Retroviral and lentiviral vectors

Abstract

To provide retroviral and lentiviral vectors.SOLUTION: The invention provides a retroviral or lentiviral vector having a viral envelope comprising: (i) a mitogenic T cell activating transmembrane protein comprising a mitogenic domain and a transmembrane domain; and / or (ii) a cytokine-based T cell activating transmembrane protein comprising a cytokine domain and a transmembrane domain, wherein the mitogenic or cytokine-based T cell activating transmembrane protein is not part of the viral envelope glycoprotein. When cells such as natural killer cells are transduced with such viral vectors, they are simultaneously activated by the mitogenic T cell activating transmembrane protein and / or the cytokine-based T cell activating transmembrane protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to retroviral and lentiviral vectors and cells for their production. The vectors can be used to transduce cells, such as T cells. In particular, the present invention relates to retroviral or lentiviral vectors that can both transduce and activate cells, such as T cells. [Background technology]

[0002] The generation of genetically engineered T cell products typically requires mitogen stimulation followed by transduction with an integrating vector, such as a lentiviral or retroviral vector.

[0003] A widely used approach is to add soluble mitogenic monoclonal antibodies (mAbs), such as anti-TCR / CD3 and anti-CD28, to cell cultures. An alternative approach is to attach anti-TCR / CD3 mAbs to beads together with anti-CD28 mAbs. The surface of the beads has improved T cell activation properties compared to soluble antibodies alone.

[0004] Additionally, cytokines (eg, IL2, IL15, or IL7) are commonly added to the cell culture.

[0005] These mitogenic antibodies and cytokines are single-use consumables and typically represent the most expensive part of the T cell production process.

[0006] Maurice et al. have developed a lentivirus in which the CD3 agonist OKT3 is displayed on the virion surface. have described the direct genetic manipulation of viral envelope proteins (Maurice et al., Blood, 2002, 99, 2342-2350). described a similar approach in which the envelope protein was engineered to incorporate IL7 (Verhoeyen et al.; Blood; 2003; 101; 2167-2174). Each of these genetic engineering approaches requires complex genetic manipulation of viral envelope proteins, which must be performed so that individual peptides are displayed on the virion surface. This approach has also been shown to reduce viral titers. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Maurice et al.; Blood; 2002; Volume 99; Pages 2342-2350 [Non-patent document 2] Verhoeyen et al.; Blood; 2003; Volume 101; Pages 2167-2174 Summary of the Invention [Means for solving the problem]

[0008] Therefore, there is a need for new approaches to generate genetically engineered T cell products that do not suffer from the drawbacks mentioned above. [Brief explanation of the drawings]

[0009] [Figure 1] Diagram of retroSTIM vector surrounded by a lipid bilayer interspersed with RD114 envelope glycoprotein and various mitogenic elements such as scFvs or membrane-bound cytokines.

[0010] [Figure 2]Demonstration that OKT3 scFv can be incorporated into lentivirus. Results show activation of T cells. (a) Unstimulated 293T cells transduced with lentiviral vector; (b) OKT3, CD28.2, and IL2-stimulated 293T cells transduced with lentiviral vector; (c) Unstimulated 293T.OKT3 transduced with supernatant and transfected with transfer vector only; (d) Unstimulated 293T.OKT3 transduced with lentiviral vector. The top panel shows a scatter plot of transduction (x-axis) and activation by CD25 expression (y-axis). The bottom panel shows a photomicrograph of T cell culture. Clumping indicated activation.

[0011] [Figure 3] Demonstration of gagpol-dependent T cell mitogenic stimulation and transduction. 293T cells stably expressing surface-bound OKT3 were transfected with all three plasmids along with gagpol, RD-PRO env, transfer vector, or rev. The resulting supernatant was applied to primary human T cells. T cells were examined by flow cytometry using the following parameters: CD25 to measure T cell activation; anti-Fc to detect the transgene, a CAR with an Fc spacer; and ki67 to determine cycling cells. Only the condition in which gagpol was provided resulted in significant mitogenic stimulation. Only the condition in which all plasmids (along with rev) were provided resulted in mitogenic stimulation and transduction of T cells.

[0012] [Figure 4]Demonstration that different lentiviral pseudotypings support mitogenic effects. 293T cells stably expressing membrane-bound OKT3 were transfected with lentiviral transfer vectors, lentiviral gagpol, rev, and different env plasmids: VSV-G, RD-PRO, Ampho, GALV, and measles M / H. The resulting supernatants were applied to primary human T cells. Cells were then stained with Ki67 and examined by flow cytometry. All pseudotypes supported mitogenic effects, although the effect appeared to be reduced with measles pseudotyping.

[0013] [Figure 5] Demonstration of mitogenic stimulation and transduction of T cells using gamma-retroviral vectors. 293T cells stably expressing membrane-bound OKT3 were transfected with a gamma-retroviral transfer vector encoding a CAR, a gamma-retroviral gagpol expression plasmid, and an RD114 expression plasmid. The resulting supernatant was applied to primary human T cells. T cells were then stained with anti-Fc, anti-CD25, and ki67 and examined by flow cytometry. Although no mitogenic stimulation was applied, T cells were activated, cycled, and expressed the transgene.

[0014] [Figure 6] Demonstration that two different mitogenic stimuli can be incorporated into a viral vector and that anti-CD3 / TCR stimulation in conjunction with anti-CD28 stimulation has improved efficacy compared to anti-CD3 / TCR alone.

[0015] [Figure 7] Low-resolution microscopy images of T cells stimulated with various lentiviral vectors generated from 293T cells expressing various elements on the cell surface.

[0016] [Figure 8]Activation of CD4 and CD8 T cells after transduction of lentiSTIM vectors displaying various combinations of mitogenic and cytokine peptides. Activation is determined by CD25 expression 120 hours post-transduction.

[0017] [Figure 9] Proliferation of CD4 and CD8 T cells after transduction of lentiSTIM vectors displaying various combinations of mitogenic and cytokine peptides. Proliferation is determined by Ki67 expression 120 hours post-transduction.

[0018] [Figure 10] Expansion of T cells following transduction of lentiSTIM vectors displaying various combinations of mitogenic and cytokine peptides. Expansion is determined by absolute cell counts 120 hours post-transduction.

[0019] [Figure 11] Examination of T cell subset phenotypes in PBMCs activated with lentiSTIM vectors expressing anti-CD3 and anti-CD28 antibodies or beads coated with anti-CD3 and anti-CD28 antibodies. NM-LV = unmodified lentivirus; STIM-LV = lentiSTIM vector; Tem = effector memory T cells; Tcm = central memory T cells; Tscm = memory T stem cells; Tn = naive T cells. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention is based on the discovery that it is possible to incorporate mitogenic and / or cytokine stimuli into the capsid of retroviruses or lentiviruses so that these viruses activate and transduce T cells. This eliminates the need for added vectors, mitogens, and cytokines. The present invention involves including a mitogenic transmembrane protein and / or cytokine-based transmembrane protein in the producer or packaging cell that is incorporated into the retrovirus as it budding from the producer / packaging cell membrane. The mitogenic transmembrane protein and / or cytokine-based transmembrane protein is expressed as a separate cell surface molecule on the producer cell, rather than as part of the viral envelope glycoprotein. This means that the reading frame of the viral envelope is not affected, thus preserving functional integrity and viral titer.

[0021] Thus, in a first aspect, the present invention provides a method for producing a medicament for the treatment of a medicament comprising: (i) a mitogenic T cell-activating transmembrane protein comprising a mitogenic domain and a transmembrane domain; and / or (ii) a cytokine-based T cell-activating transmembrane protein comprising a cytokine domain and a transmembrane domain; wherein the mitogenic or cytokine-based T cell activating transmembrane protein is not part of the viral envelope glycoprotein.

[0022] Retroviral or lentiviral vectors may contain a separate viral envelope glycoprotein encoded by the env gene. therefore, (i) viral envelope glycoproteins; and (ii) Structure: Mitogenic T-cell activating transmembrane protein with MS-TM (wherein M is a mitogenic domain, S is an optional spacer, and TM is a transmembrane domain); and / or (iii) a cytokine-based T cell-activating transmembrane protein comprising a cytokine domain and a transmembrane domain; A retroviral or lentiviral vector having a viral envelope comprising:

[0023] The mitogenic T cell activation transmembrane proteins and / or cytokine-based T cell activation transmembrane proteins are not part of the viral envelope glycoproteins. They are present in the viral envelope as separate proteins and are encoded by separate genes. The mitogenic T cell activating transmembrane protein may have the structure: MS-TM, where M is a mitogenic domain, S is an optional spacer, and TM is a transmembrane domain.

[0024] The mitogenic T cell activating transmembrane protein can bind to an activating T cell surface antigen, such as CD3, CD28, CD134, or CD137. The mitogenic T cell activating transmembrane protein may comprise an agonist for such an activating T cell surface antigen.

[0025] Mitogenic T cell activating transmembrane proteins may include binding domains from antibodies such as OKT3, 15E8, TGN1412; or costimulatory molecules such as OX40L or 41BBL.

[0026] The viral vector may contain two or more mitogenic T cell activating transmembrane proteins in the viral envelope. For example, the viral vector may contain a first mitogenic T cell activating transmembrane protein that binds to CD3 and a second mitogenic T cell activating transmembrane protein that binds to CD28.

[0027] The cytokine-based T cell activation transmembrane protein may, for example, comprise a cytokine selected from IL2, IL7 and IL15. especially, (ia) the first mitogenic T cell activation transmembrane protein that binds to CD3; and (ib) A second mitogenic T cell activation transmembrane protein that binds to CD28 A retroviral or lentiviral vector having a viral envelope comprising:

[0028] (ia) the first mitogenic T cell activation transmembrane protein that binds to CD3; (ib) a second mitogenic T cell activation transmembrane protein that binds to CD28; and (ii) cytokine-based T cell activation transmembrane proteins, including IL2; Also provided is a retroviral or lentiviral vector having a viral envelope comprising:

[0029] (ia) the first mitogenic T cell activation transmembrane protein that binds to CD3; (ib) a second mitogenic T cell activation transmembrane protein that binds to CD28; (iia) cytokine-based T cell activation transmembrane proteins, including IL7; and (iib) cytokine-based T cell activation transmembrane proteins, including IL15. Also provided is a retroviral or lentiviral vector having a viral envelope comprising:

[0030] The viral vector may contain a heterologous viral envelope glycoprotein to provide a pseudotyped viral vector. For example, the viral envelope glycoprotein may be derived from RD114 or one of its mutants, VSV-G, gibbon ape leukemia virus (GALV), or amphotropic envelope, measles envelope, or baboon retrovirus envelope glycoprotein.

[0031] In a second embodiment of the first aspect of the present invention, the viral envelope of the viral vector also comprises: (iv) a binding domain that binds to the capture moiety spacer; and transmembrane domain The tagged protein may include a tagged protein comprising the following:

[0032] The binding domain of the tagged protein can include one or more streptavidin-binding epitopes, which can be biotin mimics, such as biotin mimics that bind to streptavidin with lower affinity than biotin, so that biotin can be used to elute streptavidin-captured retroviral vectors produced by packaging cells.

[0033] Examples of suitable biotin mimetics include StreptagII (SEQ ID NO: 36), Flankedccstretag (SEQ ID NO: 37) and ccstreptag (SEQ ID NO: 38).

[0034] The viral vector of the first aspect of the invention may comprise a nucleic acid sequence encoding a T cell receptor or a chimeric antigen receptor.

[0035] The viral vector may be a virus-like particle (VLP).

[0036] In a second aspect, the present invention provides a method for producing a protein comprising, at the cell surface: (ii) a mitogenic T cell-activating transmembrane protein comprising a mitogenic domain and a transmembrane domain; and / or (ii) a cytokine-based T cell-activating transmembrane protein comprising a cytokine domain and a transmembrane domain; wherein the packaging cell expresses a vector encoding a retroviral or lentiviral vector, such that the retroviral or lentiviral vector produced by the packaging cell is as defined in the first aspect of the invention. In a second embodiment of the second aspect of the invention, the host cell also comprises at the cell surface: (iii) a binding domain that binds to the capture moiety; and Transmembrane domain wherein the tagged protein comprises a tagged protein that facilitates purification of the viral vector from the cell supernatant via binding of the tagged protein to the capture moiety, such that the retroviral or lentiviral vector produced by the packaging cell may be as defined in the second embodiment of the first aspect of the present invention.

[0037] The tagged protein may also include a spacer between the binding domain and the transmembrane domain.

[0038] The term host cell may be a packaging cell or a producer cell.

[0039] The packaging cells may contain one or more of the following genes: gag, pol, env and / or rev.

[0040] The producer cells contain the gag, pol, env and optionally rev genes, and also contain the retroviral or lentiviral genome.

[0041] In this regard, the host cell can be any suitable cell line stably expressing mitogenic and / or cytokine transmembrane proteins, which can be transiently transfected with the transfer vectors, gagpol, env (and rev in the case of lentivirus) to produce replication-incompetent retroviral / lentiviral vectors.

[0042] In a third aspect, there is provided a method for producing a host cell according to the second aspect of the invention, comprising the step of transducing or transfecting the cell with a nucleic acid encoding a mitogenic T cell activation transmembrane protein and / or a cytokine-based T cell activation transmembrane protein.

[0043] In a fourth aspect, there is provided a method for producing a viral vector according to the first aspect of the invention, comprising expressing a retroviral or lentiviral genome in a cell according to the second aspect of the invention.

[0044] In a fifth aspect, there is provided a method for generating activated transgenic T cells or natural killer (NK) cells, comprising the step of transducing T cells or NK cells with a viral vector according to the first aspect of the invention, such that the T cells or NK cells are activated by one or more mitogenic T cell activating transmembrane proteins and / or one or more cytokine-based T cell activating transmembrane proteins.

[0045] In a sixth aspect, (i) a host cell as defined in the second aspect of the invention; (ii) a nucleic acid comprising gag, pol, env, and optionally rev; and (iii) Retroviral genome There is provided a kit for producing a retroviral or lentiviral vector as defined in the first aspect of the invention, comprising:

[0046] (i) a packaging cell as defined in the second aspect of the present invention; and (ii) Retroviral genome There is also provided a kit for producing a retroviral or lentiviral vector as defined in the first aspect of the invention, comprising:

[0047] (i) one or more nucleic acids encoding mitogenic T cell activation transmembrane proteins and / or cytokine-based T cell activation transmembrane proteins; and (ii) a nucleic acid containing the gag, pol, and env genes of a retrovirus There is also provided a kit for producing a packaging cell according to the second embodiment of the second aspect of the invention, comprising:

[0048] (i) one or more nucleic acids encoding mitogenic T cell activating transmembrane proteins and / or cytokine-based T cell activating transmembrane proteins; (ii) a nucleic acid comprising the gag, pol, and env genes of a retrovirus; and (iii) a retroviral or lentiviral vector genome There is also provided a kit for producing a producer cell according to the second aspect of the invention comprising:

[0049] Thus, the present invention provides a viral vector with built-in mitogenic and / or cytokine stimulation (see FIG. 1). This vector has the ability to stimulate T cells and also function as a gene insert. This has many advantages: (1) it simplifies the process of T cell genetic engineering, since only one component needs to be added; (2) it avoids bead removal and the associated yield loss, since the virus is unstable and does not need to be removed. (3) Reduces the cost of T cell genetic engineering, since only one component needs to be manufactured; (4) Allows greater design flexibility: each T cell genetic engineering process will involve the creation of a gene transfer vector, and the product can be "fitted" with a mitogenic stimulus to produce the same product; (5) Allows for a shortened production process: in soluble antigen / bead-based approaches, the mitogen and vector are typically given separately, sequentially, over 1, 2, or sometimes 3 days, but with the retroviral vectors of the present invention, this can be avoided because the mitogenic stimulus and viral entry occur synchronously and simultaneously; (6) Genetic engineering is easier, since there is no need to test many different fusion proteins for expression and functionality; (7) It is possible to add two or more signals simultaneously; and (8) It is possible to regulate the expression and / or expression level of each signal / protein separately.

[0050] Because the mitogenic and / or cytokine stimulus is provided to molecules separate from the viral envelope glycoprotein, the integrity of the viral envelope glycoprotein is maintained and there is no adverse effect on viral titer.

[0051] (Detailed explanation) retrovirus Retroviruses are double-stranded RNA enveloped viruses characterized primarily by their ability to "reverse transcribe" their genome from RNA to DNA. Virions are 100-120 nm long and contain a dimeric genome of identical positive-stranded RNA complexed with nucleocapsid proteins. The genome is enclosed in a proteic capsid, which also contains the enzyme proteins required for viral infection: reverse transcriptase, integrase, and protease. Matrix proteins surround the viral nucleoparticle and act as a host. The capsid core forms an outer layer that interacts with the envelope, a lipid bilayer derived from the main cell membrane. This bilayer anchors viral envelope glycoproteins, which are responsible for recognizing specific receptors on the host cell and initiating the infection process. The envelope protein is formed by two subunits: a transmembrane (TM) subunit that anchors the protein within the lipid membrane and a surface (SU) subunit that binds to cellular receptors.

[0052] Based on their genome structure, retroviruses are classified as simple retroviruses, such as MLV and murine leukemia virus; or complex retroviruses, such as HIV and EIAV. Retroviruses encode four genes: gag (group-specific antigen), pro (protease), pol (polymerase), and env (envelope). The gag sequence encodes three major structural proteins: matrix protein, nucleocapsid protein, and capsid protein. The pro sequence encodes the protease responsible for cleaving Gag and Gag-Pol during particle assembly, budding, and maturation. The pol sequence encodes the enzymes reverse transcriptase and integrase; the former catalyzes the reverse transcription of the viral genome from RNA to DNA during the infection process, and the latter is responsible for integrating proviral DNA into the host cell genome. The env sequence encodes both the SU and TM subunits of the envelope glycoprotein. Furthermore, the retroviral genome contains two LTRs (long terminal repeats), which contain elements necessary for facilitating gene expression, reverse transcription, and integration into host cell chromosomes; a sequence termed the packaging signal (Ψ), which is necessary for the specific packaging of viral RNA into newly forming virions; and sequences such as the polypurine tract (PPT), which serve as sites for initiating plus-strand DNA synthesis during reverse transcription. In addition to gag, pro, pol, and env, complex retroviruses such as lentiviruses possess accessory genes, including vif, vpr, vpu, nef, tat, and rev, that regulate viral gene expression, infectious particle assembly, and modulate viral replication in infected cells.

[0053] During the infection process, retroviruses first attach to specific cell surface receptors. Upon entry into a susceptible host cell, the retroviral RNA genome is copied into DNA by the virally encoded reverse transcriptase carried within the parent virus. This DNA is transported to the host cell nucleus and then integrated into the host genome. At this stage, it is typically called a provirus. The provirus is stable in the host chromosome during cell division and is transcribed like other cellular proteins. The provirus encodes the proteins and packaging machinery required to make more virus and can leave the cell by a process known as "budding."

[0054] When enveloped viruses such as retroviruses and lentiviruses bud from host cells, they occupy part of the host cell lipid membrane. In this way, host cell-derived membrane proteins become part of the retroviral particle. The present invention utilizes this process to introduce a protein of interest into the viral particle envelope. Retroviral vectors

[0055] Retrovirus and lentivirus can be used as vector or delivery system to introduce one or more nucleotides of interest (NOI) into target cells.Transduction can occur in vitro, ex vivo or in vivo.When used in this manner, virus is typically referred to as viral vector.

[0056] In the viral vector of the present invention, the NOI may encode a T cell receptor or a chimeric antigen receptor and / or a suicide gene.

[0057] Gamma-retroviral vectors, commonly referred to as retroviral vectors, were the first viral vectors used in gene therapy clinical trials in 1990 and remain one of the most widely used. Recently, interest in lentiviral vectors, derived from complex retroviruses such as human immunodeficiency virus (HIV), has increased due to their ability to transduce non-dividing cells. The most attractive features of retroviral and lentiviral vectors as gene transfer tools include their large genetic payload capacity (up to 9 kb), minimal patient immune response, high transduction efficiency in vivo and in vitro, and the ability to permanently alter the genetic content of target cells and sustain long-term expression of the delivered gene.

[0058] Retroviral vector can be based on any suitable retrovirus that can deliver genetic information to eukaryotic cells.For example, retroviral vector can be alpha retroviral vector, gamma retroviral vector, lentiviral vector or spuma retroviral vector.This type of vector has been widely used in gene therapy treatment and other gene delivery applications.

[0059] The viral vector of the present invention may be a retroviral vector, such as a gamma-retroviral vector. The viral vector may be based on the human immunodeficiency virus.

[0060] The viral vector of the present invention may be a lentiviral vector, which may be based on a non-primate lentivirus, such as equine infectious anemia virus (EIAV).

[0061] The viral vector of the present invention comprises a mitogenic T cell activating transmembrane protein and / or a cytokine-based T cell activating transmembrane protein in the viral envelope, as illustrated in FIG.

[0062] Mitogenic T cell activation transmembrane proteins and / or cytokine-based T cell activation transmembrane proteins are derived from the host cell membrane, as explained above. Virus-like particles (VLPs)

[0063] For retroviral and lentiviral vectors, expression of the Gag precursor is sufficient to mediate virion assembly and release. Gag proteins, and even fragments of Gag, have been shown to be capable of assembling in vitro to form various structures resembling virion cores. These particles, which lack viral genetic material and are therefore noninfectious, are called virus-like particles (VLPs). Like intact virus particles, they contain an outer viral envelope made of the host cell lipid-bilayer (membrane) and therefore contain host cell transmembrane proteins.

[0064] The viral vector of the first aspect of the present invention may be or may comprise a virus-like particle. Nucleotide of Interest (NOI)

[0065] The viral vectors of the present invention are capable of delivering a nucleotide of interest (NOI) to target cells such as T cells or natural killer (NK) cells.

[0066] The NOI may encode all or part of a T cell receptor (TCR) or chimeric antigen receptor (CAR) and / or a suicide gene.

[0067] CARs are chimeric type I transmembrane proteins that connect an extracellular antigen-recognition domain (binder) to an intracellular signaling domain (endodomain). The binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but can also be based on other formats that contain antibody-like antigen-binding sites. A spacer domain is usually required to isolate the binder from the membrane and properly orient it. The transmembrane domain anchors the protein in the cell membrane. CARs may contain or be associated with an intracellular T cell signaling domain or endodomain.

[0068] The nucleic acid encoding CAR can be introduced into cells such as T cells using the retroviral vector or lentiviral vector of the present invention.In this way, a large number of cancer-specific T cells can be generated for adoptive cell transfer.When CAR binds to the target antigen, it transmits an activation signal to the T cell that expresses it.Therefore, CAR directs the specificity and cytotoxicity of T cells against tumor cells that express the target antigen.

[0069] Suicide genes encode polypeptides that allow cells expressing such polypeptides to be deleted, for example by inducing apoptosis. Examples of suicide genes are described in WO 2013 / 153391. host cell

[0070] In a second aspect, the present invention provides a host cell that expresses a mitogenic T cell activation transmembrane protein or a cytokine-based T cell activation transmembrane protein at the cell surface.

[0071] The host cell may be for the production of a viral vector according to the first aspect of the invention.

[0072] The host cell may be a packaging cell and may contain one or more of the following genes: gag, pol, env and rev.

[0073] Packaging cells for retroviral vectors may contain the gag, pol and env genes.

[0074] Packaging cells for lentiviral vectors may contain the gag, pol, env and rev genes.

[0075] The host cell may be a producer cell and may contain the gag, pol, env and optionally rev genes and the retroviral or lentiviral vector genome.

[0076] In a typical recombinant retroviral or lentiviral vector for use in gene therapy, at least a portion of one or more of the gag-pol and env protein coding regions may be removed from the virus and provided by the packaging cell. Although the virus is capable of integrating its genome into the host genome, this renders the viral vector replication-deficient because the modified viral genome cannot propagate due to the lack of structural proteins.

[0077] The packaging cells are used to grow and isolate quantities of the viral vector, ie, to prepare an appropriate titer of retroviral vector for transduction of target cells.

[0078] In some instances, propagation and isolation may involve isolating the retroviral gagpol and env (and in the case of lentiviruses, rev) genes and separately introducing them into host cells to produce packaging cell lines. Packaging cell lines produce the proteins necessary for packaging retroviral DNA but are unable to induce encapsidation due to a lack of the psiregion. However, when a recombinant vector carrying the psi region is introduced into a packaging cell line, the helper proteins can package the psi-positive recombinant vector to produce a recombinant virus stock.

[0079] An overview of available packaging lines is given in "Retroviruses" (eds. JM Offin, S.M. Hughes, H.E. Varmus, Cold Spring Harbor Laboratory Press, 1997). (p. 449).

[0080] Packaging cells have also been developed in which the gag, pol, and env (and rev, in the case of lentiviral vectors) viral coding regions are carried on separate expression plasmids that are independently transfected into the packaging cell line, so that three recombination events are required for wild-type virus production.

[0081] Transient transfection avoids the longer time required to generate stable vector-producing cell lines and is used when vector or retroviral packaging components are toxic to cells.The components typically used to generate retroviral / lentiviral vectors include a plasmid encoding Gag / Pol proteins, a plasmid encoding Env proteins (and rev proteins in the case of lentiviral vectors) and a retroviral / lentiviral vector genome.Vector production involves transiently transfecting one or more of these components into cells that contain other necessary components.

[0082] The packaging cells of the present invention may be any mammalian cell type capable of producing retroviral / lentiviral vector particles. The packaging cells may be 293T cells or variants of 293T cells adapted to grow in suspension and grow without serum. The packaging cells a) Transfer vector b) gagpol expression vector

[0083] c) env expression vector The vector may be produced by transient transfection of The env gene may be heterologous, resulting in a pseudotyped retroviral vector. For example, the env gene may be from RD114 or one of its mutants, VSV-G, gibbon ape leukemia virus (GALV), amphotropic envelope or measles envelope or baboon retrovirus envelope glycoprotein.

[0084] In the case of lentiviral vectors, transient transfections with the rev vector are also performed. Mitogenic T cell activation transmembrane protein

[0085] The viral vector of the present invention may contain a mitogenic T cell activating transmembrane protein in the viral envelope. The mitogenic T cell activating transmembrane protein is derived from the host cell during the production of the retroviral vector. The mitogenic T cell activating transmembrane protein is produced by the packaging cell and expressed on the cell surface. When the nascent retroviral vector buds from the host cell membrane, the mitogenic T cell activating transmembrane protein The protein is incorporated into the viral envelope as part of the packaging cell-derived lipid bilayer.

[0086] The term "host cell-derived" indicates that the mitogenic T cell activating transmembrane protein 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, which encodes the major structural protein; or env, which encodes the envelope protein.

[0087] The envelope protein is formed by two subunits: a transmembrane (TM) that anchors the protein in the lipid membrane and a surface (SU) that binds to the cellular receptor. The mitogenic T cell activating transmembrane proteins derived from the packaging cells of the present invention do not contain a surface envelope subunit (SU).

[0088] The mitogenic T cell activating transmembrane protein may comprise one of the following sequences or variants thereof: SEQ ID NO: 1 (OKT3-CD8STK-TM-A)

[0089] [ka] SEQ ID NO: 2 (15E8-CD8STK-TM-A)

[0090] [ka] SEQ ID NO: 3 (TGN1412-CD8STK-TM-A)

[0091] [ka]

[0092] The mitogenic T cell activating transmembrane protein may comprise a variant of the sequence set forth in SEQ ID NO: 1, 2 or 3 having at least 80, at least 85, at least 90, at least 95, at least 98 or at least 99% sequence identity, provided that the variant sequence is a mitogenic T cell activating transmembrane protein that has the required property, i.e., the ability to activate T cells when present in the envelope protein of a retroviral vector.

[0093] Methods for sequence alignment are well known in the art and are performed using appropriate alignment programs. % sequence identity refers to the percentage of amino acid or nucleotide residues that are identical in two sequences when the two sequences are optimally aligned. Homology or identity of nucleotide and protein sequences can be determined using the BLAST program (Basic Local Alignment Search Tool at the National Center for Biotechnology Information) with default parameters, as published at http: / / blast.ncbi.nlm.nih.gov. Other algorithms for determining sequence identity or homology include LALIGN (http: / / www.ebi.ac.uk / Tools / psa / lalign / and http: / / www.ebi.ac.uk / Tools / psa / lalign / nucleotide.html), AMAS (Analysis of Multiply Aligned Sequences, available at http: / / www.compbio.dundee.ac.uk / Software / Amas / amas.html), FASTA (http: / / www.ebi.ac.uk / Tools / sss / fasta / ), Clus Examples include tal Omega (http: / / www.ebi.ac.uk / Tools / msa / clustalo / ), SIM (http: / / web.expasy.org / sim / ), and EMBOSS Needle (http: / / www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html).

[0094] The mitogenic T cell activating transmembrane protein may have the structure: MS-TM, where M is the mitogenic domain; S is an optional spacer domain, and TM is the transmembrane domain. Mitogenic domain

[0095] The mitogenic domain is a part of the mitogenic T cell activation transmembrane protein that causes T cell activation.It can directly or indirectly bind or otherwise interact with T cells, resulting in T cell activation.In particular, the mitogenic domain can bind to T cell surface antigens such as CD3, CD28, CD134 and CD137.

[0096] CD3 is a T cell coreceptor. It is a protein complex consisting of four separate chains. In mammals, the complex includes 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 an activation signal in T lymphocytes. The TCR, ζ chain, and CD3 molecule together comprise the TCR complex.

[0097] The mitogenic domain may be bound to the CD3 epsilon chain.

[0098] CD28 is one of the proteins expressed on T cells that provides costimulatory signals necessary for T cell activation and survival. T cell stimulation via CD28 in addition to the T cell receptor (TCR) can provide a powerful signal for the production of various interleukins (especially IL-6).

[0099] CD134, also known as OX40, is a member of the TNFR superfamily of receptors that, unlike CD28, is not constitutively expressed on resting naive T cells. OX40 is a secondary costimulatory molecule that is expressed 24 to 72 hours after activation; its ligand, OX40L, is also not expressed on resting antigen-presenting cells but is expressed after their activation. OX40 expression is dependent on full activation of T cells; in the absence of CD28, OX40 expression is delayed and at four-fold lower levels.

[0100] CD137, also known as 4-1BB, is a member of the tumor necrosis factor (TNF) receptor family. CD137 can be expressed by activated T cells, but to a greater extent on CD8 than on CD4 T cells. Furthermore, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and vascular wall cells at sites of inflammation. The best-characterized activity of CD137 is its costimulatory activity on activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity.

[0101] The mitogenic domain may comprise all or part of an antibody or other molecule that specifically binds to a T cell surface antigen. The antibody may activate TCR or CD28. The antibody may bind to TCR, CD3, or CD28. Examples of such antibodies include OKT3, 15E8, and TGN1412. Other suitable antibodies include: Anti-CD28:CD28.2, 10F3 Anti-CD3 / TCR: UCHT1, YTH12.5, TR66.

[0102] The mitogenic domain may include a binding domain from OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5, or TR66.

[0103] The mitogenic domain can include all or part of a costimulatory molecule, such as OX40L and 41BBL. For example, the mitogenic domain can include the binding domain from OX40L or 41BBL.

[0104] OKT3, also known as Muromonab-CD3, is a monoclonal antibody that targets the CD3 epsilon chain. It is used clinically to reduce acute rejection in organ transplant patients. It was the first monoclonal antibody approved for clinical use in humans. The CDRs of OKT3 are as follows: [ka] 15E8 is a mouse monoclonal antibody against human CD28. Its CDRs are as follows: [ka]

[0105] TGN1412 (also known as CD28-SuperMAB) is a humanized monoclonal antibody that not only binds to the CD28 receptor but is also a potent agonist of the CD28 receptor. Its CDRs are as follows: [ka]

[0106] OX40L is a ligand for CD134 and is expressed on cells such as DC2s (a subtype of dendritic cells) that enable the amplification of Th2 cell differentiation. OX40L is also referred to as CD252 (cluster of differentiation 252). OX40L sequence (SEQ ID NO: 22) [ka]

[0107] 41BBL is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This transmembrane cytokine is a bidirectional signal transducer that acts as a ligand for the costimulatory receptor molecule 4-1BB on T lymphocytes. 41BBL has been shown to promote T lymphocyte proliferation and reactivate anergic T lymphocytes. 41BBL sequence (SEQ ID NO: 23)

[0108] [ka] spacer domain

[0109] The mitogenic T cell activating transmembrane proteins and / or cytokine-based T cell activating transmembrane proteins may include a spacer sequence connecting the antigen-binding domain to the transmembrane domain, where the flexible spacer allows the antigen-binding domain to be oriented in various directions to facilitate binding.

[0110] The spacer sequence may comprise, for example, an IgG1 Fc region, an IgG1 hinge, or a human or mouse CD8 stalk. may contain alternative linker sequences with similar length and / or domain spacing characteristics to the IgG1 Fc region, IgG1 hinge, or CD8 stalk. The human IgG1 spacer can be altered to remove the Fc binding motif. Examples of amino acid sequences for these spacers are shown below:

[0111] [ka]

[0112] The spacer sequence may be derived from a human protein. The spacer sequence may not be derived from a viral protein. In particular, the spacer sequence may not be part of, be derived from, or include a part of the surface envelope subunit (SU) of a retroviral env protein. Transmembrane domain

[0113] The transmembrane domain is a membrane-spanning sequence of a mitogenic T cell activation transmembrane protein and / or a cytokine-based T cell activation transmembrane protein. The transmembrane domain may contain a hydrophobic alpha helix. The transmembrane domain may be derived from CD28, thereby providing good receptor stability.

[0114] The transmembrane domain may be derived from a human protein. The transmembrane domain may not be derived from a viral protein. In particular, the transmembrane domain may not be, be derived from, or include a portion of the transmembrane envelope subunit (TM) of a retroviral env protein.

[0115] An alternative to a transmembrane domain is a membrane-targeting domain such as a GPI anchor.

[0116] GPI anchoring is a post-translational modification that occurs in the endoplasmic reticulum. A preassembled GPI anchor precursor is introduced into a protein with a C-terminal GPI signal sequence. During processing, the GPI anchor replaces the GPI signal sequence and is linked to the target protein via an amide bond. The GPI anchor targets the mature protein to the membrane.

[0117] The tagged proteins of the present invention may include a GPI signal sequence. Cytokine-based T cell activation transmembrane proteins

[0118] The viral vector of the present invention may contain a cytokine-based T cell activation transmembrane protein in the viral envelope. The cytokine-based T cell activation transmembrane protein is derived from the host cell during viral vector production. The cytokine-based T cell activation transmembrane protein is produced by the host cell and expressed on the cell surface. When the nascent viral vector buds from the host cell membrane, the cytokine-based T cell activation transmembrane protein is incorporated into the viral envelope as part of the lipid bilayer derived from the packaging cell.

[0119] The cytokine-based T cell activating transmembrane protein is not produced from one of the viral genes, such as gag, which encodes the major structural protein, or env, which encodes the envelope protein.

[0120] The cytokine-based T cell activation transmembrane protein may comprise a cytokine domain and a transmembrane domain. It may have the structure CS-TM, where C is the cytokine domain, S is an optional spacer domain, and TM is the transmembrane domain. The spacer domain and the transmembrane domain are as defined above. Cytokine Domain

[0121] The cytokine domain may include part or all of a T cell activating cytokine, such as from IL2, IL7, and IL15. The cytokine domain may include any part of the cytokine, so long as it retains the ability to bind to its particular receptor and activate T cells.

[0122] IL2 is one of the factors secreted by T cells to regulate the proliferation and differentiation of T cells and certain B cells. IL2 is a lymphokine that induces proliferation of responding T cells. It is secreted as a single glycosylated polypeptide and requires cleavage of a signal sequence for its activity. Solution NMR suggests that the structure of IL2 contains a bundle of four helices (designated A–D) flanked by two short helices and several incompletely defined loops. Residues in helix A and the loop region between helices A and B are important for receptor binding. The sequence of IL2 is shown as SEQ ID NO:29. SEQ ID NO: 29 [ka]

[0123] IL7 is a cytokine that acts as a growth factor for early lymphoid cells of both B and T cell lineages. The sequence of IL7 is shown as SEQ ID NO:30. SEQ ID NO: 30 [ka]

[0124] IL-15 is a cytokine with structural similarity to IL-2. Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain and common gamma chain. IL-15 is secreted by mononuclear phagocytes and several other cells after infection with a virus(ies). This cytokine induces cell proliferation of natural killer cells, cells of the innate immune system whose primary role is to kill virus-infected cells. The sequence of IL-15 is shown as SEQ ID NO: 31. SEQ ID NO: 31 [ka] The cytokine-based T cell activating transmembrane protein may comprise one of the following sequences or variants thereof: SEQ ID NO: 32 (membrane-IL7)

[0125] [ka] SEQ ID NO: 33 (membrane-IL15)

[0126] [ka]

[0127] The cytokine-based T cell activating transmembrane protein may comprise a variant of the sequence set forth in SEQ ID NO: 32 or 33 having at least 80, at least 85, at least 90, at least 95, at least 98 or at least 99% sequence identity, provided that the variant sequence is a cytokine-based T cell activating transmembrane protein that has the required property, i.e., the ability to activate T cells when present in the envelope protein of a retroviral or lentiviral vector. Tagged Proteins

[0128] The viral envelope of the viral vector may also comprise a tagged protein that comprises a binding domain that binds to the capture moiety and the transmembrane domain.

[0129] The tagged protein is A binding domain that binds to the capture moiety spacers; and Transmembrane domain may include:

[0130] The tagged protein facilitates purification of the viral vector from the cell supernatant via binding to the capture moiety of the tagged protein.

[0131] "Binding domain" refers to an entity, eg, an epitope, that is capable of recognizing and specifically binding to a target entity, eg, a capture moiety.

[0132] The binding domain may contain one or more epitopes capable of specifically binding to the capture moiety. For example, the binding domain may contain at least one, at least two, at least three, at least four, or at least five epitopes capable of specifically binding to the capture moiety. When the binding domain contains two or more epitopes, each epitope may be separated by a linker sequence, as described herein.

[0133] The binding domain may be releasable from the capture moiety upon the addition of an entity that has a higher binding affinity for the capture moiety compared to the binding domain. Streptavidin-binding epitope

[0134] A binding domain can comprise one or more streptavidin-binding epitopes, for example, a binding domain can comprise at least 1, at least 2, at least 3, at least 4, or at least 5 streptavidin-binding epitopes.

[0135] Streptavidin is a 52.8 kDa protein purified from the bacterium Streptomyces avidinii. The streptavidin homotetramer has a very high affinity for biotin (vitamin B7 or vitamin H), with a dissociation constant (Kd) of approximately 10 -15 Streptavidin is well known in the art and is widely used in molecular biology and bionanotechnology due to the streptavidin-biotin complex's resistance to organic solvents, denaturants, proteolytic enzymes, and extreme temperatures and pH. The strong streptavidin-biotin bond can be used to attach various biomolecules to each other or to solid supports. However, harsh conditions are required to disrupt the streptavidin-biotin interaction, which can denature the target protein being purified.

[0136] The binding domain may be, for example, a biotin mimetic. A "biotin mimetic" may refer to a short peptide sequence (e.g., 6 to 20, 6 to 18, 8 to 18, or 8 to 15 amino acids) that specifically binds to streptavidin.

[0137] As noted above, the affinity of the biotin / streptavidin interaction is very high. Thus, an advantage of the present invention is that the binding domain can comprise a biotin mimic that has a lower affinity for streptavidin compared to biotin itself.

[0138] In particular, biotin mimics can bind to streptavidin with lower binding affinity than biotin, so that biotin can be used to elute streptavidin-captured retroviral vectors. For example, biotin mimics can bind to streptavidin with a Kd of 1 nM to 100 uM.

[0139] The biotin mimic may comprise the sequences shown in Table 1.

[0140] [Table 1]

[0141] The biotin mimic may be selected from the group of StreptagII, Flankedccstreptag and ccstreptag.

[0142] A binding domain may comprise more than one biotin mimic, for example, a binding domain may comprise at least one, at least two, at least three, at least four, or at least five biotin mimics.

[0143] When a binding domain comprises two or more biotin mimetics, each mimic can be the same or a different mimic. For example, a binding domain may comprise two StreptagII biotin mimetics separated by a linker (e.g., as shown by SEQ ID NO: 43) or two Flankedccstreptag biotin mimetics separated by a linker (e.g., as shown by SEQ ID NO: 44). [ka] Glutathione S-transferase

[0144] The binding domain may comprise a glutathione S-transferase (GST) domain.

[0145] GSTs comprise a family of eukaryotic and prokaryotic phase II metabolic isozymes that catalyze the conjugation of reduced glutathione (GSH) to xenobiotic substrates for detoxification purposes. The GST family consists of three superfamilies: cytosolic, mitochondrial, and microsomal (also known as MAPEG) proteins (Udomsinpraser et al. Biochem. J. (2005) 388(Pt 3):763-71). page).

[0146] GST proteins have a strong binding affinity for GSH, and this interaction is commonly used in molecular biology to allow the isolation of GST-tagged proteins from protein mixtures.

[0147] The amino acid sequence of GST is shown in SEQ ID NO:45. SEQ ID NO: 45 (GST)

[0148] [ka] Rituximab-binding epitope

[0149] The tagged proteins of the invention may comprise a binding domain comprising a rituximab binding epitope (R epitope) and / or a Qbend10 epitope (Q epitope).

[0150] Rituximab-binding epitope refers to an epitope that specifically binds to rituximab. For example, the rituximab-binding epitope may be based on the CD20 B-cell antigen. The rituximab binding epitope sequence from CD20 is CEPANPSEKNSPSTQYC (SEQ ID NO: 46).

[0151] Perosa et al. (2007, J. Immunol. 179:7967-7974) reported that anti-CD2 describe a series of cysteine-constrained heptameric cyclic peptides containing an antigenic motif recognized by the mAb rituximab, but with amino acids surrounded by different motifs. A total of 11 peptides are described, as shown in the table below: [Table 2]

[0152] Li et al. (2006, CellImmunol 239:136-43) also reported the following sequence: The present application describes mimetopes of rituximab, including:

[0153] [ka]

[0154] The polypeptides of the present invention comprise a rituximab-binding epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 46 to 58, or a variant thereof that retains rituximab-binding activity. QBend10

[0155] The CliniMACS CD34 selection system utilizes the QBEnd10 monoclonal antibody to achieve cell selection. The inventors previously mapped the QBEnd10 binding epitope within the CD34 antigen (see WO 2013 / 153391) and determined that it has the amino acid sequence shown as SEQ ID NO: 59.

[0156] [ka]

[0157] The binding domain of the tagged protein of the present invention may comprise a QBEnd10 binding epitope having the amino acid sequence as shown as SEQ ID NO: 59 or a variant thereof that retains QBEnd10 binding activity. RQR8

[0158] The tagged protein may comprise a binding domain comprising or consisting of a 136 amino acid sequence as shown as SEQ ID NO:60. SEQ ID NO: 60 (RQR8) [ka] nucleic acid

[0159] The present invention also relates to nucleic acids encoding cytokine-based T cell activation transmembrane proteins or nucleic acids encoding mitogenic T cell activation transmembrane proteins, which may be in the form of a construct comprising multiple sequences encoding mitogenic T cell activation transmembrane proteins and / or cytokine-based T cell activation transmembrane proteins.

[0160] As used herein, the terms "polynucleotide," "nucleotide," and "nucleic acid" are intended to be synonymous with each other.

[0161] It will be understood by those skilled in the art that many different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, it should be understood that those skilled in the art can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptide is expressed.

[0162] Nucleic acids may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides containing synthetic or modified nucleotides. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. It should be understood that for use as described herein, polynucleotides may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or lifespan of the polynucleotide of interest.

[0163] The terms "variant," "homologue" or "derivative" in relation to a nucleotide sequence include any substitution, mutation, modification, replacement, deletion or addition of one or more nucleic acid(s) from or to the sequence.

[0164] The nucleic acid can produce a polypeptide comprising one or more sequences encoding a mitogenic T cell activation transmembrane protein and / or one or more sequences encoding a cytokine-based T cell activation transmembrane protein. The cleavage site can be self-cleaving, such that once the polypeptide is produced, it is immediately cleaved into receptor and signaling components without the need for external cleavage activity.

[0165] Various autocleavage sites are known, including the foot-and-mouth disease virus (FMDV) 2a autocleavage peptide and various variants, as well as 2A-like peptides. This peptide may have the sequence shown as SEQ ID NO: 34 or 35.

[0166] [ka]

[0167] The co-expression sequence may be an internal ribosome entry sequence (RES). The co-expression sequence may be an internal promoter. vector

[0168] The present invention also provides vectors or kits of vectors comprising one or more sequences encoding mitogenic T cell activating transmembrane proteins and / or one or more sequences encoding cytokine-based T cell activating transmembrane proteins. Such vectors can be used to introduce the nucleic acid sequence(s) into host cells, such as producer cells or packaging cells.

[0169] The vector may be, for example, a plasmid or a viral vector, such as a retroviral or lentiviral vector, or a transposon-based vector or synthetic mRNA.

[0170] The vector is capable of transfecting or transducing a host cell. method

[0171] The present invention also provides a method for generating activated transgenic T cells or natural killer (NK) cells, comprising the step of transducing T cells or NK cells with a retroviral or lentiviral vector described in the present invention, such that the T cells or NK cells are activated by one or more mitogenic T cell activating transmembrane proteins and, optionally, one or more cytokine-based T cell activating transmembrane proteins.

[0172] The method for transducing and activating T cells or NK cells (NK call) may take 48 hours or less, for example, between 24 and 48 hours.

[0173] The present invention will now be further described by way of examples, which are meant to serve to aid those skilled in the art in practicing the invention and are not intended to limit the scope of the invention in any way. [Example]

[0174] Example 1: Production of a viral vector displaying OKT3 on the virion surface Initial proof-of-concept experiments were performed demonstrating that expression of OKT3 scFv on packaging cells results in the production of a viral vector that causes mitogenic activation of T cell targets.

[0175] OKT3 scFv 293T cells produced lentiviral vectors that activated and transduced target T cells. This mitogenic property was dependent on the presence of lentiviral helper components; this effect was not due to nonspecific properties of the packaging cell supernatant (Figure 2).

[0176] A comparison of OKT3 scFv attached to the membrane via the CD8 stalk or via the ability of the IgG1 hinge to be incorporated into lentivirus and cause mitogenic stimulation was also performed, and no differences were observed between the two spacers.

[0177] 293T cells stably expressing surface-bound OKT3 were transfected with all three plasmids, along with lentiviral gagpol, RD-PRO env, transfer vector, or lentiviral rev expression plasmids. The resulting supernatant was applied to primary human T cells. T cells were then examined by flow cytometry using the following parameters: CD25 to measure T cell activation; anti-Fc to detect the transgene (CAR with an Fc spacer); and ki67 to determine cycling cells (Figure 3). Only the gagpol-supplied condition resulted in significant mitogenic stimulation. Only the condition in which all plasmids were supplied (together with rev) resulted in mitogenic stimulation and transduction of T cells.

[0178] Further experiments were performed to determine whether different lentiviral pseudotypings supported mitogenic effects. 293T cells stably expressing membrane-bound OKT3 were transfected with lentiviral transfer vectors, lentiviral gagpol and rev, and different env plasmids: VSV-G, RD-PRO, Ampho, GALV, and measles M / H. The resulting supernatants were applied to primary human T cells. Cells were then stained with Ki67 and examined by flow cytometry. All pseudotypes supported mitogenic effects, although the effect appeared to be reduced with measles pseudotyping (Figure 4). Example 2: Two distinct mitogenic stimuli can be incorporated into a viral vector

[0179] Additional constructs were generated containing anti-CD28 activating scFvs from antibody 15E8: the OKT3 scFv cassette (described above) expressing eGFP, and the 15E8 scFv cassette expressing the blue fluorescent protein eBFP2.

[0180] We generated 293T cells that co-expressed high levels of eGFP and eBFP2 and demonstrated successful expression of both OKT3 and 15E8 on the surface of 293T cells.

[0181] Lentiviral supernatants were generated from wild-type 293T cells, 293T cells expressing only OKT3 scFv, and 293T cells expressing both OKT3 and 15E8. The activation level and transduction efficiency were greater with the two stimuli (Figure 6). Example 3: Demonstration of functionality in gamma-retroviral vectors

[0182] 293T cells stably expressing membrane-bound OKT3 were transfected with a gamma-retroviral transfer vector encoding CAR, a gamma-retroviral gagpol expression plasmid, and an RD114 expression plasmid. The resulting supernatant was applied to primary human T cells. T cells were stained with anti-Fc, anti-CD25, and Ki67 and examined by flow cytometry. Although no mitogenic stimulation was applied, T cells were activated, cycling, and expressed the transgene (Figure 5). Example 4: Combinations of peptides incorporated into lentiviral vectors

[0183] Different combinations of elements were incorporated into the packaging cell lines, including the superagonist anti-CD28 mAb TGN1412 scFv. The cytokines IL7 and IL15, as well as OX40L and 41BBL, were also incorporated in various combinations as follows: 1. (None) 2.OKT3 3.OKT3+15E8 4.OKT3+TGN1412 5. OKT3+15E8+OX40L+41BBL 6. OKT3+15E8+OX40L+41BBL+mIL15 7. OKT3+15E8+OX40L+41BBL+mIL7

[0184] Lentiviral vectors generated from these different 293T cells were used to stimulate / transduce T cells.

[0185] Vectors produced from unengineered 293T cells combined with the mitogenic soluble antibody OKT3 and CD28.2+ / -IL2 were used as controls. Activation (CD25), proliferative fraction (Ki67), and absolute counts on day 5 were measured (Figures 7-10).

[0186] The significant advantage of incorporating two signals instead of one was again noted, and it was also noted that activation with virion-surface-displayed mitogenic peptides was significantly superior to activation achieved by adding soluble antibody to T cells.

[0187] Proliferation levels similar to those of mAb activation with cytokines were also achieved. methodology The VH and VL of the mitogenic antibody were cloned as scFv and connected with a spacer domain, a TM domain and a polar anchor (SEQ ID NOs: 1 to 3 above).

[0188] The cytokine was joined in frame with a spacer, a TM domain and a polar anchor (SEQ ID NOs: 32 and 33 above).

[0189] For natural costimulatory molecules such as 41BBL and OX40L, these are cloned in their native form.

[0190] Each of the above types of membrane-bound proteins could then be stably expressed at high levels in 293T cells.

[0191] Viral vectors were generated from these 293T cells using standard transient transfection. For lentiviral vectors, the transfer vector, rev expression vector, lenti-gagpol expression vector, and RD-PRO expression vector were co-transfected. For gammaretroviral vectors, 293T cells were co-transfected with the transfer vector, MoMLV gagpol, and RD114 expression plasmids. The supernatant was clarified by centrifugation and filtration through a 0.45 μM filter. The virus was applied to primary human T cells on retronectin plates. IL2 was added in some conditions, or no cytokine was added in others. Example 5 Comparison of T cell subset phenotypes from cells stimulated with lentiviral vectors and cells stimulated with anti-CD3 / anti-CD28 antibody-coated beads

[0192] Mononuclear cells were isolated from peripheral blood using standard techniques. Peripheral blood mononuclear cells (PBMCs) were then (i) beads coated with anti-CD3 / anti-CD28 antibodies at a ratio of 3:1 (Dynabeads® human T activator CD3 / CD28) in the presence of unmodified lentiviral vector and IL15 / IL7; or (ii) Lentiviral vectors expressing OKT3 and 15E8B on retronectin-coated plates in the presence of IL15 and IL7 (Combination 3 described in Example 4) were treated with either After 48 hours, cells were harvested and stained with a panel of T cell phenotypic antibodies as follows: aCD4-BV650 aCD8-PE.Cy7 aCD45RO-BV605 aCD45RA-FITC aCD95-PB aCD197-BV685

[0193] T cell subsets were analyzed by FACS, and the results are summarized in Figure 11. For both CD4+ and CD8+ T cell subsets, virus-stimulated cells displayed a higher percentage of naive T cells (Tn and Tscm) than did antibody-coated bead-stimulated cells.

[0194] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology, cellular immunology, or related fields are intended to be within the scope of the following claims. The present invention provides, for example, the following items. (Item 1) (i) a mitogenic T cell-activating transmembrane protein comprising a mitogenic domain and a transmembrane domain; and / or (ii) a cytokine-based T cell-activating transmembrane protein comprising a cytokine domain and a transmembrane domain; wherein said mitogenic or cytokine-based T cell activation transmembrane protein is not part of the viral envelope glycoprotein. (Item 2) 2. The viral vector of item 1, comprising a mitogenic T-cell activating transmembrane protein that binds to CD3, CD28, CD134, or CD137. (Item 3) 3. The viral vector of item 2, wherein the mitogenic T cell activating transmembrane protein comprises a binding domain from OKT3, 15E8, TGN1412, OX40L, or 41BBL. (Item 4) 2. The viral vector of item 1, comprising two or more mitogenic T cell-activating transmembrane proteins in the viral envelope. (Item 5) 5. The viral vector of item 4, comprising a first mitogenic T cell-activating transmembrane protein that binds to CD3 and a second mitogenic T cell-activating transmembrane protein that binds to CD28. (Item 6) The viral vector of any of the preceding items, comprising a cytokine-based T cell activating transmembrane protein, comprising a cytokine selected from IL2, IL7, and IL15. (Item 7) The viral vector of any of the preceding items, comprising a heterologous viral envelope glycoprotein that confers a pseudotyped viral vector. (Item 8) 8. The viral vector of item 7, wherein the envelope glycoprotein is VSV-G, Gibbon Ape Leukemia Virus (GALV), RD114 or one of its mutants, or amphotropic envelope, measles envelope, or baboon retrovirus envelope glycoprotein. (Item 9) The viral envelope also (iii) a binding domain that binds to the capture moiety; and Transmembrane domain wherein the tagging protein also comprises a tagging protein that facilitates purification of the viral vector from a cell supernatant via binding of the tagging protein to the capture moiety. (Item 10) 10. The viral vector of item 9, wherein the binding domain of the tagged protein comprises one or more streptavidin-binding epitopes. (Item 11) 11. The viral vector of item 10, wherein the streptavidin-binding epitope is a biotin mimic. (Item 12) 12. The viral vector of item 11, wherein the biotin mimic binds to streptavidin with a lower affinity than biotin, such that biotin can be used to elute streptavidin-captured retroviral vectors produced by packaging cells. (Item 13) 13. The viral vector of item 12, wherein the biotin mimetic is selected from the group consisting of StreptagII (SEQ ID NO: 36), Flankedccstretag (SEQ ID NO: 37), and ccstreptag (SEQ ID NO: 38). (Item 14) The viral vector of any of the preceding items, comprising a nucleic acid sequence encoding a T cell receptor or a chimeric antigen receptor. (Item 15) Item 16. The viral vector according to any one of the preceding items, which is a virus-like particle (VLP). On the cell surface, (i) a mitogenic T cell-activating transmembrane protein comprising a mitogenic domain and a transmembrane domain; and / or (ii) a cytokine-based T cell-activating transmembrane protein comprising a cytokine domain and a transmembrane domain; 9. A host cell which expresses a vector encoding a retroviral or lentiviral vector, wherein the vector is a vector encoding ... (Item 17) On the cell surface, (iii) a binding domain that binds to the capture moiety; and Transmembrane domain wherein the tagging protein facilitates purification of the viral vector from a cell supernatant via binding of the tagging protein to the capture moiety, such that the retroviral or lentiviral vector produced by the packaging cell is as defined in any of items 9 to 14. (Item 18) 18. A packaging cell that is a host cell as defined in item 16 or 17, which also contains one or more of the following genes: gag, pol, env and / or rev. (Item 19) 18. A producer cell which is a host cell as defined in item 16 or 17, comprising the gag, pol, env and optionally rev genes, and also comprising a retroviral or lentiviral genome. (Item 20) 19. A method for producing the host cell of claim 16 or 17, the packaging cell of claim 18, or the producer cell of claim 19, comprising transducing or transfecting the cell with a nucleic acid encoding a mitogenic T cell activating transmembrane protein and / or a cytokine-based T cell activating transmembrane protein. (Item 21) 20. A method for producing the viral vector of any one of items 1 to 15, comprising expressing a retroviral genome or a lentiviral genome in the cell of any one of items 17 to 19. (Item 22) 16. A method for generating activated transgenic T cells or natural killer (NK) cells, comprising transducing T cells or NK cells with the viral vector of any of items 1 to 15, such that the T cells or NK cells are activated by one or more mitogenic T cell-activating transmembrane proteins and / or one or more cytokine-based T cell-activating transmembrane proteins. (Item 23) (i) A host cell as defined in items 16 or 17; (ii) a nucleic acid comprising gag, pol, env, and optionally rev; and (iii) Retroviral genome 16. A kit for producing a retroviral or lentiviral vector as defined in any of items 1 to 15, comprising: (Item 24) (i) packaging cells as defined in item 18; and (ii) a retroviral or lentiviral vector genome 16. A kit for producing a retroviral or lentiviral vector as defined in any of items 1 to 15, comprising: (Item 25) (i) one or more nucleic acids encoding mitogenic T cell activation transmembrane proteins and / or cytokine-based T cell activation transmembrane proteins; and (ii) a nucleic acid comprising the retroviral gag, pol, env, and optionally rev genes; 19. A kit for producing the packaging cell of Item 18, comprising: (Item 26) (i) one or more nucleic acids encoding mitogenic T cell activating transmembrane proteins and / or cytokine-based T cell activating transmembrane proteins; (ii) a nucleic acid comprising retroviral gag, pol, env, and optionally rev genes; and (iii) a retroviral or lentiviral vector genome 20. A kit for producing the producer cell of item 19, comprising:

Claims

[Claim 1] The method described in the specification.

Images