by using the series of filters described above. In some embodiments, chromatography is used not only to further remove impurities, such as HCP and hcDNA, from the filtered preparation of particles, but also to concentrate the viral particles in the preparation.

[0173] Chromatography techniques well known to those of ordinary skill in the art can be used in the methods of the disclosure. These techniques can involve separating vector particles from the cellular environment and, if necessary, further purifying the vector particles. One or more of a variety of chromatography methods can be used for purification. In some embodiments, chromatography is performed after clarification of the recovered material.

[0174] In some embodiments, residual host cell protein (HCP) is measured after chromatography. HCP may be measured by ELISA and normalized against the amount of viral particles. In some embodiments, HCP is measured as units / dose, where 1 dose is defined using viral transduction units (TU) of the virus. In some embodiments, HCP is measured as μg / dose, where 1 dose is 1E9 TU.

[0175] In some embodiments, 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 after chromatography. In some embodiments, HCP is less than about 3000 μg / 1E9 TU after chromatography.

[0176] In some embodiments, HCP 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 after chromatography compared to before chromatography. In some embodiments, HCP is at least about 40-fold lower after chromatography compared to before chromatography.

[0177] Exemplary chromatography techniques are described below.

[0178] a. Ion exchange chromatography In some embodiments, chromatography includes ion exchange chromatography. Ion exchange chromatography utilizes the fact that a charged species, such as a biomolecule and a viral vector, can reversibly bind to a stationary phase (e.g., a membrane or a column packing) having groups of opposite charge fixed to its surface. There are two types of ion exchangers. An anion exchanger is a stationary phase having groups with a positive charge and thus can bind species with a negative charge. A cation exchanger has a negative charge and thus has groups that can bind species with a positive charge. The pH of the medium has an important influence on this because it can change the charge of the species. Thus, for a species such as a protein, when the pH exceeds the pI, the effective charge is negative, whereas when it is below the pI, the effective charge is positive.

[0179] The displacement (elution) of the bound species can be affected by the use of an appropriate buffer. Generally, the ionic concentration of the buffer is increased until buffer ions compete for the ionic sites on the stationary phase and displace the species. An alternative method of elution requires changing the pH of the buffer until the effective charge of the species is no longer favorable for binding to the stationary phase. An illustrative example is lowering the pH until the species has an effective positive charge and no longer binds to the anion exchanger.

[0180] Purification can be achieved when the impurities are uncharged or when the impurities have a charge of the opposite sign to that of the desired species but the same sign as the charge on the ion exchanger. This is because uncharged species and species having the same sign of charge as the ion exchanger usually do not bind. For different bound species, the strength of binding varies depending on factors such as the charge density and distribution of the charges of the various species. Thus, by applying an ionic gradient or a pH gradient (either as a continuous gradient or as a series of steps), the desired species can be eluted separately from the impurities.

[0181] In some embodiments, ion exchange chromatography is performed after clarification of the recovered material. In some embodiments, anion exchange chromatography is performed after clarification of the recovered material.

[0182] In some embodiments, the chromatography is anion exchange (AEX) chromatography. In some embodiments, AEX is performed using a membrane having a surface coating of positively charged functional groups, the membrane allowing convective flow of negatively charged viral vectors therethrough, where the viral vectors can bind to the positively charged coating surface. In some embodiments, the membrane has a nominal pore size of about 0.8 μM.

[0183] In some embodiments, AEX utilizes a commercially available membrane. Illustrative examples of commercially available products for AEX include, but are not limited to, the Mustang® Q chromatography membrane. In certain embodiments, the AEX membrane binds lentivirus while impurities do not. The lentivirus can then be eluted by increasing the concentration of salt (e.g., NaCl).

[0184] In some embodiments, using AEX allows not only larger impurities but also impurities having a binding ability different from that of the viral vector, mainly positively charged impurities, to pass through the membrane freely during loading. The loosely bound impurities are then removed in the washing step. In some embodiments, the washing step is performed using a salt buffer having the same pH and conductivity as the filtered formulation, typically added as a bulk load.

[0185] In some embodiments, ion exchange chromatography, such as anion exchange chromatography, includes a step of eluting virus vectors bound to a membrane. In some embodiments, elution is performed by increasing the salt concentration on both sides of the membrane, which can result in dissociation of the virus vector from the membrane. In some embodiments, a low concentration of salt (e.g., NaCl) is used during the equilibration, loading, and washing steps of anion exchange chromatography, and then the concentration of salt (e.g., NaCl) in the buffer added for elution is increased.

[0186] In some embodiments, the methods provided herein involve eluting surface engineered lentiviral particles by adding a high salt buffer, such as one containing sodium chloride (NaCl), potassium chloride (KCl), sodium acetate (NaOAc), or tetramethylammonium chloride. In some embodiments, the method of eluting surface engineered lentiviral particles involves NaCl.

[0187] In some embodiments, the NaCl concentration in the salt buffer for elution is about 0.5 M to 3 M. In some embodiments, the NaCl concentration in the salt buffer for elution is about 0.75 M to 2 M. In some embodiments, the NaCl concentration in the salt buffer for elution is about 0.75 M. In some embodiments, the NaCl concentration in the salt buffer for elution is about 1 M. In some embodiments, the NaCl concentration in the salt buffer for elution is about 2 M.

[0188] In some embodiments, the present disclosure herein surprisingly shows that the concentration of the salt buffer for elution can affect the yield of lentivirus produced by the process. In particular, the results show a tendency for a decrease in yield with changes to surface engineering and / or transgene payload. In some embodiments, an improvement in the yield of lentivirus can be achieved by using a higher salt concentration for elution when the surface engineered protein is larger or the transgene payload sequence is longer.

[0189] In one exemplary method, lentiviral particles generated to express a surface engineering protein containing a single-domain fusion protein can be eluted in a buffer containing 0.5 M to 1.5 M NaCl, such as 0.5 M to 0.75 M NaCl. In some embodiments, lentiviral particles generated to express a surface engineering protein containing a single-domain fusion protein can be eluted in a buffer containing 0.75 M NaCl.

[0190] In another exemplary method, lentiviral particles generated to express a surface engineering protein containing a multi-domain fusion protein can be eluted in a buffer containing 1.5 M NaCl to 2.5 M NaCl, such as 1.75 M to 2.25 M NaCl. In some embodiments, lentiviral particles generated to express a multi-domain fusion protein are eluted in a buffer containing 2 M NaCl.

[0191] b. Size-exclusion chromatography In some embodiments, chromatography includes size-exclusion chromatography. Size-exclusion chromatography is a technique that separates species by their size. Typically, this is done by use of a column packed with particles having pores of a specified size. For chromatographic separation, particles with an appropriate pore size are selected with respect to the size of the species in the mixture to be separated. When the mixture is applied to the column as a solution (or a suspension in the case of viruses) and then eluted with a buffer, the largest particles elute first because access to the pores is limited (or there is no access at all). Smaller particles can enter the pores and thus take a longer path through the column and elute more slowly.

[0192] Thus, considering the use of size-exclusion chromatography for purification of viral vectors, it is expected that the vector elutes before smaller impurities such as proteins.

[0193] In some embodiments, size exclusion chromatography is performed after clarification of the recovered material.

[0194] c. Hydrophobic interaction chromatography (HIC) In some embodiments, chromatography includes hydrophobic interaction chromatography. Species, such as proteins, have hydrophobic regions on their surface that can reversibly bind to weak hydrophobic sites on the stationary phase. In a medium having a relatively high salt concentration, this binding is promoted. Typically in HIC, the sample to be purified is bound to the stationary phase in a high salt environment. Elution is then achieved by application of a gradient of decreasing salt concentration (either continuously or as a series of steps). The salt commonly used is ammonium sulfate. Species having different levels of hydrophobicity tend to elute at different salt concentrations, and thus the target species can be purified from impurities. Other factors, such as pH, temperature, and additives to the elution medium, such as surfactants, chaotropic salts, and organics, can also affect the strength of the binding 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.

[0195] Viral vectors have hydrophobic moieties, such as proteins, on their surface and thus HIC can also be used as a purification means.

[0196] In some embodiments, HIC is performed after clarification of the recovered material.

[0197] d. Reverse phase chromatography (RPC) In some embodiments, chromatography includes reverse-phase chromatography (RPC). Like HIC, RPC separates species by differences in their hydrophobicity levels. A stationary phase with higher hydrophobicity than that used in HIC is employed. The stationary phase often consists of a substance to which a hydrophobic moiety, such as an alkyl or phenyl group, is attached, typically silica. Alternatively, the stationary phase can be an organic polymer without a bonding group. A sample containing a mixture of species to be separated is applied to the stationary phase in a relatively high-polarity aqueous medium that promotes binding. Elution is then achieved by decreasing the polarity of the aqueous medium by adding an organic solvent such as isopropanol or acetonitrile. Usually, a gradient (continuous or as a series of steps) of increasing organic solvent concentration is used, and the species elute in order of their respective hydrophobicity levels.

[0198] Other factors, such as the pH of the elution medium and the use of additives, can also affect the strength of binding of species to the RPC stationary phase. One or more of these factors can be adjusted or exploited to optimize the elution and purification of the product. A common additive is trifluoroacetic acid (TFA). This suppresses the ionization of acidic groups such as carboxyl moieties in the sample. It also lowers the pH of the elution medium, which suppresses the ionization of free silanol groups that may be present on the surface of the stationary phase having a silica matrix. TFA is one of a class of additives known as ion-pairing agents. These interact with ionic groups present on species in the sample having opposite charges. This interaction shields the charge and tends to increase the hydrophobicity of the species. Anionic ion-pairing agents, such as TFA and pentafluoropropionic acid, interact with positively charged groups of the species. Cationic ion-pairing agents, such as triethylamine, interact with negatively charged groups.

[0199] Viral vectors have hydrophobic moieties, such as proteins, on their surface, and thus RPC can potentially also be used as a purification means.

[0200] In some embodiments, RPC is performed after clarification of the recovered material.

[0201] e. Affinity chromatography In some embodiments, chromatography includes affinity chromatography. Affinity chromatography takes advantage of the fact that certain ligands that specifically bind to biomolecules, such as proteins or nucleotides, can be immobilized on a stationary phase. The modified stationary phase can then be used to separate the relevant biomolecules from a mixture. Examples of highly specific ligands are antibodies for the purification of a target antigen and enzyme inhibitors for the purification of an enzyme. More general interactions, such as the use of protein A ligands for the isolation of a wide range of antibodies, can also be utilized.

[0202] Typically, affinity chromatography is performed by application of a mixture containing the species of interest to a stationary phase to which the relevant ligand is bound. Under appropriate conditions, this results in binding of the species to the stationary phase. Unbound components are then washed away before applying an elution medium. The elution medium is selected to disrupt the binding of the ligand to the target species. This is usually accomplished by selection of an appropriate ionic strength, pH, or by use of a substance that competes with the ligand site for the target species. For some bound species, chaotropic agents, such as urea, are used to cause displacement from the ligand. However, this can result in irreversible denaturation of the species.

[0203] Viral vectors have on their surface moieties such as proteins that can potentially specifically bind to an appropriate ligand. This means that affinity chromatography can potentially also be used for their isolation.

[0204] In some embodiments, affinity chromatography is performed after clarification of the harvest.

[0205] f. Immobilized metal ion affinity chromatography (IMAC) In some embodiments, chromatography includes immobilized metal ion affinity chromatography. Biomolecules such as proteins can have on their surface an electron-donating moiety that can form a coordination bond with a metal ion. This can facilitate their binding to a stationary phase carrying immobilized metal ions such as Ni2+, Cu2+, Zn2+ or Fe3+. The stationary phase used in IMAC has a chelating agent, typically nitrilotriacetic acid or iminodiacetic acid, covalently bound to its surface, and it is the chelating agent that holds the metal ion. The chelated metal ion needs to have at least one coordination site remaining available for forming a coordination bond with a biomolecule. Potentially, there are several moieties on the surface of a biomolecule that can bind to the immobilized metal ion. These include not only histidine, tryptophan and cysteine residues, but also phosphate groups. However, the main donor to proteins appears to be the imidazole group of histidine residues. Native proteins can be separated using IMAC when they present appropriate donor moieties on their surface. IMAC can also be used for the separation of recombinant proteins having a chain of several linked histidine residues.

[0206] Typically, IMAC is performed by application of a mixture containing the species of interest to the stationary phase. Under appropriate conditions, this results in coordination bonding of the species to the stationary phase. Unbound components are then washed away before an elution medium is applied. For elution, a gradient (continuous or as a series of steps) of increasing salt concentration or decreasing pH can be used. Also, a commonly used procedure is the application of a gradient of increasing imidazole concentration. Biomolecules having different donor characteristics, e.g., having histidine residues in different environments, can be separated by use of gradient elution.

[0207] Viral vectors have on their surface moieties such as proteins that can potentially bind to an IMAC stationary phase. This means that IMAC can potentially also be used for their isolation.

[0208] In some embodiments, the IMAC is performed after clarification of the recovered material.

[0209] 3. Ultrafiltration / diafiltration In some embodiments, the method of the present disclosure includes a step of concentrating a filtered formulation of viral particles, the step including ultrafiltration. In some embodiments, the method of the present disclosure includes a step of purifying a filtered formulation of viral particles, the step including ultrafiltration. In some embodiments, the ultrafiltration is performed before chromatography. In some embodiments, the ultrafiltration is performed after chromatography. In some embodiments, the ultrafiltration also acts to remove impurities, such as HCP and HcDNA, from the filtered formulation of the particles. In some embodiments, the ultrafiltration is used not only to further remove impurities, such as HCP and hcDNA, from the filtered formulation of the particles and / or from the chromatography eluate, but also to concentrate the viral particles in the formulation. In some embodiments, the method of the present disclosure includes clarifying the suspension mixture by using a chromatographic step followed by an ultrafiltration step following the series of filters described above.

[0210] According to an aspect of the disclosure, the filtered formulation of viral particles is subjected to ultrafiltration (also referred to as diafiltration when used for buffer exchange) at least once during the process, for example, for vector concentration and / or buffer exchange.

[0211] The processes used to concentrate virus particles by the methods of the present disclosure include any filtration process (e.g., ultrafiltration (UF)) in which the concentration of virus particles is increased by forcing a diluent through a filter such that the diluent is removed from the virus particle preparation while the virus particles cannot pass through the filter and thereby remain in a concentrated form in the virus particle preparation. UF is described in detail, for example, in 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).

[0212] In some embodiments, for example, tangential flow filtration (“TFF”) as described in the MILLIPORE catalogue (Bedford, Massachusetts, 1995 / 96) entitled “Pharmaceutical Process Filtration Catalogue” pp. 177-202 is used. TFF is widely used in the bioprocessing industry for cell harvesting, clarification, purification and concentration of virus-containing products. The system consists of three separate process streams: a feed stream, a permeate stream and a retentate stream. Depending on the application, filters with different pore sizes can be used for TFF. In some embodiments, the retentate contains the product (e.g., lentiviral particles).

[0213] In some embodiments, TFF is used to purify and concentrate virus particles using ultrafiltration and diafiltration (UF / DF). In embodiments using TFF, the virus vector particles are retained in the system while smaller components and impurities permeate through the membrane and are removed from the system.

[0214] In some embodiments, the particular ultrafiltration membrane selected has a pore size small enough to retain viral particles but large enough to effectively purify impurities. Depending on the manufacturer and type of membrane, membranes with a nominal molecular weight cut-off (NMWC) of 100 - 1000 kDa, such as 300 kDa or 500 kDa NMWC, may be suitable. The composition of the membrane can be, but is not limited to, regenerated cellulose, polyethersulfone, polysulfone, or derivatives thereof. The membrane can be a flat sheet (also called a flat screen) or a hollow fiber. UF generally refers to filtration using a filter with a pore size smaller than 0.1 μm. The product is generally retained while the volume can be reduced by permeation (or kept constant by adding buffer at the same rate as the rate at which the permeate containing buffer and impurities is removed on the permeate side during diafiltration).

[0215] The two most widely used geometries for TFF in the biopharmaceutical industry are plate and frame (flat screen) and hollow fiber modules. The hollow fiber module is composed of an array of self-supporting fibers with a dense skin layer. The diameter of the fibers ranges from 0.5 mm to 3 mm. The advantage of the hollow fiber module is that filters with a very small membrane area (about 16 cm2) to a very large membrane area (about 20 m2) can be utilized, allowing for linear and simple scale-up.

[0216] In some embodiments, hollow fibers are used for TFF. These are reported to give lower shear and a better virus particle / infectious unit (VP / IU) ratio than flat screen membranes. Additionally, the transmembrane pressure is generally lower for hollow fibers than for flat screens.

[0217] In certain embodiments, hollow fibers with a 500 kDa (0.05 μm) pore size are used. Ultrafiltration may include diafiltration (DF) using an ultrafiltration device and is ideal for the removal and exchange of salts, sugars, non-aqueous solvents, separation without bound species, removal of low molecular weight substances, or rapid changes in ionic and / or pH environment. Trace solutes are most efficiently removed by adding solvent to a solution that is being ultrafiltered at a rate equal to the UF rate. This purifies the retained virus particles and washes trace species from a fixed volume of solution.

[0218] UF / DF can be used to concentrate and / or buffer exchange virus particle suspensions according to the present disclosure at different stages of the concentration process. In some embodiments, the methods of the present disclosure utilize a DF step to exchange the buffer of the supernatant after chromatography or other purification steps. In some embodiments, the eluate from the chromatography step according to the present disclosure is concentrated and further purified by ultrafiltration-diafiltration. During this process, the virus particles are exchanged into the formulation buffer.

[0219] In some embodiments, the ultrafiltration / diafiltration can be tangential flow diafiltration, stirred cell diafiltration, and dialysis. In some embodiments, the ultrafiltration / diafiltration can include multiple tangential flow diafiltrations (TFF). In some embodiments, the multiple can be 1, 2, 3, 4 or more separate tangential flow diafiltrations. In some embodiments, the process includes two TFF stages and systems. In some embodiments, the first TFF stage can be used to concentrate the chromatography eluate (e.g., AEX pool), exchange the buffer to the diafiltration buffer, and for concentration. In some embodiments, the second TFF can be used to further concentrate the pool from the first TFF, e.g., to achieve the final target titer of the drug substance. In some embodiments, the first TFF and the second TFF are the same. In some embodiments, the first and second TFFs 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.

[0220] In some embodiments, the TFF, e.g., each of the TFFs individually, is performed with a hollow fiber filter. In some embodiments, the hollow fiber filter has a nominal molecular weight cut-off (NMWC) of 100 kDa to 1000 kDa. In some embodiments, the NMWC is 100 kDa to 500 kDa. In some embodiments, the NMWC is 300 kDa to 750 kDa. In some embodiments, the NMWC is 500 kDa to 1000 kDa.

[0221] In some embodiments, the NMWC is 500 kDa. In some embodiments, the NMWC is 300 kDa. In some embodiments, the NMWC is 100 kDa.

[0222] In some embodiments, the hollow fiber filter has a pore size of 0.1 μm to 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.

[0223] In some embodiments, the hollow fiber filter has a length of 20 cm, 41.5 cm, 65 cm, 50 cm, 68 cm, or 108 cm. In some embodiments, the hollow fiber filter has an inner lumen of 0.50 mm, 0.63 mm, 0.75 mm, 1 mm, 2 mm, or 3 mm.

[0224] In some embodiments, the hollow fiber filter is 235 cm 2 ~1600 cm 2 in surface area. 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 .

[0225] In some embodiments, the hollow fiber filter accommodates a volume of 300 mL.

[0226] In some embodiments, the hollow fiber filter is a commercially available hollow fiber filter. Exemplary commercially available hollow fiber filters include, but are not limited to, the Spectrum® hollow fiber filter (Repligen).

[0227] In some embodiments, each of the TFFs is performed using a hollow fiber filter that includes different cassettes or modules where one or more of the batch volume, pore size, membrane cut-off, inner lumen diameter, or length are different. In some embodiments, the hollow fiber filters for each TFF have the same nominal molecular weight cut-off (NMWC) or pore size, but may have different surface areas or volumes. In some embodiments, the hollow fiber filters for each TFF have different NMWCs or pore sizes and may also have different surface areas or volumes.

[0228] In some embodiments, the HCP is measured after the UF / DF step. The HCP is measured by ELISA and can be normalized against the amount of viral particles. In some embodiments, the HCP is measured as units / dose, where one dose is defined using the viral transduction units (TU) of the virus. In some embodiments, the HCP is measured as μg / dose, where one dose is 1E9 TU.

[0229] In some embodiments, the 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 after UF / DF. In some embodiments, the HCP is less than about 1500 μg / 1E9 TU after the first UF / DF step. In some embodiments, the HCP is undetectable after the second UF / DF step.

[0230] 4. Filtration Sterilization In some embodiments, the disclosed method includes the step of sterile filtering the filtered and concentrated virus particle formulation described herein. Filter sterilization is common in processes for pharmaceutical-grade substances and is known to those skilled in the art. Filter sterilization renders the resulting formulation substantially free of contaminants. The level of contaminants after filter sterilization is at a level suitable for clinical use. Sterile filters suitable for use according to the present disclosure are well known to those skilled in the art.

[0231] In some embodiments, the mixture concentrated after UF / DF is filter sterilized to produce a sterile formulation. In some embodiments, the sterile filter has a maximum pore size of 0.22 μm. In some embodiments, the sterile filter has a retention threshold of 0.2 μm.

[0232] In some embodiments, the sterile formulation is formulated in a buffer, thereby producing the active pharmaceutical ingredient. In some embodiments, the amount of contaminants in the active pharmaceutical ingredient is at a level acceptable 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.

[0233] In some embodiments, the clarification, chromatography, UF / DF, and filter sterilization steps are carried out over about 5 to 10 hours. In some embodiments, the clarification, chromatography, UF / DF, and filter sterilization steps are carried out over about 5 to 8 hours. In some embodiments, the clarification, chromatography, UF / DF, and filter sterilization steps are carried out over about 5 to 6 hours. In some embodiments, the clarification, chromatography, UF / DF, and filter sterilization steps are carried out over about 6 to 7 hours. In some embodiments, the clarification, chromatography, UF / DF, and filter sterilization steps are carried out over about 6 hours.

[0234] In some embodiments, each of the clarification, chromatography, UF / DF, and filtration sterilization steps is performed at a pH of 6 to 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.

[0235] In some embodiments, the viral vector formulation prepared by the above method is free of impurities. In some embodiments, the impurities include host cell DNA (hcDNA). In some embodiments, the impurities include host cell protein (HCP). In some embodiments, the viral vector formulation is substantially pure. In some embodiments, the viral vector formulations 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 formulations provided herein contain less than 1% hcDNA. In some embodiments, the method used to prepare the viral vector formulation achieves a 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%, greater than 99% reduction in hcDNA compared to, for example, a suspension mixture. In some embodiments, the method used to prepare the viral vector formulation achieves a 99% reduction in hcDNA compared to, for example, a suspension mixture.

[0236] In some embodiments, the viral vector formulations 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 formulations described herein contain less than 1% HCP. In some embodiments, the method used to prepare the viral vector formulation achieves a reduction of 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%, more than 99% in HCP compared to, for example, a suspension mixture. In some embodiments, the method used to prepare the viral vector formulation achieves a 99% reduction in HCP compared to, for example, a suspension mixture.

[0237] In some embodiments, the yield of viral vector achieved by the above method of preparing the viral vector formulation is at least 10% or about 10%, at least 12% or about 12%, at least 15% or about 15%, at least 18% or about 18%, at least 20% or about 20%, at least 22% or about 22%, at least 25% or about 25%, at least 28% or about 28%, or at least 30% or about 30%. In some embodiments, the yield of viral vector achieved by the above method of preparing the viral vector formulation is 10% - 30%, for example, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28% or 30%, or about 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28% or 30%, or any value between any of the foregoing. In some embodiments, the yield of viral vector achieved by the above method of preparing the viral vector formulation is at least 15% or about 15%, or about 15%.

[0238] In some embodiments, the downstream process produces a viral vector formulation (which may be referred to as the active pharmaceutical ingredient) having a viral vector titer of 0.5×10 7 TU / mL - 5×10 8 TU / mL. In some embodiments, the viral vector titer is 1×107 TU / mL to 3×10 7 TU / mL, 2×10 7 TU / mL to 4×10 7 TU / mL, 3×10 7 TU / mL to 5×10 7 TU / mL, 4×10 7 TU / mL to 6×10 7 TU / mL, 5×10 7 TU / mL to 7×10 7 TU / mL, 6×10 7 TU / mL to 8×10 7 TU / mL, 7×10 7 TU / mL to 9×10 7 TU / mL, 8×10 7 TU / mL to 1×10 8 TU / mL, 9×10 7 TU / mL to 2×10 8 TU / mL, 1×10 8 TU / mL to 3×10 8 TU / mL, 2×10 8 TU / mL to 4×10 8 TU / mL, 3×10 8 TU / mL to 5×10 8 is TU / mL. In some embodiments, the viral vector titer is 2.5×10 8 TU / mL to 4.7×10 8 TU / mL. In some embodiments, the downstream process produces a viral vector formulation (or drug substance) having a viral vector titer of 2.5×10 8 TU / mL to 4.7×10 8 TU / mL. In some embodiments, the viral vector titer comprises at least about 2.5×10 8 TU / mL. In some embodiments, the viral vector titer comprises at least about 4.7×10 8 TU / mL. In some embodiments, the viral vector titer is 2.5×10 8 TU / mL. In some embodiments, the viral vector titer is 4.7×10 8 TU / mL.

[0239] In some embodiments, the viral vector formulation has a volume of about 1 to 500 mL. In some embodiments, the viral vector formulation has a volume of about 1 to 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 mL. In some embodiments, the viral vector formulation comprises at least 10 mL, 50 mL, 100 mL, 200 mL, or 300 mL. In some embodiments, the viral vector formulation is 10 mL. In some embodiments, the viral vector formulation is 100 mL. In some embodiments, the viral vector formulation is 200 mL. In some embodiments, the viral vector formulation is 300 mL. In some embodiments, the viral vector formulation is 500 mL. In some embodiments, the viral vector is a retrovirus (e.g., lentivirus).

[0240] In some embodiments, the downstream process produces a viral vector formulation or a drug substance having a viral vector titer of 5×10 10 TU to 5×10 12 TU. In some embodiments, the viral vector titer is 1×10 11 TU to 3×10 11 TU, 2×10 11 TU to 4×10 11 TU, 3×10 11 TU to 5×10 11 TU, 4×10 11 TU to 6×10 11 TU, 5×10 11 TU to 7×10 11 TU, 6×10 11 TU to 8×10 11 TU, 7×10 11 TU to 9×10 11 TU, 8×10 11 TU to 1×10 12 TU, 9×10 11 TU to 2×10 12 TU, 1×10 12 TU to 3×10 12 TU, 2×1012 TU ~ 4×10 12 TU, 3×10 12 TU ~ 5×10 12 It is TU. In some embodiments, the viral vector titer is 2.5×10 12 TU ~ 4.7×10 12 TU. In some embodiments, the viral vector titer is at least about 2.5×10 12 TU. In some embodiments, the viral vector titer is at least about 4.7×10 12 TU. In some embodiments, the viral vector titer is 2.5×10 12 TU. In some embodiments, the viral vector titer is 4.7×10 12 TU.

[0241] In some embodiments, the downstream process produces a viral vector formulation or active pharmaceutical ingredient having a viral vector titer of 5×10 8 TU ~ 5×10 10 TU. In some embodiments, the viral vector titer is 1×10 9 TU ~ 3×10 9 TU, 2×10 9 TU ~ 4×10 9 TU, 3×10 9 TU ~ 5×10 9 TU, 4×10 9 TU ~ 6×10 9 TU, 5×10 9 TU ~ 7×10 9 TU, 6×10 9 TU ~ 8×10 9 TU, 7×10 9 TU ~ 9×10 9 TU, 8×10 9 TU ~ 1×10 10 TU, 9×10 9 TU ~ 2×10 10 TU, 1×10 10 TU ~ 3×10 10 TU, 2×10 10 TU ~ 4×10 10 TU, 3×10 10 TU ~ 5×10 10It is TU. In some embodiments, the viral vector titer is 2.5×10 10 TU to 4.7×10 10 TU. In some embodiments, the viral vector titer is at least about 2.5×10 10 TU. In some embodiments, the viral vector titer is at least about 4.7×10 10 TU. In some embodiments, the viral vector titer is 2.5×10 10 TU. In some embodiments, the viral vector titer is 4.7×10 10 TU. In some embodiments, the viral vector titer is 6×10 10 TU.

[0242] In some embodiments, the downstream process produces a viral vector formulation or a drug substance having a viral vector titer of 1×10 9 TU to 1×10 11 TU. In some embodiments, the viral vector titer is 2×10 9 TU to 6×10 9 TU, 4×10 9 TU to 8×10 9 TU, 6×10 9 TU to 1×10 10 TU, 8×10 9 TU to 1.2×10 10 TU, 1×10 10 TU to 1.4×10 10 TU, 1.2×10 10 TU to 1.6×10 10 TU, 1.4×10 10 TU to 1.8×10 10 TU, 1.6×10 10 TU to 2×10 10 TU, 1.8×10 10 TU to 4×10 10 TU, 2×10 10 TU to 6×10 10 TU, 4×10 10 TU to 8×10 10 TU, 6×10 10 TU to 1×10 11 TU. In some embodiments, the viral vector titer is 5×1010 TU ~ 9.4×10 10 It is TU. In some embodiments, the viral vector titer is at least about 5×10 10 including TU. In some embodiments, the viral vector titer is at least about 9.4×10 10 including TU. In some embodiments, the viral vector titer is 5×10 10 It is TU. In some embodiments, the viral vector titer is 9.4×10 10 It is TU. In some embodiments, the viral vector titer is 6×10 10 It is TU.

[0243] In some embodiments, the downstream process produces a viral vector formulation or a drug substance having a viral vector titer of 1.5×10 9 TU ~ 1.5×10 11 TU. In some embodiments, the viral vector titer is 3×10 9 TU ~ 9×10 9 TU, 6×10 9 TU ~ 1.2×10 10 TU, 9×10 9 TU ~ 1.5×10 10 TU, 1.2×10 10 TU ~ 1.8×10 10 TU, 1.5×10 10 TU ~ 2.1×10 10 TU, 1.8×10 10 TU ~ 2.4×10 10 TU, 2.1×10 10 TU ~ 2.7×10 10 TU, 2.4×10 10 TU ~ 3×10 10 TU, 2.7×10 10 TU ~ 6×10 10 TU, 3×10 10 TU ~ 9×10 10 TU, 6×10 10 TU ~ 1.2×10 11 TU, 9×10 10 TU ~ 1.5×10 11 It is TU. In some embodiments, the viral vector titer is 7.5×10 10 TU ~ 1.4×1011 It is TU. In some embodiments, the viral vector titer is at least about 7.5×10 10 contains TU. In some embodiments, the viral vector titer is at least about 1.4×10 11 contains TU. In some embodiments, the viral vector titer is 7.5×10 10 is TU. In some embodiments, the viral vector titer is 1.4×10 11 is TU. In some embodiments, the viral vector titer is 6×10 10 is TU.

[0244] C. Process Management and Characterization In some embodiments, methods for preparing viral particle formulations are provided herein. In some embodiments, the method for preparing a viral particle formulation includes a manufacturing process. In some embodiments, an exemplary manufacturing process is shown in the upstream and downstream processes in Figure 1C. In some embodiments, the manufacturing process provided herein includes the evaluation of one or more manufacturing process controls shown in Figure 1C. In some embodiments, manufacturing process control includes measuring one or more characteristics during the manufacturing process for the purpose of ensuring consistency between different manufacturing runs. For example, determining the cell number at the start of the manufacturing process can ensure that similar cell yields are consistent between different manufacturing runs, which ultimately results in consistent, predictable, and stable virus production.

[0245] In some embodiments, the manufacturing processes provided herein include an evaluation of the characterization of one or more manufacturing processes shown in FIG. 1C. In some embodiments, the characterization of a manufacturing process is a characteristic that is measured to retrospectively provide information about the performance of the manufacturing process. For example, measuring process impurities (i.e., impurities introduced due to reagents or other materials used in the manufacturing process) indicates how well a particular stage of the manufacturing process is functioning. Measuring process impurities at the clarification stage of the harvest or at the anion exchange chromatography stage indicates how well the materials (e.g., columns or membranes) are functioning to remove process impurities.

[0246] In some embodiments, manufacturing process control in the upstream process includes measuring the cell number or cell viability during cell expansion (e.g., seed train); or the cell number, cell viability, pH, dissolved oxygen (DO), or temperature in the production bioreactor. In some embodiments, the cell number is also used to determine whether the cells are growing and to enable subculturing of the cells at non-limiting densities during seed train expansion. In some cases, higher cell densities can induce stress in the cells. In some embodiments, the cell viability is used to confirm whether the cell number contains predominantly viable and non-dead cells. In some embodiments, the pH is used to confirm that the cells remain viable. In some cases, pH < 6.8 negatively affects the growth and transfection of virus producer cells. In some embodiments, the DO represents the total amount of oxygen available to the virus producer cells.

[0247] In some embodiments, manufacturing process control in the downstream process includes measuring the pressure or turbidity of the viral particle product in the recovered product clarification stage; the pH, conductivity, or flow rate of the viral particle product in the AEX chromatography stage; the viral particle count or p24 of the viral particle product in the UF / DF stage; the pressure between the API (DS) filtration stages or the loading rate of the viral particle product; or the fill weight of the frozen formulation. 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 can cause problems in the membrane chromatography stage. P24 is a capsid protein expressed by the virus. Thus, in some embodiments, measuring p24 indicates the presence of cells producing viral particles. In some embodiments, the fill weight is the amount of formulation in a given container (e.g., vial). In some embodiments, the formulation is evaluated for whether it conforms to an acceptable weight range, otherwise the formulation may be rejected during clinical use.

[0248] In some embodiments, characterizing the manufacturing process in the upstream process includes measuring metabolites, p24, titer, turbidity, hcDNA between cell expansion (e.g., seed train) and production bioreactor stages. In some embodiments, metabolites are used to determine whether the cells are healthy.

[0249] In some embodiments, characterizing the manufacturing process in the downstream process includes measuring p24, titer, turbidity, or process impurities during the recovered product clarification stage; p24, titer, or process impurities between the AEX and UF / DF stages; or the complete release, characterization, or stability between the DS filtration stages.

[0250] In addition to stepwise manufacturing process control and characterization, an analytical platform can be used to assist in the rapid scale-up of lentiviral particle formulations. In some embodiments, the analytical platform includes methods for (i) identifying the viral particle formulation; (ii) determining the purity of the viral particle formulation; (iii) assessing the potency of the viral particle formulation; and (iv) evaluating the safety of the viral particle formulation.

[0251] In some embodiments, methods used to identify viral particle formulations include sequencing to detect the presence of transgenes expressed by the virus or Western blot / ELISA to detect the presence of proteins expressed by the virus.

[0252] In some embodiments, methods used to assess the purity of viral particle formulations 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 indicative of purity include the presence of plasmid DNA, E1A DNA, or SV40 DNA.

[0253] In some embodiments, methods used to assess the potency of viral particle formulations include physical titers determined by measuring the number of p24, RNA genomes, and physical particles. In some embodiments, another method used to assess potency includes transduction titers required to generate viral particle products. In some embodiments, the transduction titer or infectivity titer is any of the infectivity titers provided herein. In some embodiments, additional methods used to assess potency include measuring the expression of cell surface markers on viral particles or measuring the functionality of cell surface markers (e.g., the functionality of CAR).

[0254] In some embodiments, the methods used to evaluate the safety of the viral particle product include measuring endotoxin, bioburden (i.e., the number of contaminating organisms found in the viral particle preparation prior to sterilization), sterility, subvisible particles (i.e., particles that are too large to be analyzed by size exclusion chromatography (SEC) (e.g., > about 0.1 μm) but too small to be seen with the naked eye (e.g., < 100 μm)), mycoplasma, adventitious viruses, and replication-competent lentiviruses.

[0255] D. Exemplary Methods In some embodiments, the present disclosure provides a method for preparing a viral particle preparation, the method comprising: (i) filtering a suspension mixture comprising a population of host cells and viral particles to remove contaminants, the filtering comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a two-layer filter component comprising a second filter and a third filter, wherein the second filter has a retention threshold smaller than that of the first filter and the third filter has a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of viral particles; (ii) purifying the filtered preparation of viral particles; and (iii) concentrating the filtered preparation of viral particles, the concentrating comprising chromatography and ultrafiltration.

[0256] In some embodiments, the present disclosure provides a method for preparing a retroviral particle preparation, comprising: (i) a step of filtering a suspension mixture comprising a population of host cells and retroviral particles to remove contaminants, the step comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a two-layer filter component comprising a second filter and a third filter, wherein the second filter has a retention threshold smaller than that of the first filter and the third filter has a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of retroviral particles; (ii) a step of purifying the filtered preparation of retroviral particles; and (iii) a step of concentrating the filtered preparation of retroviral particles, the step comprising chromatography and ultrafiltration.

[0257] In some embodiments, the present disclosure provides a method for preparing a lentiviral preparation, comprising: (i) a step of filtering a suspension mixture comprising a population of host cells and lentiviral particles to remove contaminants, the step comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a two-layer filter component comprising a second filter and a third filter, wherein the second filter has a retention threshold smaller than that of the first filter and the third filter has a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of lentiviral particles; (ii) a step of purifying the filtered preparation of lentiviral particles; and (iii) a step of concentrating the filtered preparation of lentiviral particles, the step comprising chromatography and ultrafiltration.

[0258] In some embodiments, the present disclosure provides a method for preparing a viral particle formulation, comprising: (i) a step of filtering a suspension mixture comprising a population of host cells and viral particles to remove contaminants, the step comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; (c) filtering the second filtrate through a two-layer filter component comprising the second filter and a third filter, the third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered viral particle formulation; (ii) a step of purifying the filtered viral particle formulation; and (iii) a step of concentrating the filtered viral particle formulation, the step comprising chromatography and ultrafiltration.

[0259] In some embodiments, the present disclosure provides a method for preparing a retroviral particle formulation, comprising: (i) a step of filtering a suspension mixture comprising a population of host cells and retroviral particles to remove contaminants, the step comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; (c) filtering the second filtrate through a two-layer filter component comprising the second filter and a third filter, the third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered retroviral particle formulation; (ii) a step of purifying the filtered retroviral particle formulation; and (iii) a step of concentrating the filtered retroviral particle formulation, the step comprising chromatography and ultrafiltration.

[0260] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, comprising: (i) filtering a suspension mixture comprising a population of host cells and lentiviral particles to remove contaminants, the filtering comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; (c) filtering the second filtrate through a two-layer filter component comprising the second filter and a third filter, the third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of viral particles; (ii) purifying the filtered preparation of lentiviral particles; and (iii) concentrating the filtered preparation of lentiviral particles, the concentrating comprising chromatography and ultrafiltration.

[0261] In some embodiments, the present disclosure provides a method for preparing a viral particle formulation, comprising: (i) filtering a suspension mixture comprising a population of host cells and viral particles to remove contaminants, the filtering comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; (c) filtering the second filtrate through a third filter, the third filter being a two-layer filter comprising a first-layer filter and a second-layer filter, the first-layer filter having the same retention threshold as the second filter and the second-layer filter having a retention threshold smaller than that of the first-layer filter, thereby producing a filtered preparation of viral particles; (ii) purifying the filtered preparation of viral particles; and (iii) concentrating the filtered preparation of viral particles, the concentrating comprising chromatography and ultrafiltration.

[0262] In some embodiments, the present disclosure provides a method for preparing a retroviral particle preparation, comprising: (i) a step of filtering a suspension mixture containing a population of host cells and retroviral particles to remove contaminants, comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (c) filtering the second filtrate through a third filter, wherein the third filter is a two-layer filter comprising a first layer filter and a second layer filter, the first layer filter has the same retention threshold as the second filter, and the second layer filter has a retention threshold smaller than that of the first layer filter; (ii) a step of purifying the filtered retroviral particle preparation; and (iii) a step of concentrating the filtered retroviral particle preparation, comprising chromatography and ultrafiltration.

[0263] In some embodiments, the present disclosure provides a method for preparing a lentiviral preparation, comprising: (i) a step of filtering a suspension mixture containing a population of host cells and lentiviral particles to remove contaminants, comprising: (a) filtering the mixture through a first filter that is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (c) filtering the second filtrate through a third filter, wherein the third filter is a two-layer filter comprising a first layer filter and a second layer filter, the first layer filter has the same retention threshold as the second filter, and the second layer filter has a retention threshold smaller than that of the first layer filter, thereby producing a filtered lentiviral particle preparation; (ii) a step of purifying the filtered lentiviral particle preparation; and (iii) a step of concentrating the filtered lentiviral particle preparation, comprising chromatography and ultrafiltration.

[0264] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, comprising: (i) contacting a population of host cells in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the population of host cells of step (i) for a period sufficient to produce a suspension mixture comprising the population of host cells and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, comprising: (a) contacting the mixture with an endonuclease; (b) filtering the mixture through a first filter, which is a depth filter having a retention threshold of at least 8 μm, resulting in a first filtrate; (c) filtering the first filtrate through a second filter having a retention threshold of 0.45 μm, resulting in a second filtrate; and (d) filtering the second filtrate through a third filter having a retention threshold of 0.2 μm, thereby producing a filtered formulation of lentiviral particles; (iv) purifying the filtered formulation of lentiviral particles; and (v) concentrating the filtered formulation of lentiviral particles, comprising chromatography and ultrafiltration.

[0265] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, comprising: (i) contacting a population of host cells in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the population of host cells of step (i) for a period of time sufficient to produce a suspension mixture comprising the population of host cells and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, comprising: (a) contacting the mixture with an endonuclease; (b) filtering the mixture through a first filter, which is a depth filter having a retention threshold of at least 8 μm, resulting in a first filtrate; (c) filtering the first filtrate through a second filter having a retention threshold of 0.45 μm, resulting in a second filtrate; and (d) filtering the second filtrate through a third filter having a retention threshold of 0.2 μm, thereby producing a filtered lentiviral particle formulation; (iv) purifying the filtered lentiviral particle formulation; and (v) concentrating the filtered lentiviral particle formulation, comprising ion exchange chromatography and two-stage ultrafiltration.

[0266] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, comprising: (i) contacting a population of host cells in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the population of host cells of step (i) for a period sufficient to produce a suspension mixture comprising the population of host cells and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, comprising: (a) contacting the mixture with an endonuclease; (b) filtering the mixture through a first filter which is a depth filter having a retention threshold of at least 8 μm, resulting in a first filtrate; (c) filtering the first filtrate through a second filter having a retention threshold of 0.45 μm, resulting in a second filtrate; and (d) filtering the second filtrate through a third filter having a retention threshold of 0.2 μm, thereby producing a filtered formulation of lentiviral particles; (iv) purifying the filtered formulation of lentiviral particles; (v) concentrating the filtered formulation of lentiviral particles, comprising ion exchange chromatography and two-stage ultrafiltration; and (vi) filter sterilizing the concentrated formulation to produce a sterile bulk drug substance.

[0267] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, comprising: (i) filtering a suspension mixture comprising a population of host cells and lentiviral particles to remove contaminants, comprising: (a) filtering the mixture through a first filter which is a depth filter having a retention threshold of at least 8 μm; (b) filtering the first filtrate through a two-layer filter component comprising a second filter and a third filter, wherein the second filter has a retention threshold of 0.45 μm and the third filter has a retention threshold of 0.2 μm, thereby producing a filtered formulation of lentiviral particles; (ii) purifying the filtered formulation of lentiviral particles; and (iii) concentrating the filtered formulation of lentiviral particles, comprising chromatography and ultrafiltration.

[0268] In some embodiments, the present disclosure provides a method for preparing a lentiviral preparation, comprising: (i) a step of filtering a suspension mixture comprising a population of host cells and lentiviral particles to remove contaminants, the step comprising: (a) filtering the mixture with a first filter which is a depth filter having a retention threshold of at least 8 μm; (b) filtering the first filtrate with a two-layer filter component comprising a second filter and a third filter, the second filter having a retention threshold of 0.45 μm and the third filter having a retention threshold of 0.2 μm, thereby producing a filtered preparation of lentiviral particles; (ii) a step of purifying the filtered preparation of lentiviral particles; and (iii) a step of concentrating the filtered preparation of lentiviral particles, the step comprising ion exchange chromatography and two-stage ultrafiltration.

[0269] In some embodiments, the present disclosure provides a method for preparing a lentiviral preparation, comprising: (i) a step of filtering a suspension mixture comprising a population of host cells and lentiviral particles to remove contaminants, the step comprising: (a) filtering the mixture with a first filter which is a depth filter having a retention threshold of at least 8 μm; (b) filtering the first filtrate with a two-layer filter component comprising a second filter and a third filter, the second filter having a retention threshold of 0.45 μm and the third filter having a retention threshold of 0.2 μm, thereby producing a filtered preparation of lentiviral particles; (ii) a step of purifying the filtered preparation of lentiviral particles; and (ii) a step of concentrating the filtered preparation of lentiviral particles, the step comprising ion exchange chromatography and two-stage ultrafiltration; and (iii) a step of filter-sterilizing the concentrated preparation to produce a sterile drug substance.

[0270] In some embodiments, the present disclosure provides a method for preparing a lentiviral preparation, comprising: (i) contacting a population of host cells in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the population of host cells of step (i) for a period sufficient to produce a suspension mixture comprising the population of host cells and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, comprising: (a) contacting the mixture with an endonuclease; (b) filtering the mixture through a first filter, which is a depth filter having a retention threshold of at least 8 μm, resulting in a first filtrate; (c) filtering the first filtrate through a second filter having a retention threshold of 0.45 μm, resulting in a second filtrate; and (d) filtering the second filtrate through a third filter, wherein the third filter has a first layer filter having a retention threshold of 0.45 μm and a second layer filter having a retention threshold of 0.2 μm, thereby producing a filtered preparation of lentiviral particles; (iv) purifying the filtered preparation of lentiviral particles; and (v) concentrating the filtered preparation of lentiviral particles, comprising chromatography and ultrafiltration.

[0271] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, comprising: (i) contacting a population of host cells in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the population of host cells of step (i) for a period of time sufficient to produce a suspension mixture comprising the population of host cells and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, comprising: (a) contacting the mixture with an endonuclease; (b) filtering the mixture through a first filter, which is a depth filter having a retention threshold of at least 8 μm, resulting in a first filtrate; (c) filtering the first filtrate through a second filter having a retention threshold of 0.45 μm, resulting in a second filtrate; and (d) filtering the second filtrate through a third filter having a first layer filter with a retention threshold of 0.45 μm and a second layer filter with a retention threshold of 0.2 μm; (iv) purifying the filtered lentiviral particle formulation; and (v) concentrating the filtered lentiviral particle formulation, comprising ion exchange chromatography and two-stage ultrafiltration.

[0272] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, comprising: (i) contacting a population of host cells in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the population of host cells of step (i) for a period of time sufficient to produce a suspension mixture comprising the population of host cells and lentiviral particles; (iii) filtering the suspension mixture to remove contaminants, comprising: (a) contacting the mixture with an endonuclease; (b) filtering the mixture through a first filter which is a depth filter having a retention threshold of at least 8 μm, resulting in a first filtrate; (c) filtering the first filtrate through a second filter having a retention threshold of 0.45 μm, resulting in a second filtrate; and (d) filtering the second filtrate through a third filter having a first layer filter with a retention threshold of 0.45 μm and a second layer filter with a retention threshold of 0.2 μm; (iv) purifying the filtered lentiviral particle formulation; (v) concentrating the filtered lentiviral particle formulation, comprising ion exchange chromatography and two-stage ultrafiltration; and (vi) filter sterilizing the concentrated formulation to produce a sterile bulk drug substance.

[0273] In some embodiments, the present disclosure provides a method for preparing a viral particle formulation, comprising: (i) expanding a population of host cells in one or more seed trains in shake flasks; (ii) inoculating a bioreactor with the population of host cells; (iii) transfecting the population of host cells in the bioreactor; (iv) adding an endonuclease (e.g., benzonase); (v) recovering the cell culture medium containing the population of host cells from the bioreactor; (vi) clarifying the cell culture medium one or more times; (vii) purifying and concentrating the cell culture medium by AEX chromatography including an elution step; (viii) filtering the cell culture medium (e.g., tangential flow filtration); (ix) filtering the bulk drug substance; and (x) storing the bulk drug substance, i.e., the viral particle formulation.

[0274] In some embodiments, the present disclosure provides a method for preparing a lentiviral formulation, comprising: (i) expanding a population of host cells in one or more seed trains in a shake flask; (ii) inoculating a population of host cells into a bioreactor; (iii) transfecting the population of host cells in the bioreactor; (iv) adding an endonuclease (e.g., benzonase); (v) recovering a cell culture medium containing the population of host cells from the bioreactor; (vi) clarifying the cell culture medium one or more times; (vii) purifying and concentrating the cell culture medium by AEX chromatography including an elution step; (viii) filtering the cell culture medium (e.g., tangential flow filtration); (ix) filtering the drug substance; and (x) storing the drug substance, i.e., the lentiviral formulation.

[0275] In some embodiments, the present disclosure provides a method for preparing a retroviral formulation, comprising: (i) expanding a population of host cells in one or more seed trains in a shake flask; (ii) inoculating a population of host cells into a bioreactor; (iii) transfecting the population of host cells in the bioreactor; (iv) adding an endonuclease (e.g., benzonase); (v) recovering a cell culture medium containing the population of host cells from the bioreactor; (vi) clarifying the cell culture medium one or more times; (vii) purifying and concentrating the cell culture medium by AEX chromatography including an elution step; (viii) filtering the cell culture medium (e.g., tangential flow filtration); (ix) filtering the drug substance; and (x) storing the drug substance, i.e., the retroviral formulation.

[0276] II. Viral Particles As is well known in the art, viral particles are tools that enable or facilitate the transfer of entities from one environment to another. According to and by way of example, some viral particles used in recombinant DNA technology transfer entities such as segments of DNA into host cells. Examples of vectors used in recombinant DNA technology 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.

[0277] A. Retroviral Particles In some embodiments, the present disclosure provides methods for preparing viral formulations. In some embodiments, the virus is a retrovirus. A 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), murine 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 Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763.

[0278] Retroviruses include lentiviruses, gamma-retroviruses, and alpha-retroviruses, each of which can be used to deliver polynucleotides into cells using methods known in the art. Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. The higher complexity allows the virus to modulate its life cycle during the process of latent infection. Some examples of lentiviruses include human immunodeficiency virus (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV). Retroviral vectors are generated by multiple attenuation of HIV pathogenic genes. For example, the genes env, vif, vpr, vpu, and nef are deleted to make the vector biologically safe.

[0279] The lentiviral vectors of the present disclosure may be derived from or be derived from any suitable lentivirus. Recombinant retroviral vector particles are capable of transducing a nucleotide of interest (NOI) into recipient cells. After entering the cell, the RNA genome from the vector particle is reverse transcribed into DNA and incorporated 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 essential for replication may be removed from the virus. This renders the virus vector defective in replication. A portion of the viral genome may also be replaced by the NOI to generate a vector containing the NOI that is capable of transducing target non-dividing host cells and / or integrating its genome into the host genome.

[0280] Exemplary 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. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, which are each incorporated herein by reference in their entirety. Generally, these vectors are constructed to have sequences essential for selecting cells containing the vector, incorporating foreign nucleic acids into lentiviral particles, and introducing the nucleic acids into target cells.

[0281] A commonly used lentiviral vector system is the so-called third-generation system. The third-generation lentiviral vector system contains four plasmids. The "transfer plasmid" encodes the polynucleotide sequence to be delivered to target cells by the lentiviral vector system. The transfer plasmid generally has one or more transfer gene sequences of interest flanked by long terminal repeat (LTR) sequences, which facilitate the incorporation of the transfer plasmid sequence into the host genome. For safety reasons, the transfer plasmid is generally designed to render the resulting vector incapable of replication. For example, the transfer plasmid lacks gene elements necessary for the production of infectious particles in the host cell. In addition, the transfer plasmid may be designed to have a deletion in the 3' LTR, rendering 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 viral particles may also contain a 3' untranslated region (UTR) and a 5' UTR. The UTRs contain retroviral regulatory elements that assist in the packaging, reverse transcription, and cellular incorporation of the proviral genome after cell contact by retroviral particles.

[0282] The third-generation system also generally includes two "packaging plasmids" and an "envelope plasmid". The "envelope plasmid" generally encodes an Env gene that is operably linked to a promoter. In an exemplary third-generation system, the Env gene is VSV-G and the promoter is the CMV promoter. The third-generation system uses two packaging plasmids, one encoding gag and pol and the other encoding rev as an additional safety feature; an improvement over the single packaging plasmid of the so-called second-generation system. Although safer, the third-generation system is more cumbersome to use due to the addition of additional plasmids and may result in lower virus titers. Exemplary filling plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0283] Many retroviral vector systems rely on the use of "packaging cell lines". Generally, a packaging cell line is a cell line capable of producing infectious retroviral particles when an introduction plasmid, a packaging plasmid, and an envelope plasmid are introduced into the cell. Various methods of introducing plasmids into cells, including transfection or electroporation, can be used. In some cases, packaging cell lines for high-efficiency packaging of retroviral vector systems into retroviral particles are applied.

[0284] As used herein, the terms "retroviral vector" or "lentiviral vector" are intended to mean a nucleic acid encoding the retroviral or lentiviral cis nucleic acid sequences necessary for genomic packaging and one or more polynucleotide sequences to be delivered to a target cell. Retroviral particles and lentiviral particles generally contain an RNA genome (derived from an introduced plasmid), a lipid bilayer envelope in which the Env protein is embedded, and other accessory proteins including integrase, protease, and matrix proteins. As used herein, the terms "retroviral particle" and "lentiviral particle" refer to virus particles containing an envelope, having one or more characteristics of lentiviruses, and being capable of entering a target host cell. Such characteristics include, for example, infecting non-dividing host cells, transducing non-dividing host cells, infecting or transducing host immune cells, containing a retroviral or lentiviral virion containing one or more of the gag structural polypeptides, containing a retroviral or lentiviral envelope containing one or more of the env-encoded glycoproteins, containing a genome comprising one or more retroviral or lentiviral cis-acting sequences that function in replication, proviral integration or transcription, containing a genome encoding a retroviral or lentiviral protease, reverse transcriptase or integrase, or containing a genome encoding a regulatory activity such as Tat or Rev. The introduced plasmid may contain the cPPT sequence described in U.S. Patent No. 8,093,042.

[0285] The efficiency of the system is an important concern in vector manipulation. The efficiency of a retroviral or lentiviral vector system can be evaluated by various methods known in the art, including, for example, measurement of vector copy number (VCN) or vector genome (vg) by quantitative polymerase chain reaction (qPCR), or viral titer as infectious units / milliliter (IU / mL). For example, the titer can be evaluated using a functional assay performed on the cultured tumor cell line HT1080 as described in 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 the titer is evaluated in a continuously dividing cultured cell line, no stimulation is required, and thus the measured titer is not affected by surface manipulation of the retroviral particles. Other methods for evaluating the efficiency of a retroviral vector system are provided in Gaererts et al. Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006).

[0286] In some embodiments, the retroviral particles and / or lentiviral particles of the disclosure comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a gating adapter. In some embodiments, the sequence encoding the receptor that specifically binds to the gating adapter is operably linked to a promoter. Exemplary promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the CAG promoter, the SV40 promoter, the SV40 / CD43 promoter, and the MND promoter.

[0287] In some embodiments, the retroviral particle comprises a transduction enhancer. In some embodiments, the retroviral particle comprises a tagging protein.

[0288] In some embodiments, each of the retroviral particles comprises, in order from 5' to 3', (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds a ligand, and (iv) a 3' LTR or UTR polynucleotide.

[0289] In some embodiments, the retroviral particle comprises a cell surface receptor that binds to a surface marker on the target host cell and enables transduction of the host cell. The viral vector may comprise a heterologous viral envelope glycoprotein that confers a pseudotyped viral vector. For example, the viral envelope glycoprotein may be derived from one of RD114 or a variant thereof, VSV-G, gibbon ape leukemia virus (GALV), or an amphotropic envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the cell surface receptor is the VSV G protein or a functional variant thereof from the Cocal strain.

[0290] 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 Cocal virus G protein. In some embodiments, the viral particle comprises a modified VSV G protein lacking LDLR binding affinity. In some embodiments, these mutations comprise mutations at position 47 (e.g., K47Q) and / or position 354 (e.g., R354A).

[0291] In some embodiments, the viral envelope protein is the VSV G protein from the Cocal strain (Cocal glycoprotein). In some embodiments, the VSV G protein is a Cocal envelope protein containing a mutation at position 354 (R354). In some embodiments, the VSV G protein is a Cocal envelope protein containing a mutation at position 47 (K47). In some embodiments, the VSV G protein is a Cocal envelope variant containing the R354Q mutation. In some embodiments, the VSV G protein is a Cocal envelope variant containing the K47Q mutation. In some embodiments, this variant may also be referred to as a "blinded" Cocal envelope. Exemplary Cocal envelope variants are provided, for example, in US2020 / 0216502A1, which is hereby incorporated by reference in its entirety.

[0292] Various fusion glycoproteins can be used to pseudotype lentiviral vectors. The most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), although many other viral proteins have also been used for pseudotyping lentiviral vectors. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present disclosure contemplates the substitution of various fusion glycoproteins. In particular, some fusion glycoproteins result in higher vector efficiency.

[0293] In some embodiments, pseudotyping a fusion glycoprotein or a functional variant thereof facilitates targeted transduction of specific cell types including, but not limited to, natural lymphoid cells or NK cells. In some embodiments, the fusion glycoprotein or a functional variant thereof is a full-length polypeptide, functional fragment, homolog, or functional variant of 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 endogenous virus (BaEV) gp70, measles virus (MV) H and F, Nipah virus (NiV) H and F, rabies virus (RabV) G, Mokola virus (MOKV) G, Ebola Zaire 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 Piry virus CNV-G and PRV-G.

[0294] In some embodiments, the fusion glycoprotein or a functional variant thereof is the full-length polypeptide, a functional fragment, a homolog, or a functional variant of the G protein of Vesicular stomatitis Araguaia virus (VSAV), Carajas vesiculovirus (CJSV), Chandipura vesiculovirus (CHPV), Cocal vesiculovirus (COCV), Vesicular stomatitis Indiana virus (VSIV), Isfahan vesiculovirus (ISFV), Maraba vesiculovirus (MARAV), Vesicular stomatitis New Jersey virus (VSNJV), or Bas-Congo virus (BASV). In some embodiments, the fusion glycoprotein or a functional variant thereof is the Cocal virus G protein.

[0295] In some embodiments, the fusion glycoprotein or a functional variant thereof is the full-length polypeptide, a functional fragment, a homolog, or a functional variant of the G protein of Vesicular stomatitis Araguaia virus (VSAV), Carajas vesiculovirus (CJSV), Chandipura vesiculovirus (CHPV), Cocal vesiculovirus (COCV), Vesicular stomatitis Indiana virus (VSIV), Isfahan vesiculovirus (ISFV), Maraba vesiculovirus (MARAV), Vesicular stomatitis New Jersey virus (VSNJV), or Bas-Congo virus (BASV). In some embodiments, the fusion glycoprotein or a functional variant thereof is the Cocal virus G protein.

[0296] The present disclosure further provides various retroviral vectors including, but not limited to, gamma-retroviral vectors, alpha-retroviral vectors, and lentiviral vectors. In some embodiments, the vector can be a viral vector, a retroviral vector, a lentiviral vector, a gamma-retroviral vector. In some embodiments, the viral vector comprises a VSV G protein or a functional variant thereof. In some embodiments, the viral vector comprises a Cocal G protein or a functional variant thereof.

[0297] 1. Engineered viral envelope In some embodiments, the viral envelope protein is engineered to express a surface engineered protein. In some embodiments, the surface engineered protein is exposed on the surface of the lentiviral particle. In some embodiments, the surface engineered protein is embedded in the lipid bilayer. In some embodiments, the surface engineered protein is composed of a single binding domain protein that binds to a target molecule on the target cell. In some embodiments, the surface engineered protein is composed of a multi-binding domain protein, where each binding domain binds to a target molecule on the target cell. In some embodiments, each binding domain of the multi-binding protein binds to a different target molecule. In some embodiments, the binding domains of the multi-binding protein are connected by linkers. 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 portion of the viral envelope as a fusion protein with the viral envelope protein.

[0298] In some embodiments, the viral envelope contains a transduction enhancer. In some embodiments, the viral envelope contains an immune cell activating protein. In some embodiments, the viral envelope contains a co-stimulatory molecule. In some embodiments, the viral envelope contains an immune cell activating protein and a co-stimulatory molecule. In some embodiments, the viral envelope contains an adhesion molecule.

[0299] In some embodiments, the transduction enhancer is a single domain binding protein or a multi-domain binding protein. In some embodiments, the transduction enhancer contains at least one binding domain that binds to a target molecule selected from an immune cell activating receptor, a T cell co-stimulatory receptor, or an adhesion molecule. In some embodiments, the immune cell activating receptor is a T cell activating receptor or an NK cell activating receptor.

[0300] In some embodiments, the transduction enhancer comprises a single binding domain that binds to one 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 single fusion protein is 200-300 amino acids in length. In some embodiments, the single fusion protein is about 240 amino acids in length.

[0301] In some embodiments, the transduction enhancer comprises a multi-binding domain that binds to two or more target molecules 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 transduction enhancer comprises a multi-binding domain that binds to different target molecules that are an immune cell activation receptor (e.g., a T cell activation receptor), a T cell co-stimulatory receptor, and an adhesion molecule, respectively. In some embodiments, the viral envelope comprises an immune cell activating protein, a co-stimulatory molecule, and an adhesion molecule. In some embodiments, the multi-domain fusion protein is encoded by a nucleic acid sequence of 1,000-3,000 nucleotides. In some embodiments, the multi-domain fusion protein is encoded by a protein that is at least 2,000 nucleotides. In some embodiments, the multi-domain fusion protein is at least 700 amino acids in length.

[0302] In some embodiments, the viral envelope comprises one or more transduction enhancers. In some embodiments, the transduction enhancer comprises a T cell activation receptor, an NK cell activation receptor, and / or a costimulatory molecule. In some embodiments, the 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.

[0303] In some embodiments, the viral particles are surface engineered with a protein that binds to a target molecule on the target cell. In some embodiments, the surface engineering protein is a fusion of one or more binding domains that bind to a 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 comprise a cell surface receptor that binds to a ligand on the target host cell to enable host cell transduction. In some embodiments, the viral particles comprise a heterologous viral envelope glycoprotein that generates pseudotyped viral particles. For example, the viral envelope glycoprotein can be derived from one of RD114 or a variant thereof, VSV-G, gibbon ape leukemia virus (GALV), or is an amphotropic envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the viral envelope glycoprotein is the VSV G protein from the coccal strain (cocal glycoprotein) or a functional variant thereof.

[0304] In some embodiments, the viral envelope contains more than one polypeptide on its surface. In some embodiments, the more than one polypeptide binds to target immune cells and replicates the immunological synapse. In some embodiments, the viral envelope contains immunocyte activation proteins, costimulatory molecules, and adhesion molecules, where the immunocyte activation proteins, costimulatory molecules, and adhesion molecules each bind to target immune cells.

[0305] a. Immunocyte activator In some embodiments, the transduction enhancer comprises a mitogenic stimulus that is incorporated into a retroviral or lentiviral capsid such that the virus performs both activation and transduction of T cells. This eliminates the need to add a vector and a mitogenic factor. In some embodiments, the transduction enhancer comprises a mitogenic transmembrane protein and / or one or more costimulatory molecules that will be incorporated into the retrovirus when budding from the production / package cell membrane. In some embodiments, the transduction enhancer is expressed as a distinct cell surface molecule on the production cell rather than as part of the viral envelope glycoprotein.

[0306] In some embodiments, the viral vectors described herein contain a mitogenic transduction enhancer in the viral envelope. In some embodiments, the mitogenic transduction enhancer is derived from the host cell during retroviral vector production. In some embodiments, the mitogenic transduction enhancer is made by the 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 the packaging cell-derived lipid bilayer. 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.

[0307] In some embodiments, the transduction enhancer is of host cell origin. The term "of host cell origin" indicates that the mitogenic transduction enhancer is derived from the host cell as described above and is not produced as a fusion or chimera from one of the viral genes such as gag encoding the major structural protein or env encoding the envelope protein.

[0308] The envelope protein is formed by two subunits, a transmembrane (TM) that anchors the protein to the lipid membrane and a surface (SU) that binds to the cell receptor. In some embodiments, the mitogenic transduction enhancer from the packaging cells of the present invention does not contain the surface envelope subunit (SU).

[0309] In some embodiments, the mitogenic transduction enhancer has the structure: M-S-TM, where M is the mitogenic domain; S is any spacer domain; and TM is the transmembrane domain.

[0310] The mitogenic domain is part of the mitogenic transduction enhancer that causes T cell activation. This can directly or indirectly bind to or otherwise interact with T cells to lead to T cell activation. In some embodiments, the mitogenic domain binds to T cell surface antigens such as CD3, CD28, CD134, and CD137.

[0311] CD3 is a T cell coreceptor. It is a protein complex composed of four separate chains. In mammals, this complex contains the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with the T cell receptor (TCR) and the ζ chain to generate activation signals in T lymphocytes. The TCR, ζ chain, and CD3 molecules together form the TCR complex. In some embodiments, the mitogenic domain binds to the CD3ε chain.

[0312] 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 the TCR or CD28. In some embodiments, the antibody binds to the TCR, CD3, or CD28. Examples of such antibodies include OKT3, 15E8, and TGN1412. Other suitable antibodies include the following: Anti-CD28: CD28.2, 10F3 Anti-CD3 / TCR: UCHT1, YTH12.5, TR66.

[0313] In some embodiments, the mitogenic domain comprises a binding domain from OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5, or TR66.

[0314] 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 may comprise a binding domain from OX40L or 41BBL.

[0315] OKT3, also known as muromonab-CD3, is a monoclonal antibody that targets the CD3e chain. It is clinically used to reduce acute rejection in organ transplant patients. It was the first monoclonal antibody approved for clinical use in humans.

[0316] In some embodiments, the viral envelope comprises an immune cell activating protein. In some embodiments, the immune cell activating protein specifically binds to a receptor on an immune cell. In some embodiments, the immune cell activating protein provides signal 1 for T cell activation.

[0317] In some embodiments, the immune cell activating 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 activating protein specifically binds to CD3γ, CD3δ, or CD3ε. In some embodiments, the immune cell activating 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 activating protein specifically binds to CD3γ, CD3δ, or CD3ε. In some embodiments, the immune cell activating protein specifically binds to CD3.

[0318] In some embodiments, the immunocyte activating protein is an antibody or an antigen-binding fragment thereof that specifically binds to a receptor on an immunocyte. In some embodiments, the immunocyte activating protein is an antibody or an antigen-binding fragment thereof that specifically binds to 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 immunocyte activating protein is an antibody or an antigen-binding fragment thereof that specifically binds to 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 immunocyte activating protein is an antibody or an antigen-binding fragment thereof that specifically binds to CD3γ, CD3δ, or CD3ε. In some embodiments, the immunocyte activating protein is an antibody or an antigen-binding fragment thereof that specifically binds to CD3.

[0319] Antibodies that target the polypeptides described herein are known to those of skill in the art. Methods for making antibodies are known to those of skill in the art.

[0320] 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 exemplary anti-CD3ε antibody is OKT3. OKT3, also known as muromonab-CD3, is a monoclonal antibody that targets the CD3ε chain.

[0321] In some embodiments, the viral envelope comprises a single-chain Fv fragment (scFv) of an anti-CD3 antibody.

[0322] 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.

[0323] 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.

[0324] 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 that consists of the nucleotide sequence of SEQ ID NO:121.

[0325] 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 that consists of the nucleotide sequence of SEQ ID NO: 123.

[0326] b. Costimulatory molecule In some embodiments, the viral envelope comprises at least one costimulatory molecule. In some embodiments, the costimulatory molecule specifically binds to a receptor on an immune cell. In some embodiments, the costimulation provides signal 2 for cell activation.

[0327] As used herein, the term "costimulatory molecule" refers to a molecule capable of generating a costimulatory signal to T cells. Lymphocytes, such as T cells and natural killer (NK) cells, typically require several signals and interactions with antigen-presenting cells (APCs) for optimal priming to acquire full effector function. For T cells, these include signal transduction through the T cell receptor (TCR), costimulatory molecules (such as CD28 and CD2), cytokines, and various adhesion molecules necessary to ensure sufficient time for proper synapse formation and signal transduction. NK cells require a similar type of stimulation but may rely on different activating receptors, such as NKG2D, NKp46, and DNAM-1. For T cells, in addition to TCR stimulation, appropriate costimulation is particularly important for effective priming, and many studies have shown that TCR stimulation alone can lead to functional anergy and unresponsiveness. Costimulatory signals enhance 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 substances that bind to any of the costimulatory molecules described herein, such as scFv, antibodies, single-domain antibodies, antibody fragments, nanobodies.In some embodiments, these binding substances may 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-TCRα chain, anti-TCRβ chain, anti-TCRγ chain, and anti-TCRδ chain agents.

[0328] In some embodiments, the costimulatory molecule is a ligand for CD28. CD28 is one of the proteins expressed on T cells that provides a costimulatory signal necessary for T cell activation and survival. T cell stimulation via CD28 in addition to the T cell receptor (TCR) can provide a strong signal for the production of various interleukins, particularly IL-6. In some embodiments, the costimulatory molecule is an antibody that binds to CD28, or a fragment thereof. Examples of such antibodies include 15E8 and TGN1412. Other suitable antibodies include CD28.2 and 10F3.

[0329] In some embodiments, the costimulatory molecule is CD86. CD86, also known as B7-2, is a ligand for CD28. In some embodiments, the ligand for CD28 is CD86. In some embodiments, the costimulatory molecule is CD80. CD80 is an additional ligand for CD28. In some embodiments, the ligand for CD28 is CD80. In some embodiments, the ligand for CD28 is an 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 surface presentation on a viral envelope.

[0330] In some embodiments, the co-stimulatory molecule is a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO:5. In some embodiments, the co-stimulatory 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.

[0331] 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.

[0332] In some embodiments, the co-stimulatory molecule is a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO:3. In some embodiments, the co-stimulatory 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.

[0333] 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.

[0334] In some embodiments, the co-stimulatory molecule is a CD80 extracellular domain polypeptide comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the co-stimulatory 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 co-stimulatory molecule is a CD86 extracellular domain polypeptide comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the co-stimulatory 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.

[0335] CD134, also known as OX40, is a member of the TNFR superfamily of receptors expressed on activated T cells. OX40 can promote cell division and survival. OX40 is a secondary co-stimulatory molecule that is expressed 24 - 72 hours after activation; its ligand, OX40L, is not expressed on resting antigen-presenting cells and is expressed following their activation. In some embodiments, the viral particle comprises a ligand for OX40 or a functional fragment thereof, coupled to its native transmembrane domain or a heterologous transmembrane domain.

[0336] 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 found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and cells of the blood vessel wall at the site of inflammation. The best-characterized activity of CD137 is its co-stimulatory activity towards activated T cells. Cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytotoxic activity. In some embodiments, the viral particle comprises a ligand for 4-1BB or a functional fragment thereof, coupled to its native transmembrane domain or a heterologous transmembrane domain. 4-1BBL is a cytokine belonging to the tumor necrosis factor (TNF) ligand family. This transmembrane cytokine is a bidirectional signaling factor that acts as a ligand for the co-stimulatory receptor molecule 4-1BB in T lymphocytes. 4-1BBL has been shown to promote T lymphocyte proliferation and to reactivate anergic T lymphocytes.

[0337] Viral particles containing one or more activation or co-stimulatory molecules may be produced by manipulating a packaging cell line by the method provided by WO 2016 / 139463; or by expression of a T cell activation or co-stimulatory molecule from a multicistronic helper vector as described in International Patent Application Publication No. WO 2020 / 106992 A1, both of which patents are hereby incorporated by reference in their entirety.

[0338] c. Adhesion molecules In some embodiments, the viral particles comprise adhesion molecules. As used herein, the term "adhesion molecule" refers to a subset of cell surface molecules involved in the binding of a cell to another cell. Adhesive cells can help form more stable interactions, such as immune synapses, between immune cells. An immune synapse is a stable adhesive junction between a polarized immune effector cell and an antigen-bearing cell. In some embodiments, the adhesion molecule may provide a co-stimulatory signal to the target cell. In some embodiments, adhesion molecules include, but are not limited to, CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOS-L, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and B7-H6.

[0339] In some embodiments, the adhesion molecule is a fragment or ectodomain of CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOS-L, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and B7-H6. In some embodiments, adhesion molecules include, but are not limited to, SLAMF2, B7-H2, B7-H5, B7-H3, B7x, and TMIGD2. In some embodiments, the adhesion molecule is a fragment or ectodomain of SLAMF2, B7-H2, B7-H5, B7-H3, B7x, and TMIGD2.

[0340] In some embodiments, the adhesion molecule includes, but is not limited to, a binding substance that binds to either the adhesion molecule or the costimulatory molecule described herein, such as an scFv, an antibody, a single-domain antibody, an antibody fragment, and a nanobody. In some embodiments, these binding substances 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-TCRα chain, anti-TCRβ chain, anti-TCRγ chain, and anti-TCRδ chain agents.

[0341] In some embodiments, the adhesion molecule binds to CD2. CD2, also known as T11, LFA-2, and erythrocyte rosette receptor, is a surface protein expressed on T lymphocytes and NK cells. CD2 is the natural ligand for CD58. In addition to performing an adhesion function, the association of CD2 provides a costimulatory signal that can enhance activation and effector functions. In some embodiments, the lentiviral particle includes a molecule that binds to CD2. In some embodiments, the lentiviral particle includes an antibody, single-domain antibody, antibody fragment, and / or nanobody specific for CD2. In some embodiments, the lentiviral particle includes CD58, or a functional portion thereof, that binds to CD2.

[0342] 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.

[0343] 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.

[0344] 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.

[0345] d. Additional non-viral proteins 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 described above.

[0346] 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 its native transmembrane domain or a heterologous transmembrane domain. In some embodiments, CD19 acts as a ligand for blinatumomab, thereby providing an adapter for coupling the particle to a T cell via the anti-CD3 portion of blinatumomab. In some embodiments, another type of particle surface ligand may be useful for coupling appropriately surface-engineered lentiviral particles to T cells using a multispecific antibody comprising a binding moiety for the particle surface ligand. In some embodiments, the multispecific antibody is a bispecific antibody, such as a bispecific T cell engager (BiTE).

[0347] In some embodiments, the non-viral protein is a cytokine. In some embodiments, the cytokine can be selected from the group consisting of 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), the protein can be tethered to the surface of the viral particle by fusion to a transmembrane domain such as the transmembrane domain of CD8. Alternatively, the protein can be indirectly tethered to the lentiviral particle by use of a transmembrane protein engineered to bind to the soluble protein. The further inclusion of one or more cytoplasmic residues can enhance the stability of the fusion protein.

[0348] The mitogenic transduction enhancer and / or cytokine-based transduction enhancer can include a "spacer sequence" for connecting the antigen-binding domain and the transmembrane domain. The flexible spacer can orient the antigen-binding domain in different directions to facilitate binding. As used herein, the term "coupled to" refers to chemical linkage, direct C-terminal to N-terminal fusion of two proteins; chemical linkage to a non-peptide spacer; chemical linkage to a polypeptide spacer; and C-terminal to N-terminal fusion of two proteins via a peptide bond to a polypeptide spacer, such as a spacer sequence.

[0349] The spacer sequence can include, for example, the IgG1 Fc region, the IgG1 hinge, or the human or mouse CD8 stalk. Alternatively, the spacer can include an alternative linker sequence having length and / or domain-spacing characteristics similar to the IgG1 Fc region, the IgG1 hinge, or the CD8 stalk. The human IgG1 spacer can be modified such that the Fc-binding motif is removed. In some embodiments, the spacer sequence can be derived from a human protein.

[0350] In some embodiments, the spacer array comprises a CD8-derived hinge.

[0351] In some embodiments, the spacer array comprises a "short" hinge. A short hinge is described as a hinge region that contains fewer nucleotides compared to a CAR hinge region known in the art.

[0352] The transmembrane domain is an array of a cleavage-promoting transduction enhancer and / or a cytokine-based transduction enhancer that spans the membrane. The transmembrane domain may include a hydrophobic alpha helix. The transmembrane domain may be derived from CD28. In some embodiments, the transmembrane domain is derived from a human protein.

[0353] The viral particles of the present invention may contain a cytokine-based transduction enhancer in the viral envelope. In some embodiments, the cytokine-based transduction enhancer is derived from the host cell during viral particle production. In some embodiments, the cytokine-based transduction enhancer is made by the host cell and expressed on the cell surface. When 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.

[0354] The cytokine-based transduction enhancer may include a cytokine domain and a transmembrane domain. The cytokine-based transduction enhancer can have a structure C-S-TM, where C is the cytokine domain, S is any spacer domain (e.g., spacer array), and TM is the transmembrane domain. The spacer domain and the transmembrane domain are as defined above.

[0355] The cytokine domain can include T cell activating cytokines, for example, from IL2, IL7, and IL15 or functional fragments thereof. As used herein, a "functional fragment" of a cytokine is a fragment of a polypeptide that retains the ability to bind to its specific receptor and activate T cells.

[0356] IL2 is one of the factors secreted by T cells to regulate the growth and differentiation of T cells and certain B cells. IL2 is a lymphokine that induces the proliferation of responsive T cells. It is secreted as a single glycosylated polypeptide, and cleavage of the signal sequence is required for its activity. Solution NMR suggests that the structure of IL2 contains a bundle of four helices (designated A - D) sandwiched between two shorter helices and several poorly defined loops. Residues in helix A and in the loop region between helices A and B are important for receptor binding.

[0357] 2. Payload In some embodiments, the viral particles include 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 particles deliver the payload to target cells.

[0358] 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 the 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.

[0359] In some embodiments, the expression of the payload is driven by a promoter. In some embodiments, the promoter is the MND promoter (myeloproliferative sarcoma virus enhancer, lacking the negative control region and with the dl587rev primer binding site replaced), which contains the U3 region of the modified Moloney murine leukemia virus (MoMuLV) LTR together with the myeloproliferative sarcoma virus enhancer 13 and has high expression in human CD34+ stem cells, lymphocytes and other tissues, and is a virus-derived synthetic promoter. In some embodiments, separate proteins separated by a 2A peptide sequence that induces ribosome skipping and cleavage during translation are expressed. In some embodiments, the promoter is the CMV promoter. In some embodiments, the promoter is the EF1a promoter. In other embodiments, the promoter is the HTLV promoter.

[0360] a. Synthetic cytokine receptor complex In some embodiments, the retroviral vector comprises a nucleotide sequence encoding a synthetic cytokine receptor complex. The synthetic cytokine receptors of the present disclosure each comprise a synthetic gamma chain and a synthetic beta chain, each comprising a dimerization domain. The dimerization domain dimerizes controllably in the presence of a non-physiological ligand, thereby activating the signaling of the synthetic cytokine receptor. In a preferred embodiment, the non-physiological ligand is rapamycin or a rapalog, and such synthetic cytokine receptors are referred to as rapamycin-activated cytokine receptors (RACRs).

[0361] 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-terminus of the transmembrane domain) or intracellular (C-terminus of the transmembrane domain and N-terminus or C-terminus of the intracellular domain of IL-2G).

[0362] The synthetic beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the intracellular domain of interleukin-2 receptor subunit beta (IL-2RB), the intracellular domain of interleukin-7 receptor subunit beta (IL-7RB), or the intracellular domain of interleukin-21 receptor subunit beta (IL-21RB). 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-terminus of the transmembrane domain) or intracellular (C-terminus of the transmembrane domain and N-terminus or C-terminus of the intracellular domain of IL-2RB or IL-7RB).

[0363] i) Intracellular domain In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises the domain of interleukin-2 receptor subunit gamma (IL2Rg).

[0364] 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.

[0365] 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-7RB intracellular domain, and a second dimerization domain.

[0366] 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-21RB intracellular domain, and a second dimerization domain.

[0367] ii) Dimerization domain The dimerization domain can be a heterodimerization domain that includes a 12 kD FK506-binding protein (FKBP) and an FKBP12-rapamycin-binding (FRB) domain that are known in the art to dimerize in the presence of rapamycin or a rapalog.

[0368] Alternatively, the first dimerization domain and the second dimerization domain can be a 12 kD FK506-binding protein (FKBP) and a calcineurin domain that are known in the art to dimerize in the presence of FK506 or an analog thereof.

[0369] In some embodiments, the dimerization domain is a homodimerization domain selected from: i) A 12 kD FK506-binding protein (FKBP); ii) Cyclophilin A (CypA); or iii) Gyrace B (CyrB); Here, the corresponding non-physiological ligands are, respectively, i) FK1012, AP1510, AP1903, or AP20187; ii) Cyclosporin-A (CsA); or iii) Coumermycin or an analog thereof.

[0370] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are the FKBP domain and the cyclophilin domain.

[0371] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are the FKBP domain and the bacterial dihydrofolate reductase (DHFR) domain.

[0372] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are the calcineurin domain and the cyclophilin domain.

[0373] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are PYR1-like 1 (PYL1) and abscisic acid-insensitive 1 (ABI1).

[0374] iii) Transmembrane domain The transmembrane domain is the sequence of the synthetic cytokine receptor that spans the membrane. The transmembrane domain may include a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein.

[0375] iv) Cytosolic FRB The FRB domain is an approximately 100 amino acid domain derived from the mTOR protein kinase. This 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 the transduced cells, giving those cells a growth advantage over native cells.

[0376] In some embodiments, the synthetic cytokine receptor complex comprises a cytosolic polypeptide that binds to a ligand or a complex comprising a ligand.

[0377] In some embodiments, the cytosolic polypeptide comprises an FRB domain. In some embodiments, the cytosolic polypeptide comprises an FRB domain and the ligand is rapamycin. Advantageously, cytosolic FRB confers resistance to the immunosuppressive effects of non-physiological ligands (e.g., rapamycin or rapalogs).

[0378] b. Chimeric antigen receptor In some embodiments, the virus particles described herein are used to transduce a cell (e.g., a T lymphocyte) with a nucleic acid sequence (polynucleotide) encoding one or more chimeric antigen receptors (CARs). In some embodiments, transduction of the virus particles results in the expression of one or more CARs in the transduced cells.

[0379] Conventionally, a CAR is made by fusing a polynucleotide encoding a VL, VH, or scFv to the 5' end of a polynucleotide encoding a transmembrane and intracellular domain, and transducing the cell with the polynucleotide and, optionally, the corresponding VH or VL. Numerous variations of CARs are well known in the art, and the present disclosure contemplates the use of any of the known variations. In addition, VL / VH pairs and scFvs for numerous haptens are known in the art or can be routinely made by conventional methods. Accordingly, the present disclosure contemplates the use of any known hapten-binding domain.

[0380] In some embodiments, the binding portion of the CAR can be, for example, a single-chain variable fragment (scFv) of an antibody, Fab, Fv, Fc, or (Fab')2 fragment. The use of an unmodified (i.e., full-size) antibody, such as IgG, IgM, IgA, IgD, or IgE, in or as a CAR is excluded from the scope of the present invention.

[0381] In some embodiments, the co-stimulatory domain serves to enhance lymphocyte proliferation and survival when the CAR binds to the targeted moiety. The identity of the co-stimulatory domain is limited only to the point of having the ability to enhance cell proliferation and survival activation when the CAR binds to the targeted moiety. Suitable co-stimulatory domains include, but are not limited to, CD28 (see, e.g., Alvarez-Vallina, L. et al., Eur J Immunol. 1996. 26(10):2304-9); CD137 (4-1BB), a member of the tumor necrosis factor (TNF) receptor family (see, e.g., Imai, C. et al., Leukemia. 2004. 18:676-84); and CD134 (OX40), a member of the TNFR superfamily of receptors (see, e.g., Latza, U. et al., Eur. J. Immunol. 1994. 24:677). Those skilled in the art will understand that sequence variants of these co-stimulatory domains can be used, where the variants in this case have the same or similar activity as the domains from which they are modeled. 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.

[0382] In some aspects of the present invention, the CAR construct includes two co-stimulatory domains. As a specific combination, all possible variations of the four described domains are included, and as specific examples, 1) CD28 + CD137 (4-1BB) and 2) CD28 + CD134 (OX40) can be mentioned.

[0383] In some aspects, the activation signaling domain serves to activate the cell when the CAR binds to the targeted moiety. The identity of the activation signaling domain is limited only in that it has the ability to induce activation of the selected cells when the CAR binds to the targeted moiety. Suitable activation signaling domains include the CD3ζ chain and the Fc receptor γ. Those skilled in the art will understand that they can use sequence variants of these described activation signaling domains without adversely affecting the present invention, where the variants in this case have the same or similar activity as the domains from which they are modeled. Such variants can 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.

[0384] In some aspects, the CAR can include additional elements, such as a signal peptide to ensure proper transport 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 that confers adaptability to the recognition region and enables strong binding to the targeted moiety.

[0385] In some embodiments, the payload may include a CAR-like construct. For example, the payload may include a nucleic acid encoding both a receptor and a reporter. In some embodiments, this construct can include the structure S-ETD-MBD-IRES-R, where S is a signal sequence, ETD includes an extracellular targeting domain, MBD includes a membrane-binding domain, IRES includes an internal ribosome entry site, and R encodes a reporter. In some embodiments, the reporter can be a fluorescent protein or an antibiotic resistance marker. In some embodiments, the extracellular targeting domain can include an antibody, an antibody fragment, a small molecule ligand, or a peptide. In some embodiments, the peptide can include part or all of a cytokine or an interleukin.

[0386] Exemplary CAR constructs suitable for CAR-NK cells are provided below: (1) scFv-CD8 TM -4-1BB IC -CD3ζs (see, e.g., Liu E, Tong Y, Dotti G, et al., Leukemia. 2018; 32: 520-531); (2) scFv-CD28 TM+IC -CD3ζs (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 U S 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-CD3ζs (see, for example, Xu Y, Liu Q, Zhong M et al., J Hematol Oncol. 2019; 12: 49); (5) scFv-2B4 TM+IC -CD3ζs (see, for example, Altvater B, Landmeier S, Pscherer S et al., Clin Cancer Res. 2009; 15: 4857-4866); (6) scFv-CD28 TM+IC -4-1BB IC -CD3ζs (see, for example, Kloss S, Oberschmidt O, Morgan M et al., Hum Gene Ther. 2017; 28: 897-913); (7) scFv-CD16 TM -2B4 IC -CD3ζs (see, for example, Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (8) scFv-NKp44 TM -DAP10 IC -CD3ζs (see, for example, Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (9) scFv-NKp46 TM -2B4 IC -CD3ζs (see, for example, Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (10) scFv-NKG2D TM -2B4 IC -CD3ζs (see, for example, Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (11) scFv-NKG2D TM -4-1BB IC -CD3ζs (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 -CD3ζs (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 -CD3ζs (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 -CD3ζS (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); and (15) scFv-NKG2D TM -CD3ζS (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192).

[0387] The affinity of the CAR expressed by lymphocytes to bind to the targeted portion can vary, and in some cases, low affinity binding (such as about 50 nM) may be preferred. However, the binding affinity of the CAR to the targeted ligand is generally 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.

[0388] i) CAR extracellular domain In some embodiments, the 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 an antibody or an antigen-binding portion thereof, or is an antibody or an antigen-binding portion thereof. In some embodiments, the extracellular domain comprises a single-chain Fv domain or is a single-chain Fv domain. The single-chain Fv domain can comprise, for example, a VL linked to a VH by a flexible linker, wherein the VL and VH are from an antibody that binds to the antigen.

[0389] In some embodiments, the extracellular domain of the CAR can contain any polypeptide that binds to a desired antigen (e.g., a prostate neoantigen). The extracellular domain can include an scFv, a portion of an antibody, or an alternative scaffold. The CAR can also be engineered to bind to two or more desired antigens, which can be arranged in series and separated by a linker sequence. For example, one or more domain antibodies, scFvs, llama VHH antibodies, or other VH-only antibody fragments can be organized in series via a linker to provide bispecific or multispecificity to the CAR.

[0390] The antigen to which the extracellular domain of the polypeptide binds can be any antigen of interest and can be, for example, 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 a cell of any tumor or cancer type, for example, an antigen expressed on cells of lymphoma, lung cancer, breast cancer, prostate cancer, adrenocortical cancer, thyroid cancer, nasopharyngeal cancer, melanoma, for example, malignant melanoma, skin cancer, colorectal cancer, desmoid tumor, fibromatosis, round cell tumor, endocrine tumor, Ewing sarcoma, peripheral primitive neuroectodermal tumor, solid embryonal tumor, hepatoblastoma, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms tumor, glioblastoma, myxoma, fibroma, lipoma, etc. In some embodiments, the lymphoma is chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, splenic marginal zone lymphoma, plasmacytic myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal 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 lymphoma, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, nasal type, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK cell lymphoma, mycosis fungoides, Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma (unspecified), anaplastic large cell lymphoma, Hodgkin lymphoma, or non-Hodgkin lymphoma. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL) and the B cells of CLL have a normal karyotype. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL) and the B cells of CLL carry a 17p deletion, 11q deletion, 12q trisomy, 13q deletion, or p53 deletion.

[0391] In some embodiments, the antigen is expressed on B cell malignant cells, recurrent / refractory CD19-expressing malignant 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 malignant cells, colon cancer cells, lung cancer cells, liver cancer cells, breast cancer cells, kidney cancer cells, prostate cancer cells, ovarian cancer cells, skin cancer cells, melanoma cells, bone cancer cells, brain tumor cells, squamous cell carcinoma cells, leukemia cells, myeloma cells, B cell lymphoma cells, kidney cancer cells, uterine cancer cells, adenocarcinoma cells, pancreatic cancer cells, chronic myelogenous leukemia cells, glioblastoma cells, neuroblastoma cells, medulloblastoma cells, or sarcoma cells.

[0392] In some embodiments, the antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In some embodiments, non-limitingly, 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, carbonic anhydrase IX (CAIX), claudin 18.2, FAP, mesothelin, IL13Ra2, Lewis Y, CCNA1, WT-1, TACI, CD38, SLAMF7, CD138, DLL3, transmembrane 4L6 family member 1 (TM4SF1), epithelial cell adhesion molecule (EpCAM), PD-1, PD-L1, CTLA-4, AXL, ROR2, glypican-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-associated antigen (MAGE), CD19, CD20, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, vascular endothelial growth factor receptor (VEGFR), dimeric pyruvate kinase isoenzyme M2 type (tumor M2-PK), abnormal ras protein, or abnormal p53 protein.

[0393] In other aspects, the CAR is a universal CAR that does not itself specifically target tumor antigens. For example, the CAR can include a tag-specific scFv, which allows an exogenous agent that includes the tag and a tumor targeting domain to direct the universal CAR T cells to the target tumor.

[0394] In some aspects, the CAR is a second-generation CAR composed of an anti-fluorescein scFv linked to a 4-1BB co-stimulatory domain and a CD3 zeta intracellular signaling domain.

[0395] In some aspects, the antigen is CD19. CARs targeting CD19 are described, for example, in U.S. Patent Application 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.

[0396] In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding. In some embodiments, the CAR is a second-generation CAR composed of an FMC63 mouse anti-human CD19 scFv linked to a 4-1BB co-stimulatory domain and a CD3 zeta intracellular signaling domain. In some embodiments, the CAR comprises a binding domain for CD19, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for CD19, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for CD19, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain.

[0397] In some embodiments, the CAR is a second-generation CAR composed of an FMC63 mouse anti-human CD19 scFv linked to a CD28 co-stimulatory domain and a CD3 zeta intracellular signaling domain. In some embodiments, the CAR is a second-generation CAR composed of an FMC63 mouse anti-human CD19 scFv linked to a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta intracellular signaling domain.

[0398] In some embodiments, the antigen is BCMA. CAR T therapies targeting BCMA are approved by the FDA and include Abecma and Carvykti. CARs targeting BCMA are described, for example, in U.S. Patent Application Publication No. 2020 / 0246381; U.S. Patent No. 10,918,665; U.S. Patent Application Publication No. 2019 / 0161553, each of which is incorporated herein by reference. In some embodiments, the CAR comprises a binding domain for BCMA, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for BCMA, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for BCMA, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain.

[0399] In some embodiments, the antigen is G protein-coupled receptor class C group 5 member D (GPRC5D). CARs targeting GRC5D are described, for example, in U.S. Patent Application Publication No. 2018 / 0118803 and No. 2021 / 10393689, each of which is incorporated herein by reference. In some embodiments, the CAR comprises a binding domain for GRC5D, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for GRC5D, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for GRC5D, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain.

[0400] In some embodiments, the antigen is Fc receptor-like 5 (FcRL5). CARs targeting FcRL5 are described, for example, in U.S. Patent Application Publication No. US 2017 / 0275362, which is incorporated herein by reference. In some embodiments, the CAR comprises a binding domain for FcRL5, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for FcRL5, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for FcRL5, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain.

[0401] In some embodiments, the antigen is receptor tyrosine kinase-like orphan receptor 1 (ROR1). CARs targeting ROR1 are described, for example, in U.S. Patent Application Publication No. 2022 / 0096651, which is incorporated herein by reference. In some embodiments, the CAR comprises a binding domain for ROR1, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for ROR1, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for ROR1, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain.

[0402] In some embodiments, the CAR is a second-generation CAR composed of an anti-BCMA scFv linked to a 4-1BB co-stimulatory domain and a CD3 zeta intracellular signaling domain. In some embodiments, the CAR is a second-generation CAR composed of an anti-GPRC5D scFv linked to a 4-1BB co-stimulatory domain and a CD3 zeta intracellular signaling domain. In some embodiments, the CAR is a second-generation CAR composed of an anti-ROR1 scFv linked to a 4-1BB co-stimulatory domain and a CD3 zeta intracellular signaling domain.

[0403] 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.

[0404] In some embodiments, the TAA or TSA is a carbohydrate or ganglioside, such as, fuc-GM1, GM2 (tumor fetal antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, and the like.

[0405] 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, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Gage 3,4,5,6,7, 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, CA 19-9, CA 72-4, CAM 17.1. It is NuMa, K-ras, β-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), prostain, TARP (T cell receptor gamma alternative reading frame protein), Trp-p8, STEAP1 (prostate transmembrane epithelial antigen 1 with six transmembrane domains), an abnormal ras protein, or an abnormal p53 protein. In some embodiments, the tumor-associated antigen or tumor-specific antigen is integrin αvβ3 (CD61), galectin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B. Other tumor-associated and tumor-specific antigens are known to those skilled in the art.

[0406] Antibodies and scFvs that bind to TSAs and TAAs include antibodies and scFvs known in the art, and the nucleotide sequences encoding them are likewise known.

[0407] In some embodiments, the antigen is not considered a TSA or TAA but is an antigen associated with tumor cells or damage caused by the tumor. In some embodiments, for example, the antigen is, for example, a growth factor, cytokine or interleukin, for example, a growth factor, cytokine or interleukin associated with angiogenesis or vasculogenesis. Such growth factors, cytokines or interleukins can 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 can also create a hypoxic environment near the tumor. Thus, in some embodiments, the antigen is a hypoxia-related factor, for example, HIF-1α, HIF-1β, HIF-2a, HIF-2β, HIF-3α, or HIF-3β. Tumors can also cause local damage to normal tissue and can cause the release of molecules known as damage-associated molecular pattern molecules (DAMPs; also known as alarmins). In some embodiments, therefore, the antigen can be a DAMP, for example, a heat shock protein, a chromatin-related protein high mobility group box 1 (HMGB1), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), serum amyloid A (SAA), or can be deoxyribonucleic acid, adenosine triphosphate, uric acid or heparan sulfate.

[0408] In some embodiments of the polypeptides described herein, the extracellular domain is connected to the transmembrane domain directly or by a linker, spacer or hinge polypeptide sequence, for example, a sequence from CD28 or a sequence from CTLA4.

[0409] In some embodiments, the extracellular domain that binds to the desired antigen can be derived from an antibody or an antigen-binding fragment thereof made using the techniques described herein.

[0410] Exemplary anti-CD19 CARs are shown in Table 1 along with their various parts including the extracellular domain.

[0411] In some embodiments, the CAR is an anti-CD20 CAR, and the extracellular binding domain of the CD20 CAR is specific for CD20, e.g., human CD20. In some embodiments, the extracellular binding domain of the CD20 CAR is derived from an antibody specific for CD20, e.g., including Leu16, IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronexamab, belzutifan, ublituximab, and ocrelizumab. In any of these embodiments, the extracellular binding domain of the CD20 CAR can comprise or consist of any of the VH, VL, and / or one or more CDRs of the antibody. Exemplary anti-CD20 CARs are shown in Tables 2 and 3 along with their various parts including the extracellular domain.

[0412] Universal CAR In some embodiments, the CAR targets a moiety that is not produced or expressed by the cells of the subject being treated. Thus, this CAR enables the focused targeting of cells to target cells such as cancer cells. Administration of a small conjugate molecule can target the CAR cell response to only the cells expressing the tumor receptor, thereby reducing off-target toxicity, and the rapid clearance of the small conjugate molecule can more readily control the activation of the CAR cells. As an additional advantage, using CAR-expressing cells as "universal" cytotoxic cells can target a wide variety of tumors without the need to prepare separate CAR constructs. The targeting moiety recognized by the CAR can also remain constant. The only part of the system that needs to be changed to enable targeting different identities of cancer cells is the ligand part of the small conjugate molecule.

[0413] Various methods targeting CARs and CAR-expressing cells have been described in the art, including, for example, US 2020 / 0123224, the disclosure of which is incorporated herein by reference. For example, a fluorescein or fluorescein isothiocyanate (FITC) moiety may be conjugated to an agent that binds to a desired target cell (such as a cancer cell), whereby CAR cells expressing an anti-fluorescein / FITC chimeric antigen receptor can selectively target the target cells labeled by the conjugate. As a variant, other haptens recognized by the CAR may be used in place of fluorescein / FITC. The CAR may be made using various scFv sequences known in the art, or scFv sequences made by conventional routine methods. Further exemplary scFv sequences against fluorescein / FITC and other haptens are provided, for example, in WO 2021 / 076788, the disclosure of which is incorporated herein by reference.

[0414] In some embodiments, the CAR is an anti-FITC CAR and the ligand consists of a fluorescein or fluorescein isothiocyanate (FITC) moiety conjugated to an agent that binds to a desired target cell (such as a cancer cell). Exemplary ligands are described below. In some embodiments, the ligand is FITC-folic acid.

[0415] An exemplary anti-FITC CAR is shown in Table 4 along with its various parts.

[0416] In some embodiments, the CAR includes an scFv domain. In some embodiments, the scFv domain includes anti-fluorescein isothiocyanate (FITC) E2. In some embodiments, the scFv domain includes a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).

[0417] 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.

[0418] 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.

[0419] 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.

[0420] 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.

[0421] 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.

[0422] 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.

[0423] 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.

[0424] 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.

[0425] In some embodiments, the CAR system utilizes the conjugate molecule as a bridge between the CAR-expressing cells and the targeted cancer cells. The conjugate molecule is a conjugate comprising a hapten and a cell targeting moiety, such as any suitable tumor cell-specific ligand. Exemplary haptens that can be recognized and bound by the CAR include low molecular weight organic molecules such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin, and digoxigenin, fluorescein and its derivatives (including FITC (fluorescein isothiocyanate), NHS-fluorescein, and pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives), notin, centyrin, and DARPin. Suitable cell targeting moieties that can themselves act as haptens for the CAR include notin (see Kolmar H. et al., The FEBS Journal. 2008. 275(11):26684-90), centyrin, and DARPin (see Reichert, J.M. MAbs 2009. 1(3):190-209).

[0426] In some embodiments, the cell targeting moiety is DUPA (DUPA-(99m)Tc), i.e., a ligand to which PSMA-positive human prostate cancer cells bind with nanomolar affinity (K D = 14 nM; see Kularatne, S.A. et al., Mol Pharm. 2009. 6(3):780-9). In one embodiment, the DUPA derivative can be a ligand of a low molecular weight ligand linked to the targeting moiety, and the DUPA derivative is described in WO 2015 / 057852, which is incorporated herein by reference.

[0427] In some embodiments, the cell targeting moiety is a CCK2R ligand, i.e., a ligand to which CCK2R-positive cancer cells (e.g., cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon; see Wayua. C. et al., Molecular Pharmaceutics. 2013. ePublication) bind.

[0428] In some embodiments, the cell targeting moiety is a folate, folic acid, or an analog thereof, i.e., a ligand that binds to folate receptors on cancer cells including cancers of the ovary, cervix, endometrium, lung, kidney, brain, breast, colon, and head and neck; see Sega, E.I. et al., Cancer Metastasis Rev. 2008. 27(4):655-64.

[0429] In some embodiments, the cell targeting moiety is an NK-1R ligand. Receptors for NK-1R ligands are found, for example, on cancers of the colon and pancreas. In some embodiments, the NK-1R ligand can be synthesized according to the method disclosed in International Patent Application No. PCT / US2015 / 044229, which is incorporated herein by reference.

[0430] In some embodiments, the cell targeting moiety can be a peptide ligand, e.g., the ligand can be a peptide ligand that is an endogenous ligand for the NK1 receptor. In some embodiments, the small conjugate molecule ligand can be a regulatory peptide belonging to the tachykinin family that targets tachykinin receptors. Such regulatory peptides include substance P (SP), neurokinin A (substance K), and neurokinin B (neuromedin K) (see Hennig et al., International Journal of Cancer: 61, 786-792).

[0431] In some embodiments, the cell targeting moiety is a CAIX ligand. The receptor for the CAIX ligand is found, for example, on the kidney, ovary, vulva, and breast cancer. The CAIX ligand may also sometimes be referred to herein as CA9.

[0432] In some embodiments, the cell targeting moiety is a ligand for gamma-glutamyl transpeptidase. This transpeptidase is overexpressed, for example, in ovarian cancer, colon cancer, liver cancer, glioblastoma, melanoma, and leukemia.

[0433] In some embodiments, the cell targeting moiety is a CCK2R ligand. The receptor for the CCK2R ligand is found, inter alia, on cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon.

[0434] 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.

[0435] In an exemplary aspect, the linkage in the conjugates described herein can be a direct linkage (e.g., a reaction between the isothiocyanate group of FITC and the free amine group of a small molecule ligand), or the linkage can be through an intervening linker. In one aspect, if present, the intervening linker can be any biocompatible linker known in the art, such as a divalent linker. In an exemplary aspect, the divalent linker can contain from about 1 to about 30 carbon atoms. In another exemplary aspect, the divalent linker can contain from about 2 to about 20 carbon atoms. In other aspects, lower molecular weight divalent linkers (i.e., those having an approximate molecular weight of about 30 to about 300 Da) are used. In another aspect, 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.

[0436] In some aspects, the hapten and the cell targeting moiety can be directly conjugated via means such as a 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 linking domain to connect the two molecules can be useful only if it provides adaptability and stability. Examples of suitable linking domains include: 1) polyethylene glycol (PEG); 2) polyproline; 3) hydrophilic amino acids; 4) sugars; 5) non-natural peptidoglycans; 6) polyvinylpyrrolidone; 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.

[0437] In some embodiments, the linker can be a bivalent linker that can include one or more spacers.

[0438] Exemplary 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.

[0439] The affinity of the ligand for binding to the cancer cell receptor can vary, and in some cases, low affinity binding (such as about 1 μM) may be preferred, but the binding affinity of the ligand for the cancer cell receptor is generally 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.

[0440] Examples of conjugates and methods of making them are provided in US Patent Applications US 2017 / 0290900, US 2019 / 0091308, and US 2020 / 0023009, all of which are incorporated herein by reference.

[0441] In some embodiments, the viral particles described herein include a nucleotide sequence encoding a universal modular anti-tag chimeric antigen receptor (UniCAR). This system enables reprogramming of UniCAR-transduced immune cells against multiple antigens (see, e.g., US Patent Application Publication US20170240612 A1, which is incorporated herein by reference in its entirety; see also Cartellieri et al., (2016) Blood Cancer Journal 6, e458, which is incorporated herein by reference in its entirety).

[0442] In some embodiments, the viral particles described herein include nucleotide sequences encoding switchable CARs and / or CAR effector cell (CAR-EC) switches. In this system, the CAR-EC switch has a first region to which the CAR on the CAR-EC binds and a second region that binds to a cell surface molecule on the 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. The activity can be “turned off” by reducing or stopping administration of the switch. These CAR-EC sw...

Claims

1. A method for preparing a lentiviral preparation comprising: (i) a step of filtering a suspension mixture comprising a population of host cells and lentiviral particles to remove contaminants, (a) filtering the mixture through a first filter which is a depth filter, resulting in a first filtrate; (b) filtering the first filtrate through a second filter having a retention threshold smaller than that of the first filter, resulting in a second filtrate; and (c) filtering the second filtrate through a third filter having a retention threshold smaller than that of the second filter, thereby producing a filtered preparation of lentiviral particles; comprising the step; and (ii) a step of concentrating the filtered preparation of lentiviral particles, the step comprising chromatography and ultrafiltration.

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

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

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

5. The method according to claim 3 or 4, wherein the cell comprises HEK293 cells.

6. The method according to claim 3 or 4, wherein the cell comprises HEK293T cells.

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

8. The method according to claim 7, wherein the first filter has a retention threshold of 60 μm.

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

10. The method according to 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 to 10, wherein the third filter has a retention threshold of 0.2 to 0.3 μm.

13. The method according to claim 12, wherein the third filter has a retention threshold of 0.2 μm.

14. The method according to any one of claims 1 to 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 according to any one of claims 1 and 3 to 14, wherein the endonuclease is present through steps (i)(a) to (i)(c).

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

17. The method according to any one of claims 1 to 16, wherein the second filter and the third filter are two layers in a two-layer filter component.

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

19. The method according to 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 filter is 0.45 μm, and the retention threshold of the second layer filter is 0.2 μm.

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

21. The method according to claim 20, wherein the AEX chromatography includes eluting the lentiviral particles with a salt buffer.

22. The method according to claim 21, wherein the salt buffer contains NaCl. **Claim 23** The method according to claim 22, wherein the NaCl has a concentration of about 0.5 M to 3 M. **Claim 24** The method according to claim 22, wherein the NaCl has a concentration of about 0.5 M to 1 M. **Claim 25** The method according to any one of claims 22 to 24, wherein the NaCl is 0.75 M or about 0.75 M. **Claim 26** The method according to claim 22, wherein the NaCl has a concentration of about 1 M to 3 M. **Claim 27** The method according to claim 22, wherein the NaCl has a concentration of about 1.5 M to 2.5 M. **Claim 28** The method according to any one of claims 22, 26 or 27, wherein the NaCl is about 2 M. **Claim 29** The method according to any one of claims 1 to 28, wherein the chromatography is performed before the ultrafiltration. **Claim 30** The method according to any one of claims 1 to 29, wherein the ultrafiltration is ultrafiltration / diafiltration (UF / DF). **Claim 31** The method according to claim 30, wherein the UF / DF is by one or more tangential flow filtration (TFF) steps. **Claim 32** The method according to claim 31, wherein one or more TFF filters are hollow fiber filters. **Claim 33** The method according to claim 32, wherein the nominal molecular weight cut-off (NMWC) of the hollow fiber filter is 500 kDa or about 500 kDa. **Claim 34** The method according to any one of claims 31 to 33, wherein the TFF includes a first tangential flow filtration (TFF) stage and a second tangential flow filtration (TFF) stage.

35. The method according to claim 33, wherein the first TFF is performed using a first hollow fiber filter, and the second TFF is performed using a second hollow fiber filter.

36. The method according to claim 35, wherein the first and second hollow fiber filters have the same nominal molecular weight cut-off (NMWC).

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

38. The method according to claim 35, wherein the first hollow fiber filter has a nominal molecular weight cut-off (NMWC) greater than that of the second hollow fiber filter.

39. The method according to claim 38, wherein the NMWC of the first hollow fiber filter is 500 kDa.

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

41. The surface area of the first hollow fiber filter is 790 cm 2 to 1600 cm 2 The method according to claim 40, wherein the surface area is as described above.

42. The method according to any one of claims 35 to 41, wherein the first hollow fiber filter accommodates a volume greater than that of the second hollow fiber filter.

43. The method according to claim 42, wherein the volume of the first hollow fiber filter is 300 mL.

44. The method according to any one of claims 1 to 43, comprising sterile filtration of the filtered formulation after concentration, thereby producing a sterile formulation. **Claim 45** The method according to claim 44, wherein the sterile filtration comprises filtering the filtered formulation through a fourth filter. **Claim 46** The method according to claim 45, wherein the fourth filter has a retention threshold of 0.2 μm. **Claim 47** A step of formulating the sterile formulation in a buffer solution, thereby producing a drug substance. The method according to any one of claims 44 to 46, comprising the step. **Claim 48** The method according to any one of claims 1 to 47, which is carried out at a pH of 6 to 8. **Claim 49** The method according to any one of claims 1 to 48, wherein the lentiviral formulation is for in vivo administration to a subject. **Claim 50** The method according to any one of claims 1 to 49, wherein the amount of contaminants in the filtered formulation is reduced compared to the amount of contaminants in the second filtrate. **Claim 51** The method according to any one of claims 44 to 50, wherein the amount of contaminants in the sterile formulation is at a level acceptable for in vivo administration to a subject. **Claim 52** The method according to any one of claims 1 to 51, wherein the contaminants include host cells, host cell DNA (hcDNA), and / or host cell proteins (HCP). **Claim 53** The method according to 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 sterile formulation. **Claim 54** The method according to claim 52, wherein the amount of hcDNA 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 the sterilized preparation.

55. The method according to 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 preparation.

56. The method according to claim 52, wherein the amount of HCP 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 the sterilized preparation.

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

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

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

60. The method according to any one of claims 52 to 59, wherein the amount of HCP after chromatography is less than about 3000 μg / 1E9 TU.

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

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

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

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

65. The method according to any one of claims 1 to 64, wherein the suspension mixture contains a medium that does not contain serum and / or animal by-products.

66. The method according to any one of claims 2 to 65, wherein the culturing step in step (ii) lasts for 40 to 48 hours.

67. The method according to any one of claims 1 to 66, wherein the filtering step and the concentrating step are carried out over a period of 5 to 8 hours.

68. The method according to any one of claims 1 to 67, wherein the suspension mixture has a volume of 3 to 50 liters.

69. The method according to any one of claims 1 to 67, wherein the suspension mixture has a volume of 5L to 200L.

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

71. The method according to any one of claims 1 to 67, wherein the suspension mixture has a volume from 180L or about 180L to 200L or about 200L.

72. The method according to any one of claims 1 to 71, wherein the lentiviral particles contain at least one payload.

73. The method according to claim 72, wherein the at least one payload contains non-coding nucleic acid, and optionally, the non-coding nucleic acid is siRNA, miRNA, or shRNA.

74. The method according to claim 72, wherein at least the payload is a polynucleotide encoding a polypeptide of interest. **Claim 75** The method according to any one of claims 2 to 74, wherein the at least one plasmid is a polynucleotide encoding a polypeptide of interest. **Claim 76** The method according to claim 74 or 75, wherein the polypeptide of interest is a chimeric antigen receptor (CAR). **Claim 77** The method according to claim 76, wherein the CAR is specific for a tumor-associated antigen. **Claim 78** The method according to claim 77, wherein the tumor-associated antigen is CD19, BCMA, GPRC5D, ROR1, FcRL5, alpha-fetoprotein, or Her2. **Claim 79** The method according to claim 76, wherein the CAR is a universal CAR. **Claim 80** The method according to claim 79, wherein the universal CAR comprises an extracellular domain comprising a tag-binding domain. **Claim 81** The method according to claim 80, wherein the tag is fluorescein. **Claim 82** The method according to claim 76, wherein the CAR comprises an extracellular domain comprising a hapten-binding domain. **Claim 83** The method according to any one of claims 1 to 82, wherein the lentiviral particles comprise a surface-engineered fusion protein exposed on the surface of the lentiviral particles, and optionally, the surface-engineered protein is embedded in the lipid bilayer. **Claim 84** The method according to claim 83, wherein the surface-engineered protein is composed of a single-binding domain protein that binds to a target molecule on the target cell. **Claim 85** The method according to claim 84, wherein the surface engineering protein is composed of a multi-binding domain protein, each binding domain binds to a target molecule on the target cell, and optionally, each binding domain binds to a different target molecule.

86. The method according to claim 84 or claim 85, wherein the single-binding domain protein or the multi-binding domain protein is a transduction enhancer protein.

87. The method according to any one of claims 83 to 86, wherein the surface engineering protein is a fusion protein comprising a transduction enhancer and a viral envelope protein.

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

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

90. The method according to any one of claims 2 to 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 according to claim 89 or claim 90, wherein the immunocyte activating protein comprises at least one binding domain that 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, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, or NKp80, or a combination thereof.

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

93. The method according to claim 89 and claim 92, wherein the costimulatory 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 according to any one of claims 86 to 90, wherein the transduction enhancer comprises at least one binding domain that binds to a target molecule selected from the group consisting of immunocyte activation receptors, T cell costimulatory receptors, or adhesion molecules.

95. Does the transduction enhancer comprise a single binding domain that binds to one target molecule selected from the group consisting of immunocyte activation receptors, T cell costimulatory receptors, or adhesion molecules; Does the transduction enhancer comprise a plurality of binding domains that bind to two or more target molecules selected from the group consisting of immunocyte activation receptors, T cell costimulatory receptors, and adhesion molecules; or The transduction enhancer includes a plurality of binding domains that each bind to different target molecules that are immune cell activation receptors, T cell co-stimulatory receptors, and adhesion molecules. The method according to any one of claims 86 to 94.

96. 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. The method according to any one of claims 94 to 95.

97. Each of the at least one binding domain is selected independently of an antibody or antigen-binding fragment of the target molecule or an ectodomain of a native ligand, the method according to any one of claims 84 to 96.

98. 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, the method according to any one of claims 87 to 97.

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

100. The helper virus protein is rev and / or gagpol, the method according to claim 99.

101. The population of host cells is contacted with a mixture of plasmids 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, the method according to any one of claims 2 to 100.

102. The method according to claim 101, wherein the mixture of plasmids comprises (v) a plasmid encoding an immunocyte activating protein, (vi) a plasmid encoding a costimulatory molecule, or (vii) any combination of (v) to (vi).

103. A lentiviral preparation produced by the method according to any one of claims 1 to 102.

104. The lentiviral preparation according to claim 103, wherein the preparation has an infectivity titer of 2.0 TU / mL to 6×10 8 TU / mL.

105. The lentiviral preparation according to claim 103 or claim 104, wherein the preparation has an infectivity titer of 2.5 TU / mL to 4.7×10 8 TU / mL.

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

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

108. The lentiviral preparation according to any one of claims 104 to 107, wherein the preparation contains 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 HCP.

109. The lentiviral preparation according to any one of claims 104 to 108, wherein the preparation contains 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 hcDNA.

110. The lentiviral formulation according to any one of claims 104 to 109, wherein the formulation comprises a reduction of 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% of HCP.

111. The lentiviral formulation according to any one of claims 104 to 110, wherein the formulation comprises a reduction of 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% of hcDNA, optionally reduced compared to the filtered formulation before the concentration step.

112. The lentiviral formulation according to any one of claims 104 to 111, wherein the formulation comprises a reduction of more than 99% of hcDNA and a reduction of more than 99% in HCP, optionally reduced compared to the filtered formulation before the concentration step.

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

114. 2.5 to 4.7×10 8 TU / mL titer lentiviral vector-containing lentiviral formulation, wherein the formulation comprises less than 1% of hcDNA and less than 1% of HCP.

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

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