Retroviral vector for universal receptor therapy

Abstract

To provide a retroviral vector for universal receptor therapy.SOLUTION: The present disclosure relates to retroviral vectors comprising polynucleotides encoding hapten-binding receptors and T cell activating receptors. The retroviral vectors may also comprise transduction enhancers. Also disclosed are adaptor molecules and their use in conjunction with the retroviral vectors and T cells transduced with the retroviral vectors. The present disclosure relates to viral vectors encoding transduction enhancers, synthetic T cell activating receptors, and / or hapten-binding receptors, compositions comprising the same, and methods of use thereof. The present disclosure also relates to use of adaptor molecules comprising haptens for use in conjunction with the viral vectors. The disclosed compositions and methods may be useful in treatment of disease, e.g., cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 916,110, filed October 16, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy, created on October 15, 2020, is entitled "UMOJ-003-01WO_SeqList_ST25.txt" and is 72KB in size.

[0003] The present disclosure relates to viral vectors encoding transduction enhancers, synthetic T cell activation receptors, and / or hapten-binding receptors, compositions comprising the same, and methods of using them. The disclosure also relates to the use of hapten-containing adapter molecules for use with viral vectors. The compositions and methods of the disclosure may be useful in treating diseases, such as cancer. [Background technology]

[0004] Chimeric antigen receptors (CARs) are engineered receptors used to engineer T cells for use in adoptive cellular immunotherapy (see Pule et al., Cytother. 5:3, 2003; Restifo et al., Nat. Rev. Immunol. 12:269, 2012). These receptors contain an extracellular ligand-binding domain, most commonly a single-chain variable fragment (scFv) of a monoclonal antibody, linked to an intracellular signaling component, most commonly CD3, alone or in combination with one or more costimulatory domains. Antigen binding stimulates the signaling domain of the intracellular segment of the CAR, thereby activating a signaling pathway. CAR-based adoptive cellular immunotherapy has been used to treat cancer patients with tumors that are resistant to conventional standard of care treatments (see Grupp et al., N. Engl. J. Med. 368:1509, 2013; Kalos et al., Sci. Transl. Med. 3:95ra73, 2011).

[0005] Autologous T cells can be genetically modified to express transgenes engineered to enhance in vivo efficacy after transplantation. Despite the success of adoptive transfer of transgene-modified T cells, for example, in the context of CD19 B-cell malignancies, a universal CAR target antigen present in all cancer types but absent from normal cells has not been identified. The field is therefore hampered by the need to identify cell surface targets naturally present on tumor cells and minimally expressed by normal cells / tissues in the body. Thus, the development of CAR T cell therapy is hindered by the prospect of potentially requiring refinement of dozens to hundreds of CAR targets and CARs to cover the vast majority of cancer types afflicting humans. Additionally, existing CAR technologies often require expensive and time-consuming ex vivo engineering steps to activate and transduce T cells with CAR vectors before reinfusing the CAR T cells into the host. Furthermore, CAR T cells can have numerous off-target effects that can cause dangerous, even fatal, adverse events. There is an unmet need for safer, more cost-effective, and more versatile CAR T cell engineering. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Pule et al.,Cytother.5:3,2003;Restifo et al.,Nat.Rev.Immunol.12:269,2012 [Non-patent document 2] Grupp et al.,N.Engl.J.Med.368:1509,2013 [Non-patent document 3] ;Kalos et al.,Sci.Transl.Med.3:95ra73,2011 Summary of the Invention [Means for solving the problem]

[0007] The present disclosure generally relates to a method comprising: (a) administering to a subject an adapter molecule comprising a targeting moiety and a hapten; and (b) administering to the subject either (i) a plurality of recombinant retroviral particles or (ii) cells generated by contacting immune cells ex vivo with a plurality of recombinant retroviral particles. Each of the retroviral particles comprises, in 5' to 3' order, a polynucleotide comprising: (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds to a hapten, and (iv) a 3' LTR or UTR. Each of the retroviral particles comprises a viral envelope. In some embodiments, the method is a method for treating cancer in a subject in need thereof. In some embodiments, the method is a method for killing tumor cells in a subject in need thereof.

[0008] In another aspect, the present disclosure provides a system or composition comprising: (a) an adapter molecule comprising a targeting moiety and a hapten; and (b) either (i) a plurality of recombinant retroviral particles or (ii) cells generated ex vivo by contacting immune cells with a plurality of recombinant retroviral particles. Each of the retroviral particles comprises, in 5' to 3' order, a polynucleotide comprising: (i) a 5' long terminal repeat (LTR) or UTR, (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds to the hapten, and (iv) a 3' LTR or UTR. Each of the retroviral particles comprises a viral envelope. In some embodiments, the system or composition is provided in a kit including instructions for use.

[0009] Further aspects and embodiments of the present invention are provided in the detailed description that follows. [Brief explanation of the drawings]

[0010] [Figure 1A] Figure 1A shows flow cytometry staining plots showing surface expression of FITC-CAR constructs on transduced T cells. Figure 1A shows a flow cytometry staining plot of lymphocytes, which was further gated to visualize single cells (Figure 1B). Single cells were further gated to visualize CD3+ cells (Figure 1C). CD3+ cells were further gated to visualize FITC-CAR+ cells (Figure 1D). [Figure 1B] Figure 1A shows flow cytometry staining plots showing surface expression of FITC-CAR constructs on transduced T cells. Figure 1A shows a flow cytometry staining plot of lymphocytes, which was further gated to visualize single cells (Figure 1B). Single cells were further gated to visualize CD3+ cells (Figure 1C). CD3+ cells were further gated to visualize FITC-CAR+ cells (Figure 1D). [Figure 1C]Figure 1A shows flow cytometry staining plots showing surface expression of FITC-CAR constructs on transduced T cells. Figure 1A shows a flow cytometry staining plot of lymphocytes, which was further gated to visualize single cells (Figure 1B). Single cells were further gated to visualize CD3+ cells (Figure 1C). CD3+ cells were further gated to visualize FITC-CAR+ cells (Figure 1D). [Figure 1D] Figure 1A shows flow cytometry staining plots showing surface expression of FITC-CAR constructs on transduced T cells. Figure 1A shows a flow cytometry staining plot of lymphocytes, which was further gated to visualize single cells (Figure 1B). Single cells were further gated to visualize CD3+ cells (Figure 1C). CD3+ cells were further gated to visualize FITC-CAR+ cells (Figure 1D). [Figure 2] Flow cytometry staining plots show how transduced T cells expressing the RACR-CAR payload are enriched by the addition of rapamycin, which activates RACR (Figures 2A and 2D), and / or by the addition of FITC antigen, which activates CAR (Figures 2B-2C and 2E-2F). Figures 2A-2C show transduced cells treated with 0 nM rapamycin, and Figures 2D-2F show transduced cells treated with 10 nM rapamycin. Figures 2B and 2E show transduced cells stimulated with FITC-coated beads, and Figures 2C and 2F show transduced cells stimulated with plate-bound FITC. [Figure 3] Graph showing the percentage of live FITC-CAR+ T cells among transduced cells that were either unstimulated, stimulated with FITC-coated beads, or stimulated with plate-bound FITC, and with or without (0 nM) rapamycin added. [Figure 4]Graph showing the total number of live FITC-CAR+ T cells among transduced cells that were either unstimulated, stimulated with FITC-coated beads, or stimulated with plate-bound FITC, and with or without (0 nM) rapamycin added. [Figure 5] Figure 1 shows a graph depicting upregulation of the activation marker CD25 in transduced cells that were unstimulated, stimulated with FITC-coated beads, or stimulated with plate-bound FITC, and with or without added rapamycin (10 nM) or (0 nM). gMFI = geometric mean fluorescence intensity. [Figure 6] The vector map of FITC CAR-Frb-RACR is shown. [Figure 7] A vector map of the CD3-cocal virion envelope is shown. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present disclosure generally relates to methods comprising: (a) administering to a subject an adapter molecule comprising a targeting moiety and a hapten; and (b) administering to the subject either (i) a plurality of recombinant retroviral particles or (ii) cells generated by contacting immune cells ex vivo with the plurality of recombinant retroviral particles. Each of the retroviral particles comprises a polynucleotide comprising a sequence encoding a receptor that specifically binds to a hapten. Each of the retroviral particles comprises a viral envelope. In some embodiments, the method is a method of treating cancer in a subject in need thereof. In some embodiments, the method is a method of killing tumor cells in a subject in need thereof.

[0012] In another aspect, the present disclosure provides a system or composition comprising: (a) an adapter molecule comprising a targeting moiety and a hapten; and (b) either (i) a plurality of recombinant retroviral particles or (ii) cells generated ex vivo by contacting immune cells with a plurality of recombinant retroviral particles. Each of the retroviral particles comprises, in 5' to 3' order, a polynucleotide comprising: (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds to the hapten, and (iv) a 3' LTR or UTR. Each of the retroviral particles comprises a viral envelope. In some embodiments, the system or composition is provided in a kit including instructions for use.

[0013] retroviral particles Retroviruses include lentiviruses, gamma-retroviruses, and alpha-retroviruses, each of which can be used to deliver polynucleotides to cells using methods known in the art. Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. This increased complexity allows the virus to regulate its life cycle, such as during latent infection. Some examples of lentiviruses include human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency viruses (SIV). Lentiviral vectors have been created by multiple attenuation of HIV pathogenic genes, e.g., deletion of genes env, vif, vpr, vpu, and nef, resulting in biologically safe vectors.

[0014] 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) No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated herein by reference in its entirety. Generally, these vectors are constructed to carry the necessary sequences for selection of cells containing the vector, for incorporation of the foreign nucleic acid into the lentiviral particle, and for introduction of the nucleic acid into target cells.

[0015] A commonly used lentiviral vector system is the so-called third-generation system. Third-generation lentiviral vector systems contain four plasmids. The "transfer plasmid" encodes the polynucleotide sequence delivered to target cells by the lentiviral vector system. The transfer plasmid generally contains one or more transgene sequences of interest flanked by long terminal repeats (LTRs), which facilitate integration of the transfer plasmid sequence into the host genome. For safety reasons, transfer plasmids are generally designed so that the resulting vector is replication-incompetent. For example, the transfer plasmid lacks the genetic elements necessary for the production of infectious particles within host cells. In addition, transfer plasmids can be designed with a deleted 3' LTR, which renders the virus "self-inactivating" (SIN). See Dull et al. (1998) J. Virol. 72:8463-71; Miyoshi et al. (1998) J. Virol. 72:8150-57. Viral particles can also contain a 3' untranslated region (UTR) and a 5' UTR. The UTRs contain retroviral regulatory elements that assist in packaging, reverse transcription, and integration of the proviral genome into a cell after contact of the cell with the retroviral particle.

[0016] Third-generation systems also typically include two "packaging plasmids" and an "envelope plasmid." The "envelope plasmid" typically encodes an Env gene operably linked to a promoter. In an exemplary third-generation system, the Env gene is VSV-G and the promoter is a CMV promoter. Third-generation systems utilize two packaging plasmids as an additional safety feature, i.e., an improvement over the single packaging plasmid of the so-called second-generation systems: one encoding gag and pol, and the other encoding rev. While safer, third-generation systems can be more cumbersome and result in lower virus titers due to the addition of an additional plasmid. Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0017] Many retroviral vector systems rely on the use of a "packaging cell line." Generally, a packaging cell line is a cell line that allows cells to produce infectious retroviral particles when a transfer plasmid, packaging plasmid(s), and envelope plasmid are introduced into the cells. Various methods of introducing the plasmid into cells can be used, including transfection or electroporation. In some instances, the packaging cell line is suitable for packaging the retroviral vector system into retroviral particles with high efficiency.

[0018] As used herein, the term "retroviral vector" or "lentiviral vector" is intended to refer to a nucleic acid encoding retroviral or lentiviral cis nucleic acid sequences required for genome packaging and one or more polynucleotide sequences to be delivered to a target cell. Retroviral or lentiviral particles generally contain an RNA genome (derived from a transfer 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 term "retroviral particle" or "lentiviral particle" is intended to refer to a viral particle that contains an envelope, possesses one or more properties of a lentivirus, and is capable of entering a target host cell. Such properties 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 that includes a gag structural polypeptide, e.g., one or more of p7, p24, and p17, containing a retroviral or lentiviral envelope that includes one or more env-encoded glycoproteins, e.g., p41, p120, and p160, containing a genome that includes one or more retroviral or lentiviral cis-acting sequences that function in replication, proviral integration, or transcription, containing a genome that encodes a retroviral or lentiviral protease, reverse transcriptase, or integrase, or containing a genome that encodes a regulatory activity such as Tat or Rev. Transfer plasmids can include cPPT sequences, as described in U.S. Patent No. 8,093,042.

[0019] The efficiency of the system is an important concern in vector engineering. The efficiency of retroviral or lentiviral vector systems can be evaluated by various methods known in the art, including measuring vector copy number (VCN) or vector genome (vg) by quantitative polymerase chain reaction (qPCR), or measuring viral titer in infectious units per milliliter (IU / mL). For example, titer can be measured by the method 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 The efficacy of retroviral vectors can be assessed using a functional assay performed on the cultured tumor cell line HT1080, as described in Therapy 24:1237-1246 (2016). When titers are assessed in constantly dividing cultured cell lines, no stimulation is required, and therefore the measured titer is not affected by surface modifications of the retroviral particles. Another method for assessing the efficiency of retroviral vector systems is described in Gaererts et al. Comparison of retroviral vector titration. methods.BMC Biotechnol.6:34(2006).

[0020] In some embodiments, the retroviral and / or lentiviral particles of the present disclosure comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a hapten. In some embodiments, the sequence encoding the receptor that specifically binds to a hapten 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.

[0021] In some embodiments, the retroviral particle comprises a transduction enhancer. In some embodiments, the retroviral particle comprises a polynucleotide comprising a sequence encoding a T cell activation protein. In some embodiments, the retroviral particle comprises a polynucleotide comprising a sequence encoding a hapten-binding receptor. In some embodiments, the retroviral particle comprises a tagging protein.

[0022] In some embodiments, each retroviral particle comprises a polynucleotide comprising, in 5' to 3' order: (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds to a hapten, and (iv) a 3' LTR or UTR.

[0023] In some embodiments, the retroviral particle contains a cell surface receptor that binds to a ligand on the target host cell, thereby enabling transduction of the host cell. The viral vector can contain a heterologous viral envelope glycoprotein, resulting in a pseudotyped viral vector. For example, the viral envelope glycoprotein can be derived from RD114 or one of its variants, VSV-G, gibbon ape leukemia virus (GALV), or from an amphotropic viral envelope, measles virus envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the cell surface receptor is the VSV G protein from the Kokal strain or a functional variant thereof. In some embodiments, the viral fusion glycoprotein comprises the amino acid sequence of SEQ ID NO: 1 (Kokal G protein). In some embodiments, the viral fusion glycoprotein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1 (Kokal G protein). In some embodiments, the viral fusion glycoprotein comprises an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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 (Cocal G protein). NFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPP QNCGYATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEF VDQDVYAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVE IGPNGILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK (SEQ ID NO: 1)

[0024] A variety of fusion glycoproteins can be used to pseudotype lentiviral vectors. The most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), although a number of other viral proteins have also been used to pseudotype lentiviral vectors. Joglekar et al. Human Gene Therapy See Methods 28:291-301 (2017). The present disclosure contemplates various fusion glycoprotein substitutions. Notably, some fusion glycoproteins result in higher vector efficiency.

[0025] In some embodiments, pseudotyping of the fusion glycoprotein or functional variant thereof facilitates targeted transduction of specific cell types, including, but not limited to, T cells or NK cells. In some embodiments, the fusion glycoprotein or functional variant thereof is selected from the group consisting of gp160 of human immunodeficiency virus (HIV), gp70 of murine leukemia virus (MLV), gp70 of gibbon ape leukemia virus (GALV), gp70 of feline leukemia virus (RD114), gp70 of amphotropic retrovirus (Ampho), gp70 of 10A1, gp70 of 10A2, gp70 of 10A3, gp70 of 10A4, gp70 of 10A5, gp70 of 10A6, gp70 of 10A7, gp70 of 10A8, gp70 of 10A9, gp70 of 10A1 ... gp70 of MLV (10A1), gp70 of ecotropic retrovirus (Eco), gp70 of baboon endogenous virus (BaEV), H and F proteins of measles virus (MV), H and F proteins of Nipah virus (NiV), G protein of rabies virus (RabV), G protein of Mokola virus (MOKV), G protein of Ebola Zaire virus (EboZ), GP1 and GP2 of lymphocytic choriomeningitis virus (LCMV), GP64 of baculovirus, E1 and E2 of chikungunya virus (CHIKV), Ross River virus (R The full-length polypeptide(s), functional fragment(s), homolog(s), or functional variant(s) of E1 and E2 of HIV-1 virus (RV), E1 and E2 of Semliki Forest virus (SFV), E1 and E2 of Sindbis virus (SV), E1 and E2 of Venezuelan equine encephalitis virus (VEEV), E1 and E2 of Western equine encephalitis virus (WEEV), HA of influenza A, B, C, or D, HA of avian plague virus (FPV), VSV-G of vesicular stomatitis virus, or CNV-G and PRV-G of Chandipura virus and Pili virus.

[0026] In some embodiments, the fusion glycoprotein or functional variant thereof is a full-length polypeptide, functional fragment, homolog, or functional variant of the G protein of vesicular stomatitis alagovirus (VSAV), Carajas vesiculovirus (CJSV), Chandipra vesiculovirus (CHPV), Cocal vesiculovirus (COCV), vesicular stomatitis Indiana virus (VSIV), Isfahan vesiculovirus (ISFV), Maraba vesiculovirus (MARAV), vesicular stomatitis New Jersey virus (VSNJV), or Bass-Congo virus (BASV). In some embodiments, the fusion glycoprotein or functional variant thereof is a cocalvirus G protein.

[0027] In some embodiments, the fusion glycoprotein or functional variant thereof is a full-length polypeptide, functional fragment, homolog, or functional variant of the G protein of vesicular stomatitis alagovirus (VSAV), Carajas vesiculovirus (CJSV), Chandipra vesiculovirus (CHPV), Cocal vesiculovirus (COCV), vesicular stomatitis Indiana virus (VSIV), Isfahan vesiculovirus (ISFV), Maraba vesiculovirus (MARAV), vesicular stomatitis New Jersey virus (VSNJV), or Bass-Congo virus (BASV). In some embodiments, the fusion glycoprotein or functional variant thereof is a cocalvirus G protein.

[0028] Additionally, the present disclosure provides various retroviral vectors, including but not limited to gamma-retroviral vectors, alpha-retroviral vectors, and lentiviral vectors.

[0029] Transduction Enhancers In some embodiments, a viral particle according to the present disclosure comprises a transduction enhancer.

[0030] As used herein, a "transduction enhancer" refers to a transmembrane protein that activates T cells. The transduction enhancer may be incorporated into the viral envelope of a viral particle according to the present disclosure. The transduction enhancer may include a mitogenic domain and / or a cytokine-based domain. The transduction enhancer may include a T cell activating receptor, an NK cell activating receptor, a costimulatory molecule, or a portion thereof.

[0031] Mitogenic Transduction Enhancer

[0032] Viral vectors of the present invention may contain a mitogenic transduction enhancer in the viral envelope. In some embodiments, the mitogenic transduction enhancer is derived from the host cell during production of the retroviral vector. In some embodiments, the mitogenic transduction enhancer 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 transduction enhancer may be incorporated into the viral envelope as part of the lipid bilayer derived from the packaging cell.

[0033] In some embodiments, the transduction enhancer is derived from a host cell. The term "derived from a host cell" indicates that the mitogenic transduction enhancer is derived from a host cell, as described above, and is not produced as a fusion or chimera from one of the viral genes, such as gag, which encodes the major structural protein, or env, which encodes the envelope protein.

[0034] Envelope proteins are formed by two subunits: a transmembrane (TM) subunit that anchors the protein to a lipid membrane and a surface (SU) subunit that binds to a cellular receptor. In some embodiments, the packaging cell-derived mitogenic transduction enhancer of the present invention does not comprise an envelope surface subunit (SU).

[0035] The mitogenic transduction enhancer may have an MS-TM structure, where M is a mitogenic domain, S is an optional spacer domain, and TM is a transmembrane domain.

[0036] Mitogenic domains of transduction enhancers

[0037] The mitogenic domain is part of a mitogenic transduction enhancer that causes T cell activation. It can directly or indirectly bind to or otherwise interact with T cells, thereby causing T cell activation. In particular, the mitogenic domain can bind to T cell surface antigens such as CD3, CD28, CD134, and CD137.

[0038] CD3 is a T cell coreceptor. It is a protein complex composed of four different chains. In mammals, this complex contains the CD3y chain, the CD35 chain, and two CD3e chains. These chains bind to the T cell receptor (TCR) and the zeta chain to generate an activation signal within the T lymphocyte. The TCR, zeta chain, and CD3 molecule together comprise the TCR complex.

[0039] In some embodiments, the mitogenic domain may be bound to the CD3 epsilon chain.

[0040] CD28 is a protein expressed on T cells that provides costimulatory signals required for T cell activation and survival. T cell stimulation by CD28 in addition to the T cell receptor (TCR) can generate a potent signal for the production of various interleukins (especially IL-6). CD134, also known as OX40, is a member of the TNFR superfamily of receptors and, unlike CD28, is not constitutively expressed on resting naive T cells. OX40 is a secondary costimulatory molecule expressed 24–72 hours after activation; its ligand, OX40L, is also not expressed on resting antigen-presenting cells (APCs), but is expressed after their activation. OX40 expression depends on full T cell activation; without CD28, OX40 expression is delayed and 4-fold reduced.

[0041] 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 is predominantly expressed on CD8 T cells compared to CD4 T cells. In addition, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and cells in the vascular wall at sites of inflammation. The best-characterized activity of CD137 is its costimulatory activity on activated T cells. Cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic responses.

[0042] 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 the TCR or CD28. The antibody may bind to the TCR, CD3, or CD28. Examples of such antibodies include: OKT3, 15E8, and TGN1412. Other suitable antibodies include:

[0043] Anti-CD28:CD28.2, 10F3

[0044] Anti-CD3 / TCR: UCHT1, YTH12.5, TR66

[0045] The mitogenic domain may comprise a binding domain from OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5, or TR66.

[0046] The mitogenic domain can comprise all or part of a costimulatory molecule, such as OX40L and 41 BBL. For example, the mitogenic domain can comprise a binding domain from OX40L or 41 BBL.

[0047] OKT3, also known as muromonab-CD3, is a monoclonal antibody that targets the CD3e chain. It is used clinically to reduce acute rejection in organ transplant patients. It was the first monoclonal antibody approved for clinical use in humans. The CDRs of OKT3 are as follows:

[0048] CDRH1: GYTFTRY (SEQ ID NO: 4)

[0049] CDRH2:NPSRGY (SEQ ID NO: 5)

[0050] CDRH3: YYDDHYCLDY (SEQ ID NO: 6)

[0051] CDRL1: SASSSVSYMN (SEQ ID NO: 7)

[0052] CDRL2: DTSKLAS (SEQ ID NO: 8)

[0053] CDRL3: QQWSSNPFT (SEQ ID NO: 9)

[0054] 15E8 is a mouse monoclonal antibody against human CD28. Its CDRs are as follows:

[0055] CDRH1:GFSLTSY (SEQ ID NO: 10)

[0056] CDRH2:WAGGS (SEQ ID NO: 11)

[0057] CDRH3: DKRAPGKLYYGYPDY (SEQ ID NO: 12)

[0058] CDRL1: RASESVEYYVTSLMQ (SEQ ID NO: 13)

[0059] CDRL2: AASNVES (SEQ ID NO: 14)

[0060] CDRL3: QQTRKVPST (SEQ ID NO: 15)

[0061] 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:

[0062] CDRH1: GYTFSY (SEQ ID NO: 16)

[0063] CDRH2: YPGNVN (SEQ ID NO: 17)

[0064] CDRH3: SHYGLDWNFDV (SEQ ID NO: 18)

[0065] CDRL1: HASQNIYVLN (SEQ ID NO: 19)

[0066] CDRL2: KASNLHT (SEQ ID NO: 20)

[0067] CDRL3: QQGQTYPYT (SEQ ID NO: 21)

[0068] OX40L is a ligand for CD134 and is expressed on cells such as DC2 (a subtype of dendritic cell) where it can enhance Th2 cell differentiation. OX40L is also called CD252 (cluster of differentiation 252).

[0069] OX40L sequence (SEQ ID NO: 22) MERVQPLEENVGNAARPRFENKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL

[0070] 4-1BBL is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This transmembrane cytokine is a bidirectional signaling molecule that acts as a ligand for the costimulatory receptor molecule 4-1BB on T lymphocytes. 4-1BBL has been shown to promote T lymphocyte proliferation and reactivate anergic T lymphocytes.

[0071] 4-1BBL sequence (SEQ ID NO: 23) MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

[0072] Spacer domain of transduction enhancer

[0073] The mitogenic and / or cytokine-based transduction enhancers may comprise a spacer sequence to link the antigen-binding domain to the transmembrane domain. The flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.

[0074] The spacer sequence can include, for example, an lgG1 Fc region, an lgG1 hinge, or a human or mouse CD8 stalk. Alternative linker sequences with similar length and / or domain spacing characteristics to the Fc region, IgG1 hinge, or CD8 stalk may be included. The human IgG1 spacer may be modified to remove the Fc binding motif.

[0075] Examples of the amino acid sequences of these spacers are shown below.

[0076] SEQ ID NO: 24 (Human IgG1 Hinge-CH2CH3) AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD

[0077] SEQ ID NO: 25 (human CD8 stalk): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI

[0078] SEQ ID NO: 26 (human lgG1 hinge): AEPKSPDKTHTCPPCPKDPK

[0079] SEQ ID NO: 27 (CD2 ectodomain): KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD

[0080] SEQ ID NO: 28 (CD34 ectodomain): SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITE TTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKT

[0081] In some embodiments, the spacer sequence may be derived from a human protein.

[0082] Transmembrane domains of transduction enhancers

[0083] The transmembrane domain is a mitogenic transduction enhancer and / or cytokine-based transduction enhancer sequence that spans the membrane. The transmembrane domain may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from CD28. In some embodiments, the transmembrane domain is derived from a human protein.

[0084] An alternative to the transmembrane domain is a membrane targeting domain such as a GPI anchor. GPI anchoring is a post-translational modification that occurs in the endoplasmic reticulum. A pre-assembled GPI anchor precursor is transferred to 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 by an amide bond. The GPI anchor targets the mature protein to the membrane. In some embodiments, the tagged protein of the present invention comprises a GPI signal sequence.

[0085] Cytokine-based transduction enhancers

[0086] The viral vectors of the present invention may comprise 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 vector production. In some embodiments, the cytokine-based transduction enhancer 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 transduction enhancer may be incorporated into the viral envelope as part of the lipid bilayer derived from the packaging cell.

[0087] The cytokine-based transduction enhancer may comprise a cytokine domain and a transmembrane domain. The cytokine-based transduction enhancer may have a CS-TM structure, 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 described above.

[0088] Cytokine domain of transduction enhancer

[0089] The cytokine domain can include part or all of a T cell activating cytokine, such as IL2, IL7, and IL15. The cytokine domain can include any part of the cytokine, so long as it retains the ability to bind to a specific receptor and activate T cells.

[0090] 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 reactive T cells. It is secreted as a single glycosylated polypeptide, and cleavage of a signal sequence is required for its activity. Solution NMR suggests that the structure of IL2 comprises a bundle of four helices (designated A-D) flanked by two shorter helices and several unstructured loops. Residues in helix A and in the loop region between helices A and B are important for receptor binding. The sequence of IL2 is shown as SEQ ID NO:29.

[0091] SEQ ID NO:29: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0092] IL7 is a cytokine that functions 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.

[0093] SEQ ID NO:30: MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

[0094] 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 main role is to kill virus-infected cells. The sequence of IL-15 is shown as SEQ ID NO: 31.

[0095] SEQ ID NO: 31 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0096] The cytokine-based transduction enhancer may comprise one of the following sequences or variants thereof:

[0097] SEQ ID NO: 32 (membrane-type IL7) MAHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPT KSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGGGSPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV

[0098] SEQ ID NO: 33 (membrane-type IL15) MGLVRRGARAGPRMPRGWTALCLLSLLPSGFMAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSS NGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV

[0099] The cytokine-based transduction enhancer may comprise a variant of the sequence set forth as SEQ ID NO: 32 or 33 having at least 80, 85, 90, 95, 98, or 99% sequence identity, provided that the variant sequence is a cytokine-based transduction enhancer 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.

[0100] Exemplary Advantages of Transduction Enhancers

[0101] In some embodiments, the present disclosure provides viral vectors incorporating transduction enhancers. The vectors can have both the ability to stimulate T cells and also the ability to deliver gene insertion. This can provide one or more advantages, including: (1) a simplified process for T cell engineering, since only one component needs to be added; (2) the virus is unstable and does not need to be removed, avoiding bead removal and the associated loss of yield; (3) a reduced cost for T cell engineering, since only one component needs to be manufactured; (4) increased design flexibility, since each T cell engineering process involves creating a gene transfer vector, and the same product can also be made with a transduction enhancer to "fit" the product; and (5) a shorter manufacturing process. (In soluble antigen / bead-based approaches, the mitogen and vector are typically administered sequentially, with intervals of 1, 2, or even 3 days between each administration. This can be avoided with the retroviral vectors of the present invention because signaling enhancement and viral entry are synchronized and simultaneous; (6) engineering is simplified since there is no need to test many different fusion proteins for expression and function; (7) simultaneous addition of two or more signals can be achieved; and (8) the expression and / or expression levels of each signal / protein can be individually regulated.

[0102] Embodiments of Viral Vectors Comprising Transduction Enhancers

[0103] In some embodiments, the viral envelope comprises one or more transduction enhancers. In some embodiments, the transduction enhancers include one or more T cell activating receptors, NK cell activating receptors, and / or costimulatory molecules. In some embodiments, the one or more transduction enhancers include one or more anti-CD3 In some embodiments, the transduction enhancer comprises all of an anti-CD3 scFv, CD86, and CD137L.

[0104] In some embodiments, the transduction enhancer comprises a mitogenic and / or cytokine stimulus that is incorporated into the retroviral or lentiviral capsid such that the virus both activates and transduces T cells. This eliminates the need for separate addition of vector, mitogen, and cytokine. In some embodiments, the transduction enhancer comprises a mitogenic and / or cytokine-based transmembrane protein contained in the producer or packaging cell, which is incorporated into the retrovirus as it buds from the membrane of the producer or packaging cell. In some embodiments, the transduction enhancer is expressed on the producer cell as a separate cell surface molecule, rather than as part of the viral envelope glycoprotein.

[0105] In some embodiments, the present disclosure provides a retroviral or lentiviral vector having a viral envelope comprising:

[0106] (i) a mitogenic transduction enhancer comprising a mitogenic domain and a transmembrane domain; and / or

[0107] (ii) Cytokine-based transduction enhancers containing a cytokine domain and a transmembrane domain.

[0108] In some embodiments, the transduction enhancer is not part of the viral envelope glycoprotein. In some embodiments, the retroviral or lentiviral vector comprises a separate viral envelope glycoprotein encoded by the env gene. Because the mitogenic and / or cytokine stimulus is provided in a molecule separate from the viral envelope glycoprotein, the integrity of the viral envelope glycoprotein is maintained and does not adversely affect viral titer.

[0109] In some embodiments, a retroviral or lentiviral vector is provided having a viral envelope comprising:

[0110] (i) viral envelope glycoproteins; and

[0111] (ii) a mitogenic transduction enhancer having an MS-TM structure,

[0112] a mitogenic transduction enhancer, wherein M is a mitogenic domain, S is an optional spacer, and TM is a transmembrane domain; and / or

[0113] (iii) Cytokine-based transduction enhancers containing a cytokine domain and a transmembrane domain.

[0114] In some embodiments, the mitogenic transduction enhancer and / or cytokine-based transduction enhancer are not part of the viral envelope glycoprotein. In some embodiments, they are present in the viral envelope as separate proteins and encoded by separate genes. In some embodiments, the mitogenic transduction enhancer is

[0115] It has an MS-TM structure,

[0116] where M is the mitogenic domain, S is an optional spacer, and TM is the transmembrane domain.

[0117] In some embodiments, the mitogenic transduction enhancer binds to an activated T cell surface antigen. In some embodiments, the antigen is CD3, CD28, CD134, or CD137. The mitogenic transduction enhancer can include an agonist of such an activated T cell surface antigen.

[0118] The mitogenic transduction enhancer may comprise a binding domain derived from an antibody, such as OKT3, 15E8, or TGN1412; or a costimulatory molecule, such as OX40L or 41BBL. The viral vector may comprise two or more mitogenic transduction enhancers in the viral envelope. For example, the viral vector may comprise a first mitogenic transduction enhancer that binds to CD3 and a second mitogenic transduction enhancer that binds to CD28. The cytokine-based transduction enhancer may comprise, for example, a cytokine selected from IL2, IL7, and IL15.

[0119] In some embodiments, a retroviral or lentiviral vector is provided having a viral envelope comprising:

[0120] (a) a first mitogenic transduction enhancer that binds to CD3; and

[0121] (b) A second mitogenic transduction enhancer that binds CD28.

[0122] In some embodiments, a retroviral or lentiviral vector is provided having a viral envelope comprising:

[0123] (a) The first mitogenic transduction enhancer that binds CD3;

[0124] (b) a second mitogenic transduction enhancer that binds CD28; and

[0125] (c) Cytokine-based transduction enhancers including IL2.

[0126] In some embodiments, a retroviral or lentiviral vector is provided having a viral envelope comprising:

[0127] (a) The first mitogenic transduction enhancer that binds CD3;

[0128] (b) a second mitogenic transduction enhancer that binds CD28;

[0129] (c) cytokine-based transduction enhancers, including IL7; and

[0130] (d) Cytokine-based transduction enhancers, including IL15.

[0131] T cell activation protein The present disclosure also provides viral vectors comprising a polynucleotide comprising a sequence encoding a T cell activation protein or a T cell activation protein complex. As referred to herein, the terms "T cell activation protein" and "T cell activation protein complex" may be used interchangeably and may refer to a single protein or a complex of separate proteins. In some embodiments, a viral vector transduces host T cells with a polynucleotide encoding a T cell activation protein, resulting in the T cells expressing the protein. The T cell activation protein may then be involved in the activation of the transduced T cells. In some embodiments, the T cell activation protein is a drug-inducible T cell activation protein. In some embodiments, the T cell activation protein forms a chemical-inducible signaling complex. In some embodiments, the T cell activation protein forms an engineered complex that induces a signal inside the cell as a direct result of ligand-induced dimerization. The T cell activation protein may be comprised in a homodimer (dimerization of two identical components) or a heterodimer (dimerization of two different components). The T cell activation protein complex may be a synthetic complex as described herein. Those skilled in the art will understand that the component parts of the T cell activation protein complex can be composed of natural or synthetic components useful for incorporation into the complex. Accordingly, the examples provided herein are not intended to be limiting. Additional T cell activation proteins that can be incorporated herein can be found in WO2016 / 139463 and WO2018 / 111834, the disclosures of which are incorporated herein in their entireties.

[0132] In some embodiments, the T cell activation protein sequence can have a first and a second sequence. The first sequence can encode a first T cell activation protein complex component, which can include a first extracellular binding domain or portion thereof, a hinge domain, a transmembrane domain, and a signaling domain or portion thereof. The second sequence can encode a second T cell activation protein complex component, which can include a second extracellular binding domain or portion thereof, a hinge domain, a transmembrane domain, and a signaling domain or portion thereof. In some embodiments, the first and second components can be configured such that, when expressed, they dimerize in the presence of a ligand.

[0133] As used herein, the terms "rapamycin-activated cytokine receptor" or "RACR" interchangeably refer to a multicomponent receptor that inducibly generates an intracellular signal that promotes cell proliferation and / or activity in the presence of rapamycin. In the presence of rapamycin, RACR can transduce an IL2-like signal within T cells via the IL-2R intracellular domain(s) or variants thereof.

[0134] In some embodiments, the present disclosure provides a protein sequence or sequences of a two-component heterodimeric T cell activation protein complex. In some embodiments, the first component is an IL2Rγ complex. In some embodiments, the IL2Rγ complex comprises the amino acid sequence set forth in SEQ ID NO: 34.

[0135] (MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET; SEQ ID NO: 34).

[0136] In some embodiments, the IL2Rγ complex comprises the amino acid sequence set forth in SEQ ID NO:36.

[0137] (MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET; SEQ ID NO: 36).

[0138] In some embodiments, the IL2Rγ complex comprises the amino acid sequence set forth in SEQ ID NO:37.

[0139] (MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET; SEQ ID NO: 37).

[0140] In some embodiments, the IL2Rγ complex comprises the amino acid sequence set forth in SEQ ID NO:38.

[0141] (MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET; SEQ ID NO: 38).

[0142] In some embodiments, the protein sequence of the first T cell activation protein complex component comprises a protein sequence encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain. Embodiments also include nucleic acid sequences encoding the extracellular binding domain, the hinge domain, the transmembrane domain, or the signaling domain. In some embodiments, the protein sequence of the first T cell activation protein complex component comprising the first extracellular binding domain, the hinge domain, the transmembrane domain, and / or the signaling domain comprises an amino acid sequence that comprises 100%, 99%, 98%, 95%, 90%, 85%, or 80% sequence identity to the sequence set forth in SEQ ID NO: 1, 3, 5, or 7, or that has sequence identity within a range defined by any two of the above percentages.

[0143] In some embodiments, the second T cell activation protein complex component is an IL2Rβ complex. In some embodiments, the IL2Rβ complex comprises the amino acid sequence set forth in SEQ ID NO:39.

[0144] (MALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAP EISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQ GEFRALNARLPLNTDAYLSLQELQGQDPTHLV; SEQ ID NO: 39).

[0145] In some embodiments, the IL2Rβ complex comprises the amino acid sequence set forth in SEQ ID NO:40.

[0146] (MALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHV FRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQ DKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV; SEQ ID NO: 40).

[0147] In some embodiments, the IL2Rβ complex comprises the amino acid sequence set forth in SEQ ID NO:41.

[0148] (MALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHV FRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQ DKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV; SEQ ID NO: 41).

[0149] In some embodiments, the IL2Rβ complex comprises the amino acid sequence set forth in SEQ ID NO:42.

[0150] (MALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHV FRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQ DKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV; SEQ ID NO: 42).

[0151] In some embodiments, the second T cell activation protein complex component is an IL7Rα complex. In some embodiments, the IL7Rα complex comprises the amino acid sequence set forth in SEQ ID NO:43.

[0152] (MALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHV FRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQ DKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV; SEQ ID NO: 43).

[0153] In some embodiments, the protein sequence of the second T cell activation protein complex component comprises a protein sequence encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain. Embodiments also include nucleic acid sequences encoding the extracellular binding domain, the hinge domain, the transmembrane domain, or the signaling domain of the second T cell activation protein complex component. In some embodiments, the protein sequence of the second T cell activation protein complex component comprising the second extracellular binding domain, the hinge domain, the transmembrane domain, and / or the signaling domain comprises an amino acid sequence that comprises 100%, 99%, 98%, 95%, 90%, 85%, or 80% sequence identity to the sequence set forth in SEQ ID NOs: 39-43, or a sequence identity within a range defined by any two of the above percentages.

[0154] In some embodiments, the protein sequence can include a linker. In some embodiments, the linker includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, e.g., glycine, or a number of amino acids within a range defined by any two of the aforementioned numbers, e.g., glycine. In some embodiments, the glycine spacer includes at least three glycines. In some embodiments, the glycine spacer includes a sequence set forth in SEQ ID NO: 44 (GGGS; SEQ ID NO: 44), SEQ ID NO: 45 (GGGSGGG; SEQ ID NO: 45), or SEQ ID NO: 46 (GGG; SEQ ID NO: 46). Embodiments also include nucleic acid sequences encoding SEQ ID NOs: 44-46. In some embodiments, the transmembrane domain is N-terminal to the signaling domain, the hinge domain is N-terminal to the transmembrane domain, the linker is N-terminal to the hinge domain, and the extracellular binding domain is N-terminal to the linker.

[0155] In some embodiments, a protein sequence or sequences of a two-component homodimeric T cell activation protein complex are provided. In some embodiments, the first T cell activation protein complex component is an IL2Rγ complex. In some embodiments, the IL2Rγ complex comprises the amino acid sequence set forth in SEQ ID NO:47.

[0156] (MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET; SEQ ID NO: 47).

[0157] In some embodiments, the protein sequence of the first T cell activation protein complex component comprises a protein sequence encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain. Embodiments also include nucleic acid sequences encoding the extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain. In some embodiments, the protein sequence of the first T cell activation protein complex component comprising the first extracellular binding domain, the hinge domain, the transmembrane domain, and / or the signaling domain comprises an amino acid sequence that comprises 100%, 99%, 98%, 95%, 90%, 85%, or 80% sequence identity to the sequence set forth in SEQ ID NO:47, or a sequence identity within a range defined by any two of the above percentages.

[0158] In some embodiments, the second T cell activation protein complex component is an IL2Rβ complex or an IL2Rα complex. In some embodiments, the IL2Rβ complex comprises the amino acid sequence set forth in SEQ ID NO:48.

[0159] (MALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHV FRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQ DKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV; SEQ ID NO: 48).

[0160] In some embodiments, the IL2Rα complex comprises the amino acid sequence set forth in SEQ ID NO:49

[0161] (MALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHV FRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQ DKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV; SEQ ID NO: 49).

[0162] In some embodiments, the protein sequence of the second T cell activation protein complex component comprises a protein sequence encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain. Embodiments also include nucleic acid sequences encoding the extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain of the second T cell activation protein complex component. In some embodiments, the protein sequence of the second T cell activation protein complex component comprising the second extracellular binding domain, hinge domain, transmembrane domain, and / or signaling domain comprises an amino acid sequence that comprises 100%, 99%, 98%, 95%, 90%, 85%, or 80% sequence identity to the sequence set forth in SEQ ID NO:48 or SEQ ID NO:49, or has sequence identity within a range defined by any two of the above percentages.

[0163] In some embodiments, the sequence for homodimerizing the two-component T cell activation protein complex incorporates the FKBP F36V domain for homodimerization with the ligand AP1903.

[0164] In some embodiments, a protein sequence or sequences of a single-component homodimerized T cell activation protein complex is provided. In some embodiments, the single-component T cell activation protein complex is an IL7Rα complex. In some embodiments, the IL7Rα complex comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0165] (MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET; SEQ ID NO: 50).

[0166] In some embodiments, at least one T cell activation protein comprises a first receptor protein comprising a first dimerization domain and a second receptor protein comprising a second dimerization domain, wherein the first dimerization domain and the second dimerization domain specifically bind to each other in response to a molecule. A molecule bound by a T cell activation protein, alternatively referred to as a "ligand" or "agent," refers to a molecule that has a desired biological effect. In some embodiments, the ligand is recognized and bound by the extracellular binding domain, forming a ternary complex comprising the ligand and two bound T cell activation protein complex components. Ligands include, but are not limited to, protein molecules, including, but not limited to, peptides, polypeptides, proteins, post-translationally modified proteins, antibodies, and the like; small molecules (less than 1000 daltons), inorganic or organic compounds; and nucleic acid molecules, including, but not limited to, double- or single-stranded DNA or double- or single-stranded RNA (e.g., antisense, RNAi, etc.), aptamers, and triple-helical nucleic acid molecules. Ligands can be derived from or obtained from any known organism, including, but not limited to, animals (e.g., mammals (human and non-human mammals)), plants, bacteria, fungi, and protists, or viruses, or libraries of synthetic molecules. In some embodiments, the ligand is a protein, antibody, small molecule, or drug. In some embodiments, the ligand is rapamycin or a rapamycin analog (rapalog). In some embodiments, rapalogs include variants of rapamycin having one or more of the following modifications compared to rapamycin: demethylation, removal, or replacement of methoxy at C7, C42, and / or C29; removal, derivatization, or replacement of hydroxy at C13, C43, and / or C28; reduction, removal, or derivatization of ketone at C14, C24, and / or C30; replacement of a 6-membered pipecolate ring with a 5-membered prolyl ring; and alteration of substituents on the cyclohexyl ring or replacement of the cyclohexyl ring with a cyclopentyl ring.Thus, in some embodiments, the rapalog is everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, CCI-779, C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-iRap, AP21967, mycophenolate sodium, benidipine hydrochloride, rapamine, AP23573, or AP1903, or metabolites, derivatives, and / or combinations thereof. In some embodiments, the ligand is an IMID class drug (e.g., thalidomide, pomalidomide, lenalidomide, or related analogs).

[0167] In some embodiments, the molecule is selected from FK1012, tacrolimus (FK506), FKCsA, rapamycin, coumermycin, gibberellin, HaXS, TMP-HTag, and ABT-737, or a functional derivative thereof.

[0168] Adaptor molecules As used herein, the term "adapter molecule" refers to any molecule having a hapten moiety recognizable by a receptor linked to a targeting moiety. A "targeting moiety" is any molecule that specifically or non-specifically binds to a target cell. In some embodiments, the targeting moiety is a protein. Exemplary proteins for use as targeting moieties include those described in U.S. Pat. No. 9,233,125, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the protein is an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is specific for a tumor antigen. Exemplary proteins include, but are not limited to, anti-cancer monoclonal antibodies, such as cetuximab (anti-EGFR), nimotuzumab (anti-EGFR), panitumumab (anti-EGFR), rituximab (anti-CD20), omalizumab (anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-Her2), gemtuzumab (anti-CD33), alemtuzumab (anti-CD52), and bevacizumab (anti-VEGF).

[0169] In some embodiments, the targeting moiety is a small molecule. Exemplary proteins for use as targeting moieties include those described in U.S. Patent Application Nos. 15 / 296,666, 16 / 092,054, and 16 / 253,562, each of which is incorporated herein by reference in its entirety. Exemplary small molecules useful as targeting moieties include, but are not limited to, folic acid, dicarboxypropyl)ureido]pentanedioic acid (DUPA), NK-1R ligand, CAIX ligand, gamma glutamyl transpeptidase ligand, NKG2D ligand, or cholecystokinin 2 receptor (CCK2R) ligand.

[0170] In some embodiments, the targeting moiety is a lipid, or more specifically, a phospholipid ether (PLE). Exemplary proteins for use as targeting moieties include those described in International Patent Application Publication Nos. WO2018148224, WO2019060425, and WO2018148224, each of which is incorporated herein by reference in its entirety. In some embodiments, the lipid comprises a polar head group and a hydrophobic group. In some embodiments, the hydrophobic group is a carbon chain or a fatty acid, e.g., an aliphatic chain. In some embodiments, the carbon chain or fatty acid is saturated or unsaturated. In some embodiments, the hydrophobic group comprises an alkyl, alkenyl, or alkynyl group. In some embodiments, the hydrophobic group comprises a terpenoid lipid, e.g., a steroid or cholesterol, or comprises an aromatic ring. In some embodiments, the hydrophobic group comprises an ether bond, wherein the ether bond is between the polar head group and the aliphatic chain. In some embodiments, the lipid is a phospholipid ether. In some embodiments, the polar head group comprises a choline, phosphatidylcholine, sphingomyelin, a phosphoethanolamine group, an oligosaccharide residue, a sugar residue, phosphatidylserine, or phosphatidylinositol. In some embodiments, the sugar is glycerol. In some embodiments, the targeting moiety is a hapten, a poly(his) tag, a Strep tag, a FLAG tag, a VS tag, a Myc tag, an HA tag, a NE tag, biotin, digoxigenin, dinitrophenol, or fluorescein. In some embodiments, the hydrophobic group comprises a carbon alkyl chain, wherein the carbon alkyl chain contains at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbons, or any number within the range defined by any two of the above values. In some embodiments, the carbon alkyl chain contains 8 to 22 carbons, e.g., 8 to 12, 12 to 14, 14 to 16, or 16 to 22 carbons. In some embodiments, the polar head group comprises a phosphocholine, a piperidine moiety, or a trimethylarseno-ethyl-phosphate moiety. In some embodiments, the lipid further comprises a spacer separating the targeting moiety from the polar head group.In some embodiments, the spacer comprises a PEG spacer, a hapten(2x) spacer, a hapten(3x) spacer, a hapten(4x) spacer, a hapten(5x) spacer, or an alkane chain. In some embodiments, the spacer comprises poly(carboxybetaine), a peptide, polyglycidol, polyethylene, a polyanhydride, a polyphosphoester, polycaprolactone, or poly(ethylene oxide). In some embodiments, the PEG spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 PEG molecules, or any amount within the range defined by any two of the above values. In some embodiments, the lipid is intercalated in the lipid bilayer of a target cell, such as a cancer cell.

[0171] In some embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof. As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or an immunoreactive portion of an intact immunoglobulin. The term is used in its broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies, as well as functional (antigen-binding) antibody fragments, including antigen-binding fragments (Fab), F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single-chain antibody fragments, including single-chain variable fragments (scFv), diabodies, and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies; multispecific, e.g., bispecific, antibodies; diabodies, triabodies, and tetrabodies; tandem di-scFvs; and tandem tri-scFvs. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable regions of the intact antibody. Examples of antibody fragments include, but are not limited to, antigen-binding fragments (Fab), F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single-chain antibody fragments including single-chain variable fragments (scFv), single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments, diabodies, and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody fragment is an scFv.Non-limiting examples of antibodies or binding fragments thereof include monoclonal antibodies, bispecific antibodies, Fab, Fab2, Fab3, scFv, bis-scFv, minibodies, triabodies, diabodies, tetrabodies, VhH domains, V-NAR domains, IgNAR, and camelid Ig. Additional examples of antibodies are IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgM, IgE, IgD, and IgA. Non-limiting examples of antibodies include human antibodies, humanized antibodies, or chimeric antibodies. Non-limiting examples of recombinant antibodies include antibodies that specifically bind to tumor antigens.

[0172] In some embodiments, the targeting moiety comprises a phospholipid ether (PLE). In some embodiments, the targeting moiety comprises folic acid. In some embodiments, the adapter molecule comprises HPF conjugated to a PLE. In some embodiments, the adapter molecule comprises HPF-FITC-PEG3-C18-alkylphospholipid. In some embodiments, the adapter molecule comprises HPF-{linker}-erufosine.

[0173] As used herein, "hapten" refers to any moiety that can be specifically recognized by a receptor, such as a chimeric antigen receptor. In some embodiments, an adapter molecule contains two or more haptens, e.g., two, three, four, five, six, or more haptens. The haptens can be the same or different from one another. Generally, the hapten (or haptens) is covalently attached to the targeting moiety directly or via a spacer. Several types of "spacers" are contemplated for use with the embodiments described herein, including, but not limited to, poly(carboxybetaine), peptides, polyglycidol, polyethylene, polyanhydrides, polyphosphoesters, polycaprolactone, poly(ethylene oxide), PEG spacers, small peptides, or alkane chains. In some embodiments, the alkane spacer can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons, or any number of carbons between the range defined by any two of the above values. In some embodiments, the PEG spacer contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 PEG molecules, or any amount of PEG molecules between the range defined by any two of the above values.

[0174] Various small molecule haptens are known in the art. Exemplary haptens for use in the compositions and methods of the present disclosure include those described in International Patent Application Publication No. WO2018148224, which is incorporated herein by reference in its entirety. In another aspect, the present disclosure contemplates selecting a novel hapten and generating an antibody specific to that hapten using anti-hapten antibody production techniques known in the art. In some embodiments, the hapten comprises fluorescein. Recombinant human antibody E2 is an antibody capable of binding to fluorescein. In some embodiments, the hapten comprises fluorescein, and the hapten-binding receptor comprises an anti-fluorescein antibody or antigen-binding fragment thereof, such as antibody E2. In some embodiments, the hapten comprises 2,4-dinitrophenol (DNP).

[0175] In some embodiments, the adapter molecule comprises one or more masking moieties covalently attached to the hapten, resulting in a "masked hapten" comprising at least one hapten and at least one masking moiety. A "masking moiety" is a chemical moiety that prevents or inhibits binding of a ligand or receptor that would normally bind to the unmasked form of the hapten to the masked form of the hapten. The masking moiety may comprise a protecting group that prevents recognition of the hapten by inhibiting binding and recognition by a hapten-specific hapten-binding receptor (e.g., a chimeric antigen receptor). When the adapter molecule is incorporated into a cell, where the cell is present in a tumor environment or a site where reactive oxygen species are present, the masking moiety may self-cleave, thereby allowing binding and recognition of the hapten by the chimeric antigen receptor. In some embodiments, the targeting moiety is a lipid that is a phospholipid ether. In some embodiments, the masking moiety comprises a phenolic hydroxyl group or PEG. In some embodiments, the phenolic hydroxyl group is attached to the hydroxyl of the xanthene moiety of fluorescein. In some embodiments, the masking moiety is attached to the adapter molecule via a cleavable moiety, optionally configured to be specifically cleavable in the tumor microenvironment. In some embodiments, the cleavable moiety configured to be cleavable in the tumor microenvironment is cleaved by reaction with reactive oxygen species, acidic pH, hypoxia, or nitrosylation. In some embodiments, the phospholipid ether comprises a hapten, and the CAR is linked to the phospholipid ether through interaction with the hapten. In some embodiments, the phospholipid ether comprises a polar head group and a carbon-alkyl chain. In some embodiments, the carbon-alkyl chain comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbons, or any number within the range defined by any two of the above values. In some embodiments, the carbon-alkyl chain comprises 8 to 22 carbons, e.g., 8 to 12, 12 to 14, 14 to 16, or 16 to 22 carbons. In some embodiments, the masking moiety is removed when the composition is in an acidic environment.In some embodiments, the acidic environment includes a pH of 4, 5, 6, or 6.5, or any pH between the range defined by any two of the above values. In some embodiments, the masking moiety is removed by nitrosylation.

[0176] In some embodiments, the masked hapten is configured to allow a chemical reaction to remove the masking moiety from the hapten. In some embodiments, the masked hapten is configured to allow a reactive oxygen species to remove the masking moiety from the hapten. In some embodiments, the masked hapten comprises a hydroxyphenyl group. In some embodiments, the masking moiety comprises a 2,4-dinitrophenol (DNP) group. In some embodiments, the hapten comprises fluorescein. In some embodiments, the masked hapten comprises hydroxyphenylfluorescein (HPF). In some embodiments, the masked hapten comprises fluorescein-DNP.

[0177] Hapten-binding receptor The present disclosure also provides receptors that bind to adapter molecules, particularly receptors that bind to haptens contained in adapter molecules. The haptens and adapter molecules can be any of those described in the previous sections. In some embodiments, the hapten-binding receptor is a cell surface receptor that naturally binds to the hapten. In some embodiments, the hapten-binding receptor is partially synthetic or entirely synthetic. In some embodiments, the hapten-binding receptor is a recombinant protein. In some embodiments, the hapten-binding receptor is a chimeric receptor.

[0178] In some embodiments, the hapten-binding receptor is a chimeric antigen receptor. The term "chimeric antigen receptor" or "CAR" or "chimeric T cell receptor" refers to an artificially designed receptor that includes a ligand-binding domain of an antibody or other protein sequence that binds to a molecule, a transmembrane domain, one or more intracellular signaling domains, and one or more costimulatory domains. The ligand-binding domain is linked to one or more intracellular signaling domains, e.g., costimulatory domains, of a T cell receptor or other receptor via a spacer domain. Chimeric receptors may also be referred to as artificial T cell receptors, chimeric T cell receptors, chimeric immune receptors, and chimeric antigen receptors (CARs). These CARs are genetically engineered receptors that can introduce any specificity into immune receptor cells. In some embodiments, the spacer of the chimeric antigen receptor is selected (e.g., a specific length of amino acids in the spacer) to achieve the desired binding properties of the CAR. CARs with spacers of different lengths, for example, displayed on cells, are then screened for their ability to bind to or interact with the adapter molecule and / or hapten of interest.

[0179] In some embodiments herein, the CAR comprises one or more intracellular signaling domains, hi some embodiments, the intracellular signaling domains are derived from a ligand that specifically binds to CD27, CD28, 4-IBB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-I (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or CD83, or a portion thereof.

[0180] In some embodiments, a CAR comprises one or more costimulatory domains. A "costimulatory domain" refers to a signaling moiety that provides a signal to a T cell that mediates a T cell response, including, but not limited to, activation, proliferation, differentiation, cytokine secretion, and the like, in addition to the primary signal provided by, for example, the CD3ζ chain of the TCR / CD3 complex. Costimulatory domains may include, but are not limited to, all or a portion of a ligand that specifically binds to CD27, CD28, 4-IBB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-I (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or CD83. In some embodiments, a costimulatory domain is an intracellular signaling domain that interacts with other intracellular mediators to mediate a cellular response, including, but not limited to, activation, proliferation, differentiation, and cytokine secretion. In some embodiments herein, the costimulatory domain comprises 4IBB and CD3ζ. In some embodiments, a T cell is provided that comprises a CAR specific for a hapten on an adapter molecule. In some embodiments, the T cell further comprises an 806 CAR (anti-EGFR(806) 41BB-CD3ζ CAR).

[0181] In some embodiments, the chimeric receptor has at least 80% amino acid identity, at least 90% amino acid identity, or at least 95% amino acid identity to: SVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGSTSGSGKPGSGEGSTKGQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTIS RDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVSSESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 53), or a variant thereof sharing the same complementarity determining regions.

[0182] In some embodiments, the chimeric antigen receptor comprises an anti-FITC scFv conjugated to a CD28 transmembrane span and a 4-1BBζ signaling tail via a short IgG4 linker.

[0183] Exemplary chimeric antigen receptors comprising anti-FITC scFvs include those described in U.S. Patent Application No. 63 / 052,806, which is incorporated herein by reference in its entirety.

[0184] In some embodiments, the hapten-binding receptor comprises a hapten-specific antigen-binding fragment of an antibody. In some embodiments, the antigen-binding fragment comprises a Fab fragment or a single-chain Fv fragment (scFv). In some embodiments, the receptor that specifically binds to a hapten comprises a hapten-specific chimeric antigen receptor.

[0185] In some embodiments, the hapten-binding receptor is a T cell receptor (TCR) or a functional portion thereof. "T cell receptor" or "TCR" refers to a molecule found on the surface of T lymphocytes or T cells that is responsible for recognizing fragments of antigens bound to major histocompatibility complex molecules.

[0186] In some embodiments, the hapten-binding receptor is a dimerization-activated receptor initiation complex (DARIC). DARIC provides the binding and signaling components, each expressed as a separate fusion protein, but contains an extracellular multimerization mechanism (a bridging agent) for recombining the two functional components on the cell surface (see U.S. Patent Application Publication No. 2016 / 0311901, expressly incorporated herein by reference in its entirety). Importantly, in the DARIC system, the bridging agent generates a heterodimeric receptor complex, which alone does not elicit significant signal transduction. The described DARIC complexes elicit physiologically relevant signals only after further colocalization with other DARIC complexes. Therefore, they do not enable the selective proliferation of desired cell types without a mechanism for further multimerization of the DARIC complex (e.g., by contact with tumor cells expressing a ligand bound by a binding domain incorporated into one of the DARIC components). Thus, as used herein, in some embodiments, a binding domain incorporated into a DARIC component binds to a hapten contained in an adapter molecule disclosed herein.

[0187] In some embodiments, the hapten-binding portion of the hapten-binding receptor can comprise the antigen-binding portion of an antibody or antigen-binding antibody derivative. The antigen-binding portion of the antibody or derivative can be a Fab, Fab', F(ab'), Fd, Fv, scFv, diabody, linear antibody, single-chain antibody, minibody, etc. In some embodiments, the hapten-binding portion of the hapten-binding receptor can comprise a DARPin or centirin.

[0188] A hapten-binding receptor can bind to a molecule associated with a disease or disorder. As used herein, the molecule can be a hapten contained in an adapter molecule. In some embodiments, the hapten to which the hapten-binding receptor binds or interacts can be displayed on a substrate, such as a membrane, bead, or support (e.g., a well), or a binding agent, such as a lipid (e.g., a PLE), a hapten, a ligand, or an antibody or binding fragment thereof. In some embodiments, the adapter molecule is a binding agent specific for an antigen present on cancer cells. In some embodiments, the adapter molecule is a binding agent specific for a pathogen, such as a virus or bacterium. According to one approach, a substrate or adapter molecule containing a desired hapten is contacted with a plurality of cells containing a hapten-binding receptor specific for the hapten, and the level or amount of binding of the cells containing the hapten-binding receptor to the hapten present on the substrate or binder is determined. Such assessment of binding can include staining of cells bound to the adapter molecule or assessment of fluorescence or loss of fluorescence. Similarly, modifications of the hapten-binding receptor structure, such as changing the length of the spacer, can be assessed in this manner. In some approaches, a target cell is also provided, such that the method involves contacting cells, such as T cells, comprising a hapten-binding receptor specific for an adapter molecule comprising a targeting moiety and a hapten, in the presence of the target cell, e.g., a cancer cell or a bacterial cell or a target virus, and assessing binding of the cells comprising the hapten-binding receptor to the adapter molecule and / or assessing binding of the cells comprising the hapten-binding receptor to the target cell or target virus. Alterations in different components of the hapten-binding receptor can result in, for example, stronger binding affinity for a specific epitope or antigen.

[0189] In some embodiments described herein, the hapten-binding receptor is specific for a lipid or peptide that targets tumor or cancer cells, where the lipid or peptide includes a hapten, and the hapten-binding receptor can specifically bind to the lipid through an interaction with the hapten. In some embodiments, the lipid is a phospholipid ether. In some embodiments described herein, the hapten-binding receptor is specific for a phospholipid ether, where the phospholipid ether includes a hapten, and the hapten-binding receptor specifically binds to the phospholipid ether through an interaction with the hapten.

[0190] In some embodiments, the hapten-binding receptor is specific for a hapten immobilized on an antibody or binding fragment thereof, where the hapten-binding receptor specifically binds to the antibody or binding fragment thereof through interaction with the hapten. Exemplary haptens that can be conjugated to the antibody or binding fragment thereof include poly(his) tag, Strep tag, FLAG tag, VS tag, Myc tag, HA tag, NE tag, biotin, digoxigenin, dinitrophenol, green fluorescent protein (GFP), yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, far-red fluorescent protein, or fluorescein (e.g., fluorescein isothiocyanate (FITC)). In some embodiments, the antibody or binding fragment thereof is specific for an antigen or ligand present on cancer cells or pathogens (e.g., viral or bacterial pathogens). In some embodiments, the antibody or binding fragment thereof is specific for an antigen or ligand present on tumor cells, viruses, preferably chronic viruses (e.g., hepatitis viruses, e.g., HBV or HCV, or HIV), or bacterial cells.

[0191] In some embodiments, the hapten-binding receptor nucleic acid comprises a polynucleotide encoding a transmembrane domain, which provides anchoring of the chimeric receptor to a membrane.

[0192] In some embodiments, a conjugate is provided that includes a hapten-binding receptor bound to a lipid, wherein the lipid includes a hapten, and the hapten-binding receptor is bound to the lipid through an interaction with the hapten.

[0193] In some embodiments, a conjugate is provided that comprises a hapten-binding receptor bound to an antibody or binding fragment thereof, wherein the antibody or binding fragment comprises a hapten (e.g., poly(his) tag, Strep tag, FLAG tag, VS tag, Myc tag, HA tag, NE tag, biotin, digoxigenin, dinitrophenol, green fluorescent protein (GFP), yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, far-red fluorescent protein, or fluorescein (e.g., fluorescein isothiocyanate (FITC)), and the hapten-binding receptor is bound to the antibody or binding fragment thereof through an interaction with the hapten. In some embodiments, the antibody or binding fragment thereof The antibody or binding fragment further binds to an antigen or ligand present on a cancer cell or pathogen (e.g., a viral or bacterial pathogen). In some embodiments, the antibody or binding fragment thereof binds to an antigen or ligand present on a tumor cell, a virus, preferably a chronic virus (e.g., a hepatitis virus, e.g., HBV or HCV, or HIV), or a bacterial cell. In some embodiments, a hapten is present on the antibody or binding fragment thereof specific for an antigen of a cancer cell or pathogen (e.g., a viral or bacterial cell), and the hapten is bound by a hapten-binding receptor present on the surface of a cell (e.g., a T cell), and the cell having the hapten-binding receptor is directed against the cancer cell or pathogen.

[0194] In some embodiments, the hapten-binding receptor or T cell activation protein of the present disclosure confers resistance to immunosuppressants or antiproliferative substances on immune cells. In some examples, the lentiviral vector confers resistance to immunosuppressants or antiproliferative substances on transduced cells, thereby promoting selective proliferation of target cells. The present disclosure provides lentiviral vectors containing any nucleic acid sequence that confers resistance to immunosuppressants or antiproliferative substances. Examples of immunosuppressants or antiproliferative substances include, but are not limited to, rapamycin or a derivative thereof, a rapalog or a derivative thereof, tacrolimus or a derivative thereof, cyclosporine or a derivative thereof, methotrexate or a derivative thereof, and mycophenolate mofetil (MMF) or a derivative thereof. Various resistance genes are known in the art. Resistance to rapamycin can be conferred by a polynucleotide sequence encoding FRb, a protein domain found in the mTOR domain and known to be a target of the FKBP-rapamycin complex. Resistance to tacrolimus can be conferred by a polynucleotide sequence encoding the calcineurin mutant CNa22 or the calcineurin mutant CNb30. Resistance to cyclosporine can be conferred by a polynucleotide sequence encoding the calcineurin mutant CNa12 or the calcineurin mutant CNb30. These calcineurin mutants are described in Brewin et al. (2009) Blood 114:4792-803. Resistance to methotrexate can be provided by various mutant forms of dihydrofolate reductase (DHFR) (Volpato et al. (2011) J Mol Recognition 24:188-198), and resistance to MMF can be provided by various mutant forms of inosine monophosphate dehydrogenase (IMPDH) (Yam et al. (2006) Mol Ther 14:236-244).

[0195] Immunosuppressants or antiproliferative substances (e.g., immunosuppressants) are commonly used before, during, and / or after ACT. In some instances, the use of immunosuppressants can improve treatment outcomes. In some instances, the use of immunosuppressants can attenuate side effects of treatment, such as, but not limited to, acute graft-versus-host disease, chronic graft-versus-host disease, and post-transplant lymphoproliferative disorder. The present disclosure contemplates the use of immunosuppressants in conjunction with any of the disclosed methods of treating or preventing a disease or condition, including, but not limited to, the disclosed methods of conferring resistance to immunosuppressants to lentiviral vector-transduced cells.

[0196] Polynucleotides The present disclosure also relates to nucleic acids and polynucleotides encoding the transduction enhancers, T cell activation proteins, adapter molecules, and hapten-binding receptors of the present disclosure. The nucleic acids may be in the form of constructs containing multiple sequences encoding any of the above proteins. As used herein, the terms "polynucleotide," "nucleotide," and "nucleic acid" are intended to be synonymous with each other.

[0197] 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. In addition, it will be understood that those skilled in the art can use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon preferences of any particular host organism in which the polypeptide is to be expressed.

[0198] Nucleic acids can include DNA or RNA. They can be single-stranded or double-stranded. They can be polynucleotides containing synthetic or modified nucleotides. Many different types of modifications of 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 the uses described herein, polynucleotides can be modified by any method available in the art. Such modifications can be performed to enhance the in vivo activity or lifespan of the polynucleotide of interest.

[0199] With respect to nucleotide sequences, the terms "variant," "homolog," or "derivative" include any substitution, mutation, modification, replacement, deletion, or addition to or from one (or more) nucleic acid. The nucleic acid may result in a polypeptide comprising one or more sequences encoding a mitogenic transduction enhancer and / or one or more sequences encoding a cytokine-based transduction enhancer. The cleavage site may be self-cleaving, such that once the polypeptide is generated, it is immediately cleaved into the receptor and signaling components without the need for any exogenous cleavage activity.

[0200] Various self-cleaving sites are known, including the foot-and-mouth disease virus (FMDV) 2a self-cleaving peptide and various variants and 2A-like peptides. The peptide may have the sequence shown as SEQ ID NO: 51 or 52.

[0201] SEQ ID NO: 51: RAEGRGSLLTCGDVEENPGP.

[0202] SEQ ID NO: 52: QCTNYALLKLAGDVESNPGP.

[0203] The co-expressed sequence may be an internal ribosome entry sequence (IRES). The co-expressed sequence may be an internal promoter.

[0204] In some embodiments, the polynucleotide encodes a protein that confers resistance to anti-angiogenic agents to immune cells transduced therewith.

[0205] Virus particle tagging proteins The viral envelope of the viral vector may also comprise a tagging protein that comprises a binding domain that binds to the capture moiety and a transmembrane domain.

[0206] The tagging protein may comprise a binding domain that binds to the capture moiety; a spacer; and a transmembrane domain.

[0207] The tagging protein facilitates the purification of viral vectors from cell supernatants by binding to the tagging protein's capture moiety. "Binding domain" refers to an entity, such as an epitope, that can recognize and specifically bind to a target entity, such as a capture moiety. The binding domain can contain one or more epitopes that can specifically bind to the capture moiety. For example, the binding domain can contain at least one, two, three, four, or five epitopes that can specifically bind to the capture moiety. When the binding domain contains two or more epitopes, each epitope can be separated by a linker sequence as described herein.

[0208] The binding domain may be releasable from the capture moiety by the addition of an entity that has a higher binding affinity for the capture moiety compared to the binding domain.

[0209] A binding domain can comprise one or more streptavidin-binding epitopes. For example, a binding domain can comprise at least one, two, three, four, or five streptavidin-binding epitopes.

[0210] 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 molecular weight of approximately 10 -15 It has a dissociation constant (Kd) of M. Streptavidin is well known in the art and is widely used in molecular biology and bionanotechnology due to the resistance of the streptavidin-biotin complex to organic solvents, denaturants, proteases, and extreme temperatures and pH. The strong streptavidin-biotin bond can be used to bind various biomolecules to each other or to solid supports. Harsh conditions are required to break the streptavidin-biotin interaction, but harsh conditions can denature the protein of interest being purified.

[0211] The binding domain can be, for example, a biotin mimetic. A "biotin mimetic" can refer to a short peptide sequence (e.g., 6-20, 6-18, 8-18, or 8-15 amino acids) that specifically binds to streptavidin. As previously mentioned, the affinity of the biotin / streptavidin interaction is very high. Thus, an advantage of the present invention is that the binding domain can include a biotin mimetic that has a lower affinity for streptavidin compared to biotin itself.

[0212] In particular, biotin mimetics can bind to streptavidin with lower binding affinity than biotin, such that biotin can be used to elute retroviral vectors captured by streptavidin. For example, biotin mimetics can bind to streptavidin with a Kd of 1 nM to 100 uM.

[0213] The biotin mimetic can be selected from the following group: Streptagll, Flankedccstreptag, and ccstreptag. The binding domain can include two or more biotin mimetics. For example, the binding domain can include at least one, two, three, four, or five biotin mimetics. When the binding domain includes two or more biotin mimetics, each mimetic can be the same or different.

[0214] The present disclosure also provides optionally purified viral particles and methods for purifying the same. In some embodiments, the viral envelope of the viral vector may comprise a tagging protein comprising a binding domain that binds to a capture moiety; a spacer; and a transmembrane domain, wherein the tagging protein facilitates purification of the viral vector from a cell supernatant by binding to the capture moiety of the tagging protein.

[0215] The binding domain of the tagging protein may comprise one or more streptavidin-binding epitopes. The streptavidin-binding epitopes may be biotin mimics, such as those that bind to streptavidin with lower affinity than biotin, so that biotin can be used to elute retroviral vectors produced by packaging cells and captured by streptavidin. Examples of suitable biotin mimics include Streptagll, Flankedccstretag, and ccstreptag. The viral vector of the first aspect of the present invention may comprise a nucleic acid sequence encoding a T cell receptor or a chimeric antigen receptor. The viral vector may be a virus-like particle (VLP).

[0216] Production / Packaging Cell Lines The present disclosure provides host cells for producing viral particles according to the present disclosure. In some embodiments, the host cells express a mitogenic transduction enhancer and / or a cytokine-based transduction enhancer on the cell surface. The host cells may be for producing viral vectors according to the foregoing embodiments. In some embodiments, the host cells may contain tagging proteins useful for purifying viral particles.

[0217] The host cell may be a packaging cell and may contain one or more of the following genes: gag, pol, env, and rev. Packaging cells for retroviral vectors may contain the gag, pol, and env genes. Packaging cells for lentiviral vectors may contain the gag, pol, env, and rev genes.

[0218] The host cell can be a producer cell and contains the gag, pol, env, and optionally rev genes, as well as the retroviral or lentiviral vector genome. 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 can be removed from the virus and provided by a packaging cell. This allows the virus to integrate its genome into the host genome, but the modified viral genome cannot propagate itself due to the lack of structural proteins, rendering the viral vector replication-deficient.

[0219] The packaging cells are used to propagate and isolate large quantities of the viral vector, ie, to prepare a suitable titer of retroviral vector for transduction of target cells.

[0220] In some cases, propagation and isolation may require isolation of the retroviral gag-pol and env (and, in the case of lentivirus, rev) genes and their separate introduction into host cells to generate a packaging cell line. The packaging cell line produces the proteins required to package retroviral DNA but is unable to induce encapsidation due to the lack of a psi region. However, when a recombinant vector carrying a psi region is introduced into the packaging cell line, helper proteins can package the psi-positive recombinant vector, resulting in a recombinant virus stock.

[0221] An overview of available packaging lines is given in "Retroviruses" (1997 Cold Spring Harbor Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 449).

[0222] Packaging cells have also been developed in which the viral gag, pol, and env (and, in the case of lentiviral vectors, rev) coding regions are carried on separate expression plasmids that are independently transfected into packaging cell lines, resulting in the three recombination events required for wild-type virus production.

[0223] Transient transfection avoids the longer time required to generate stable vector-producing cell lines and is used when the vector or retroviral packaging components are toxic to cells. Components typically used to produce retroviral / lentiviral vectors include a plasmid encoding the Gag / Pol proteins, a plasmid encoding the Env protein (and, in the case of lentiviral vectors, the Rev protein), and the retroviral / lentiviral vector genome. Vector production involves transiently transfecting one or more of these components into cells containing the other necessary components. The packaging cells of the present invention can be any mammalian cell type capable of producing retroviral / lentiviral vector particles. The packaging cells can be 293T cells or variants of 293T cells that are adapted to growth in suspension and grow without serum.

[0224] Packaging cells can be generated by transient transfection with:

[0225] a) Transfer vector

[0226] b) gag-pol expression vector

[0227] c) env expression vector. The env gene can be heterologous, resulting in a pseudotyped retroviral vector. For example, the env gene can be derived from RD114 or one of its variants, VSV-G, gibbon ape leukemia virus (GALV), amphotropic viral envelope, measles virus envelope, or baboon retrovirus envelope glycoprotein.

[0228] In the case of lentiviral vectors, transient transfection with the rev vector is also performed.

[0229] The present disclosure provides a host cell expressing a viral particle according to the foregoing embodiments. In some embodiments, the host cell expresses one or more transduction enhancers on the cell surface. In some embodiments, the present invention provides a host cell expressing a viral particle according to the foregoing embodiments.

[0230] (a) a mitogenic transduction enhancer comprising a mitogenic domain and a transmembrane domain; and / or

[0231] (b) expressing a cytokine-based transduction enhancer on the cell surface, the cytokine-based transduction enhancer comprising a cytokine domain and a transmembrane domain;

[0232] As a result, the retroviral or lentiviral vector produced by the packaging cells is provided as described in the previous embodiments.

[0233] In some embodiments, the host cells may also express on their cell surface a tagging protein comprising a binding domain that binds to the capture moiety; and a transmembrane domain, where the tagging protein facilitates purification of the viral vector from the cell supernatant by binding of the tagging protein to the capture moiety, such that the retroviral or lentiviral vector produced by the packaging cells has the properties described in the preceding section.

[0234] The tagged protein may also include a spacer between the binding domain and the transmembrane domain.

[0235] The term host cell can be used to describe packaging cells or producer cells. Packaging cells can contain one or more of the following genes: gag, pol, env, and / or rev. Producer cells can contain the gag, pol, env, and optionally rev genes, and also contain the retroviral or lentiviral genome. In some embodiments, host cells can be any suitable cell line that stably expresses mitogenic and / or cytokine-based transduction enhancers. Host cells can be transiently transfected with transfer vectors, gag-pol, env (and rev, in the case of lentivirus) to generate replication-incompetent retroviral / lentiviral vectors.

[0236] The present disclosure also provides a method for producing the host cell described above, comprising transducing or transfecting the cell with a nucleic acid encoding one or more transduction enhancers. Also provided is a method for producing the viral vector described in the previous embodiment, comprising expressing a retroviral or lentiviral genome in a cell according to the second aspect of the present invention.

[0237] Systems and Kits The present disclosure provides:

[0238] (a) an adapter molecule comprising a targeting moiety and a masked hapten, the masked hapten comprising a masking moiety linked to the hapten;

[0239] (b) a plurality of recombinant retroviral particles;

[0240] wherein each of the retroviral particles comprises, in 5' to 3' order:

[0241] (i) a 5' long terminal repeat (LTR) or untranslated region (UTR);

[0242] (ii) a promoter; and

[0243] (iii) a sequence encoding a receptor that specifically binds to the hapten; and

[0244] (iv) a 3' LTR or a UTR,

[0245] Each of the retroviral particles comprises:

[0246] (i) a viral fusion glycoprotein;

[0247] (ii) one or more transduction enhancers;

[0248] Each of the transduction enhancers is optionally selected from the group consisting of a T cell activating receptor, an NK cell activating receptor, and a costimulatory molecule. In some embodiments, the polynucleotide additionally comprises a sequence encoding a T cell activating protein.

[0249] The present disclosure also provides a kit that includes the system and instructions for using the system.

[0250] Transgenic immune cells The present disclosure provides a method for generating transgenic activated immune cells, comprising contacting immune cells with a viral vector according to any of the preceding embodiments. The immune cells may be transduced in vivo or ex vivo. In some embodiments, the viral vector is administered to a living subject such that the immune cells are transduced in vivo without the need for isolation and ex vivo manipulation of host cells. In some embodiments, the immune cells are manipulated ex vivo and then returned to the subject in need thereof.

[0251] Immune cells are generally mammalian cells, usually human cells, and more typically primary human cells, e.g., allogeneic or autologous donor cells. Cells can be isolated from a sample, such as a biological sample, e.g., obtained from or derived from a subject. In some embodiments, the subject from whom the cells are isolated has a disease or condition or is in need of or will be administered cell therapy. In some embodiments, the subject is a human in need of a particular therapeutic intervention, e.g., adoptive cell therapy, in which cells are isolated, treated, and / or manipulated. In some embodiments, the cells are derived from blood, bone marrow, lymph, or lymphoid organs and are cells of the immune system, e.g., myeloid or lymphoid cells, including cells of the innate or adaptive immune system, e.g., lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, e.g., multipotent stem cells and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). Cells are typically primary cells, e.g., isolated directly from a subject and / or isolated and frozen from a subject. In some embodiments, the cells include one or more subsets of T cells or other cell types, e.g., the total T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, e.g., those defined by function, activation state, maturity, differentiation potential, proliferation, recirculation, localization, and / or persistence capacity, antigen specificity, antigen receptor type, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation.

[0252] Subtypes and subpopulations of T cells and / or CD4+ T cells and / or CD8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells, and their subtypes, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, natural and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0253] In some embodiments, the cells provided herein are cytotoxic T lymphocytes. "Cytotoxic T lymphocytes" (CTLs) include, but are not limited to, T lymphocytes that express CD8 on their surface (e.g., CD8+ T cells). In some embodiments, such cells are preferably antigen-experienced "memory" T cells (TM cells). In some embodiments, the cells are T progenitor cells. In some embodiments, the T progenitor cells are hematopoietic stem cells. In some embodiments, the cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells.

[0254] As used herein, any reference to transgenic or transduced T cells, or their use, may also apply to any of the other immune cell types disclosed herein.

[0255] The present disclosure also provides transgenic immune cells comprising one or more exogenous nucleic acid molecules. In some embodiments, the transgenic immune cells comprise a polynucleotide encoding a hapten-binding receptor. In some embodiments, the transgenic immune cells comprise a polynucleotide encoding a transduction enhancer. In some embodiments, the transgenic immune cells comprise a polynucleotide encoding a T cell activation protein. In some embodiments, the transgenic immune cells comprise a polynucleotide encoding a hapten-binding receptor and a polynucleotide encoding a T cell activation protein.

[0256] Methods of treating subjects with compositions of the present disclosure The present disclosure provides methods of treating a subject in need thereof with the compositions, therapeutic compositions, cells, vectors, and polynucleotides disclosed herein. In some embodiments, the present disclosure provides a method of treating cancer and / or killing cancer cells in a subject, comprising administering to the subject a therapeutically effective amount of a viral particle of the present disclosure, wherein before, during, or after the administering step, the subject has been or is administered an adapter molecule comprising a targeting moiety and a masked hapten in a dose effective to label cancer cells with the hapten. Also provided is a method of treating tumors and / or killing tumor cells in a subject, comprising administering to the subject an effective amount of an adapter molecule, wherein the adapter molecule labels tumor cells with the masked hapten, and the masked hapten is activated by reactive oxygen species, thereby generating the hapten, wherein before, during, or after the administering step, the subject has been or is administered a retroviral particle according to any of the preceding embodiments.

[0257] In some embodiments, the methods disclosed herein may be used to treat cancer and / or kill cancer cells in a subject by administering a therapeutically effective amount of a lentiviral particle according to any of the preceding embodiments, wherein prior to the administering step, the subject has been administered a dose of a targeting moiety and an adapter molecule comprising the hapten effective to label cancer cells with the hapten. In some embodiments, the methods disclosed herein may be used to treat cancer and / or kill cancer cells by administering a system.

[0258] The present disclosure also provides a method of treating cancer and / or killing cancer cells in a subject, comprising administering to the subject a system of any of the preceding embodiments.

[0259] In some embodiments, the present disclosure provides a method for treating cancer using any of the compositions provided herein. "Cancer," when read in light of the present specification, has its common and ordinary meaning, and may include, but is not limited to, a group of diseases involving abnormal cell proliferation that may invade or spread to other parts of the body. Subjects that may be treated using the methods described herein include subjects identified or selected as having cancer, including, but not limited to, colon cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, ovarian cancer, skin cancer (including melanoma), bone cancer, and brain cancer. Such identification and / or selection may be made by clinical or diagnostic evaluation. In some embodiments, tumor-associated antigens or molecules are known, such as those associated with melanoma, breast cancer, brain cancer, squamous cell carcinoma, colon cancer, leukemia, myeloma, and / or prostate cancer. Examples include, but are not limited to, B-cell lymphoma, breast cancer, brain cancer, prostate cancer, and / or leukemia. In some embodiments, the one or more oncogenic polypeptides are associated with kidney cancer, uterine cancer, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer (including melanoma), bone cancer, brain cancer, adenocarcinoma, pancreatic cancer, chronic myeloid leukemia, or leukemia. In some embodiments, methods are provided for treating, ameliorating, or inhibiting cancer in a subject. In some embodiments, the cancer is breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, kidney cancer, pancreatic cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, liver cancer, colon cancer, skin cancer (including melanoma), bone cancer, or brain cancer.

[0260] In some embodiments, the target cell is a tumor cell. In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is a T cell or a B cell. In some embodiments, the target cell is present in the tumor microenvironment.

[0261] In some embodiments, the transduced T cells are provided to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 24, 36, 48, 60, or 72 hours after administration of the adapter molecule composition, or any time within a range defined by any two of the above values. In some embodiments, the cells are provided to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 24, 36, or 48 hours before administration of the composition, or any time within a range defined by any two of the above values. In some embodiments, the cells are provided to the subject within seconds or minutes, e.g., less than an hour, of providing the composition to the subject. In some embodiments, additional cells and / or compositions are provided to the subject. In some embodiments, the viral vector is administered directly to the subject. In some embodiments, the viral vector is administered together with the T cells. In some embodiments, the viral vector and the T cells are administered separately. In some embodiments, T cells are activated and transduced in vivo by the administered viral vector.

[0262] In some embodiments, additional cancer treatments are provided, such as small molecules, e.g., compounds, antibody therapies, e.g., humanized monoclonal antibodies, with or without conjugation to radionuclides, toxins, or drugs, surgery, and / or radiation.

[0263] In some embodiments, the subject is selected to receive additional cancer therapy, which may include cancer therapy, radiation, chemotherapy, or drugs for the treatment of cancer, such as abiraterone, alemtuzumab, anastrozole, aprepitant, arsenic trioxide, atezolizumab, azacitidine, bevacizumab, bleomycin, bortezomib, cabazitaxel, capecitabine, carboplatin, cetuximab, chemotherapy combinations, cisplatin, crizotinib, cyclophosphamide, cytarabine, denosumab, docetaxel, doxorubicin, eribulin, erlotinib, etoposide, everolimus, exemestane, filgrastim, fluorouracil, fulvestra, or the like. These include methadone, gemcitabine, imatinib, imiquimod, ipilimumab, ixabepilone, lapatinib, lenalidomide, letrozole, leuprolide, mesna, methotrexate, nivolumab, oxaliplatin, paclitaxel, palonosetron, pembrolizumab, pemetrexed, prednisone, radium-223, rituximab, sipuleucel-T, sorafenib, sunitinib, intrapleural talc, tamoxifen, temozolomide, temsirolimus, thalidomide, trastuzumab, vinorelbine, or zoledronic acid.

[0264] Mode of administration and dosage The viral particles, adapter molecules, and immune cells of the present disclosure can be administered in a number of ways depending on whether local or systemic treatment is desired.

[0265] In the case of adoptive cell therapy, methods for administering cells for adoptive cell therapy are known and can be used with the provided methods and compositions. For example, adoptive T cell therapy is described, for example, in U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85; Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) See Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

[0266] Generally, administration can be topical, parenteral, or enteral. The compositions of the present disclosure are generally suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any administration route characterized by physically breaking through the target tissue and administering the pharmaceutical composition through the break in the tissue, generally resulting in direct administration to the bloodstream, muscle, or internal organs. Thus, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injecting the composition, administering the pharmaceutical composition by applying the composition through a surgical incision, administering the pharmaceutical composition by applying the composition through a non-surgical wound that penetrates the tissue, and the like. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intratumoral, intrasynovial injection or infusion; and kidney dialysis infusion. In a preferred embodiment, parenteral administration of the compositions of the present disclosure includes intravenous administration.

[0267] Pharmaceutical compositions suitable for parenteral administration typically contain the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as ampoules or multidose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further contain one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions, which may contain excipients such as salts, carbohydrates, and buffers (preferably to a pH of 3 to 9); however, for some applications, they may be more appropriately formulated as sterile nonaqueous solutions or as a dry form for use with a suitable vehicle, such as sterile, pyrogen-free water. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, such as aqueous propylene glycol or dextrose solutions. Such dosage forms may be suitably buffered, if necessary. Other useful parenterally administrable formulations include those containing the active ingredient in microcrystalline form or in a liposomal formulation. Formulations for parenteral administration may be formulated for immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0268] The compositions of the present invention may additionally contain other auxiliary ingredients typically found in pharmaceutical compositions. Thus, for example, the compositions may contain additional compatible pharmaceutically active substances, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional substances useful for the physical formulation of various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, when added, such substances should not unduly interfere with the biological activity of the components of the compositions of the present invention. The formulations may be sterilized and, if necessary, mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors, and / or fragrances, which do not adversely interact with the nucleic acid(s) of the formulation.

[0269] The viral particle, adapter molecule, and / or immune cell compositions of the present invention can be administered in an amount effective to treat or prevent a disease or condition, e.g., a therapeutically or prophylactically effective amount. In some embodiments, therapeutic or prophylactic effectiveness is monitored by periodic evaluation of the treated subject. In the case of repeated administration over several days or longer, depending on the condition, treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosing regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0270] In certain embodiments, in the context of infusion of immune cells or transgenic immune cells according to the present disclosure, a subject is administered a total of about 1 million to about 100 billion cells, e.g., about 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 50 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, or about 90 million cells). cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and optionally about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), etc., or any value therebetween, and / or such number of cells per kg of body weight of the subject. For example, in some embodiments, administration of the cells or cell population is at a rate of about 10 per kg of body weight. 3 ~about 10 9 cells (including all integer numbers of cells within these ranges).

[0271] In the context of administering viral particles, the amount of viral particles and the number of times such particles are administered are within the purview of those skilled in the art who have the benefit of the teachings of the present invention. In some embodiments, administration of a therapeutically effective amount of a composition of the present disclosure can be achieved by a single administration, such as, for example, a single injection of a sufficient number of viral particles to provide a therapeutic effect to a patient undergoing such treatment. In some embodiments, multiple or continuous administrations of a lentiviral vector composition are provided to a subject over either a relatively short or a relatively long period of time, as can be determined by a medical professional overseeing the administration of such compositions. For example, the number of infectious particles administered to a mammal can be about 10, which may be required to achieve treatment for the particular disease or disorder being treated. 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 The viral vector composition may be administered in the amount of about 100 or even more viral particles / ml, either as a single dose or divided into two or more administrations. In some embodiments, a subject may be administered two or more different viral vector compositions, either alone or in combination with one or more other therapeutic agents, to achieve the desired effect of a particular treatment regimen. In some embodiments, the viral vector is administered in combination with transgenic immune cells. In some embodiments, the viral vector is administered in combination with previously untransduced immune cells. The phrase "in combination" can include simultaneously or within a short period of time, e.g., within 1 week, 1 day, 12 hours, 6 hours, 1 hour, 30 minutes, 10 minutes, 5 minutes, or 1 minute of each other.

[0272] In the context of administering an adapter molecule, the dosage depends on the type of target cell, the targeting moiety included in the adapter molecule, and the hapten molecule included in the adapter molecule. Depending on the type and severity of the disease, exemplary dosages of the adapter molecule can range from about 1 μg / kg to about 50 mg / kg or from about 5 mg / kg to about 15 mg / kg, including, but not limited to, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, or 15 mg / kg. The frequency of administration varies depending on the type and severity of the disease. In the case of repeated administration over several days or longer depending on the condition, treatment can be continued until the condition, e.g., cancer, is treated or the desired therapeutic effect is achieved as measured by methods known in the art. In some embodiments, the adapter molecule is administered once. In some embodiments, the adapter molecule is administered once every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or year. The adapter molecule may be administered in combination with the viral particles and / or transgenic immune cells disclosed herein. The phrase "in combination" can include simultaneously or within a short period of time, e.g., within 1 week, 1 day, 12 hours, 6 hours, 1 hour, 30 minutes, 10 minutes, 5 minutes, or 1 minute.

[0273] **** All publications and patents mentioned herein are incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be considered as, an admission or any form of suggestion that they constitute valid prior art or form part of the general knowledge in any country in the world.

[0274] As used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the stated range, and, where appropriate, fractions thereof (e.g., tenths and hundredths of an integer), unless otherwise specified. The term "about," when immediately preceding a number or numeral, means that the number or numeral is within a range of plus or minus 10%. As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the indicated components, unless otherwise specified. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. The term "and / or" should be understood to mean either one or both of the alternatives. As used herein, the terms "include" and "comprise" are used interchangeably.

[0275] The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0276] While illustrative embodiments have been shown and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. [Example]

[0277] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be merely exemplary of the invention and are not intended to limit the scope of what is regarded as the invention.

[0278] Example 1: Transduction, enrichment, and activation of FITC CAR This example demonstrates enhanced proliferation and activation of CAR-expressing T cells in the presence of the antigen fluorescein isothiocyanate (FITC).

[0279] Thawing of PBMCs and culture in the absence of stimulation On day 0 of the protocol, frozen peripheral blood mononuclear cells (PBMCs) were quickly thawed in a 37°C water bath and slowly added to 9 mL of warm RPMI-C (RPMI 1640 + P / S + 10% FBS) medium. The thawed PBMCs were then centrifuged at 200 × g for 10 minutes. After centrifugation, the medium was aspirated, and the cells were counted and collected at 1 × 10 6 Cells were plated at a cell density of 1.5 × 10 cells / mL in approximately 250 mL flasks in RPMI-C medium. 50 U / mL of IL-2 was also added to the flasks. The total number of viable cells after plating was 2.32 × 10 cells in 20 mL of RPMI-C medium containing IL-2. 7 It was a single cell.

[0280] Transduction with FITC CAR vector On day 1 of the protocol, 1 × 10 7 PBMC cells were collected in a 50 mL conical tube and centrifuged at 400 × g for 5 minutes. The cells were then diluted to 2.5 × 10 in a total of 2.5 mL of RPMI-C medium containing 50 U / mL of IL-2 in a 6-well plate. 6 PBMCs were plated at a concentration of 1.5 × 10 cells / well. Lentiviral particles modified with a CD3-cocal envelope and containing the FITC CAR-Frb-RACR polycistronic vector were used to transduce PBMCs at a multiplicity of infection (MOI) of 10. The lentiviral titer used for transduction was 1.61 × 10 cells / well. 8 TU / mL, 2.5 x 10 7 The target titer was TU / well. The volume of particles added to each well was 155 μL. The transduced cells were maintained in an incubator for 6 days, and 50 U / mL of IL-2 was added to the cells every 3 days. The transduced cells were monitored daily, and when the cell culture medium was observed to have an orange-yellow color, RPMI-C medium containing 50 U / mL of IL-2 was added to the well.

[0281] Splitting cells into rapamycin-supplemented conditions On day 7 of the protocol, 1 × 10 7PBMC cells were collected in a 15 mL conical tube, washed with RPMI-C medium, and centrifuged at 400 × g for 5 minutes. The cells were diluted to 2 × 10 in 2 mL of RPMI-C medium containing 50 U / mL of IL-2. 6 The cells were split into two wells of a 6-well plate at a cell density of 10 cells / well. 10 nM rapamycin was added to one well and 0 nM to the second well. The rapamycin-treated and untreated cells were maintained in an incubator for 4 days, and 50 U / mL IL-2 was added to the cells every 3 days. The cells were monitored daily, and when the cell culture medium was observed to take on an orange-yellow color, RPMI-C medium containing 50 U / mL IL-2 + / - 10 nM rapamycin was added to the well.

[0282] Coating of plates with FITC-OVA On day 10 of the protocol, a 5 μg / mL solution of fluorescently labeled ovalbumin (FITC-OVA) in PBS was prepared. 125 μL of the FITC-OVA solution was added per well of a 48-well plate. The plate, wrapped in aluminum foil, was incubated with the solution overnight at 4°C.

[0283] Conjugation of FITC-biotin to beads On day 10 of the protocol, vortex the anti-biotin MACSiBead particles and add 30 µg of FITC-biotin primary antibody to 10 x 10 8 The total volume was brought to 1 mL using a buffer containing PBS with 0.5% BSA and 2 nM EDTA.

[0284] Cell division under antigen (FITC) stimulation conditions On day 11 of the protocol, a total of 4.8 x 10 cells were incubated in 0 nM rapamycin. 6 A total of 1.9 x 10 live cells were incubated in 10 nM rapamycin. 6 Viable cells were collected in a 15 mL conical tube, washed with RPMI-C medium, and centrifuged at 400 × g for 5 minutes. The washed cells were collected at a concentration of 2.5 × 10 per mL.6 The cells were resuspended in RPMI-C medium without IL-2 at a cell density of 100 cells / ml.

[0285] The FITC-OVA solution was aspirated from the treated plates and washed three times with cold PBS. Cells were plated at 2.5 x 10 in 500 μL of RPMI-C medium + / - 10 nM rapamycin. 5 The cells were divided into 48-well plates at 100 cells / well. Table 1 shows the stimulation conditions for the samples. For cells treated under FITC-biotin bead conditions, a 1:1 ratio of beads to cells was used. The cells were maintained in an incubator for 5 days. The cells were monitored daily, and when the cell culture medium was observed to take on an orange-yellow color, RPMI-C medium + / - 10 nM rapamycin was added to the wells. [Table 1]

[0286] Assessment of CAR expression and activation by flow cytometry Cells were counted for each stimulation condition on day 16 of the protocol, and the cell count results are shown in Table 2. [Table 2]

[0287] The cells were transferred to individual wells of a 96-well V-bottom plate, the plate was centrifuged at 400×g for 5 minutes, the cells were washed with 200 μL of PBS, and centrifuged again at 400×g for 5 minutes. The cells were incubated in 50 μL / well of Zombie NIR Fixable Viability Dye (diluted 1:3000 in PBS) at room temperature for 10 minutes. The cells were then washed with 150 μL of FACS buffer (1X PBS + 2% FBS), and the plate was centrifuged at 400×g for 5 minutes. Next, 50 μL / well of surface stain in FACS buffer was added to the cells and incubated at 4°C for 45 minutes.

[0288] The cells were washed with 150 μL of FACS buffer and the plates were centrifuged at 400×g for 5 minutes. The cells were then fixed with 100 μL / well of BD Cytofix / Cytoperm buffer for 20 minutes at 4° C. The cells were then fixed with 100 μL of BD Cytofix / Cytoperm buffer diluted 1:10 in water for 20 minutes. The cells were washed with Perm / Wash, and the plate was centrifuged at 400 x g for 5 minutes. The cells were washed again with 200 μL of BD Perm / Wash diluted 1:10 in water, and the plate was centrifuged again at 400 x g for 5 minutes. 50 μL / well of intracellular stain in 1X BD Perm / Wash was added to the cells and incubated at 4°C for 30 minutes. The cells were washed with 150 μL of FACS buffer, and the plate was centrifuged at 400 x g for 5 minutes. The cells were resuspended in 200 μL of FACS buffer for analysis using a Beckman Coulter CytoFLEX S cytometer. As shown in Figures 2A-2F, CAR-expressing T cells proliferate in the presence of the antigen FITC, and this proliferation is enhanced by rapamycin.

[0289] Antibodies and fluorescently labeled molecules for flow cytometry analysis were used at the following dilutions: FITC dextran (1:10) CD3-AF700(1:100) CD25-BV421(1:100) PD1-BV650(1:100) Lag3-PECy7(1:100) 2A-AF647(1:100) In certain embodiments, for example, the following are provided: (Item 1) (a) administering to a subject an adapter molecule comprising a targeting moiety and a hapten; (b) administering to the subject either (i) a plurality of recombinant retroviral particles or (ii) immune cells that have been contacted with a plurality of recombinant retroviral particles ex vivo, each of said retroviral particles comprises a polynucleotide comprising a sequence encoding a receptor that specifically binds to said hapten; The method, wherein each of the retroviral particles comprises a viral envelope. (Item 2) 2. The method of claim 1, wherein the immune cells are T cells. (Item 3) Item 4. The method according to item 1 or 2, wherein the retroviral particle is a lentiviral particle. 4. The method according to any one of items 1 to 3, wherein the viral envelope comprises a cell surface receptor that specifically binds to the immune cell. (Item 5) 5. The method of claim 4, wherein the cell surface receptor comprises a multi-component signaling complex. (Item 6) 6. The method of claim 5, wherein the multi-component signaling complex forms a functional multi-component signaling complex in the presence of a cross-linking agent. (Item 7) 7. The method according to any one of items 1 to 6, wherein the viral envelope comprises one or more transduction enhancers. (Item 8) 8. The method of claim 7, wherein each of the transduction enhancers is a T cell activating receptor, an NK cell activating receptor, or a costimulatory molecule. (Item 9) 9. The method of any one of items 1 to 8, wherein the adapter molecule comprises a masked hapten comprising one or more masking moieties covalently attached to the hapten. (Item 10) 8. The method of claim 7, wherein the masked hapten is configured to allow a chemical reaction to remove the masking moiety from the hapten. (Item 11) 11. The method of any of items 9 or 10, wherein the masked hapten is configured to allow reactive oxygen species to remove the masking moiety from the hapten. (Item 12) 12. The method according to any one of items 9 to 11, wherein the masked hapten comprises a hydroxyphenyl group. (Item 13) 13. The method according to any one of items 9 to 12, wherein the hapten comprises a 2,4-dinitrophenol (DNP) group. (Item 14) 14. The method according to any one of items 1 to 13, wherein the hapten comprises fluorescein. (Item 15) 15. The method of claim 14, wherein the hapten is a masked hapten comprising hydroxyphenylfluorescein (HPF). (Item 16) 15. The method of claim 14, wherein the hapten is a masked hapten comprising fluorescein-DNP. (Item 17) 17. The method of any one of items 1 to 16, wherein the targeting moiety comprises a phospholipid ether (PLE). (Item 18) 17. The method of any one of items 1 to 16, wherein the targeting moiety comprises folic acid. (Item 19) 16. The method of claim 15, wherein the adapter molecule comprises a HPF conjugated to a PLE. (Item 20) The adapter molecule is HPF-PEG3-C 18 - The method according to item 15, comprising an alkyl phospholipid. (Item 21) The adapter molecule has Formula I: [ka] 18. The method of claim 17, comprising the molecule of (Item 22) 18. The method of claim 17, wherein the adapter molecule comprises HPF-{linker}-erufosine. (Item 23) 23. The method according to any one of items 1 to 22, wherein the viral envelope comprises a viral fusion glycoprotein derived from the Cocal strain or a functional variant thereof. (Item 24) 24. The method according to any one of items 1 to 23, wherein the viral envelope comprises a viral fusion glycoprotein comprising an amino acid sequence at least 95% identical to SEQ ID NO: 1 (Cocal G protein). (Item 25) Item 26. The method of any one of Items 1 to 24, wherein the one or more transduction enhancers include one or more of anti-CD3 scFv, CD86, and CD137L. 26. The method of any one of items 1 to 25, wherein the transduction enhancer comprises all of anti-CD3 scFv, CD86, and CD137L. (Item 27) 27. The method according to any one of items 1 to 26, wherein the polynucleotide comprises a sequence encoding at least one T cell activation protein. (Item 28) 28. The method of claim 27, wherein the at least one T cell activation protein is a dimeric T cell activation receptor. (Item 29) 29. The method of claim 27 or 28, wherein the at least one T cell activation protein comprises a first receptor protein comprising a first dimerization domain and a second receptor protein comprising a second dimerization domain, and the first dimerization domain and the second dimerization domain specifically bind to each other in response to a molecule. (Item 30) 30. The method of claim 29, wherein the molecule is selected from the list consisting of FK1012, tacrolimus (FK506), FKCsA, rapamycin, coumermycin, gibberellin, HaXS, TMP-HTag, ABT-737, and functional derivatives thereof. (Item 31) 31. The method according to any one of items 1 to 30, wherein the receptor that specifically binds to the hapten comprises a hapten-specific antigen-binding fragment of an antibody. (Item 32) 32. The method of claim 31, wherein the antigen-binding fragment comprises a Fab fragment, a single-chain Fv fragment (scFv), or a single-chain heavy-chain antibody. (Item 33) 33. The method according to any one of items 1 to 32, wherein the receptor that specifically binds to the hapten comprises a hapten-specific chimeric antigen receptor. (Item 34) 34. The method of claim 33, wherein the hapten-specific chimeric antigen receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence hapten:NN. (Item 35) 35. The method of any one of items 1 to 34, wherein T cells in the subject are transduced with the retroviral particles. (Item 36) 36. The method according to any one of items 1 to 35, wherein T cells in the subject express a receptor that specifically binds to the hapten. (Item 37) 37. The method according to any one of items 1 to 36, wherein the adapter molecule specifically binds to and / or labels cancer cells in the subject. (Item 38) 38. The method according to any one of items 1 to 37, wherein the masked hapten is removed by a chemical reaction in the subject. (Item 39) 39. The method according to any one of items 1 to 38, wherein T cells transduced by said retroviral particles specifically kill cancer cells containing the unmasked hapten. (Item 40) 40. The method of any one of items 1 to 39, wherein the subject is suffering from cancer and the method treats the cancer. (Item 41) 41. The method of any one of items 1 to 40, wherein said method kills tumor cells. (Item 42) (a) an adapter molecule comprising a targeting moiety and a masked hapten, the masked hapten comprising a masking moiety linked to the hapten; (b) a plurality of recombinant retroviral particles, each of said retroviral particles comprising, in 5' to 3' order: (i) a 5'UTR; (ii) a promoter; and (iii) a sequence encoding a receptor that specifically binds to the hapten; (iv) a 3'UTR; and a polynucleotide comprising: each of said retroviral particles (i) a cell surface receptor; (ii) one or more transduction enhancers; The system, the therapeutic system, or the composition, wherein each of the transduction enhancers is optionally selected from the group consisting of a T cell activating receptor, an NK cell activating receptor, and a costimulatory molecule. (Item 43) Item 43. The system of item 42, wherein the retroviral particle is a lentiviral particle. (Item 44) 44. The system of item 42 or 43, wherein the viral envelope comprises a cell surface receptor that specifically binds to an immune cell. (Item 45) 45. The system of claim 44, wherein the cell surface receptor comprises a multi-component signaling complex. (Item 46) 46. The system of claim 45, wherein the multi-component signaling complex forms a functional multi-component signaling complex in the presence of a cross-linking agent. (Item 47) 47. The system according to any one of items 42 to 46, wherein the viral envelope comprises one or more transduction enhancers. (Item 48) 48. The system of claim 47, wherein each of the transduction enhancers is a T cell activating receptor, an NK cell activating receptor, or a costimulatory molecule. (Item 49) 49. The system of any one of items 42 to 48, wherein the adapter molecule comprises a masked hapten comprising one or more masking moieties covalently attached to the hapten. (Item 50) 50. The system of claim 49, wherein the masked hapten is configured to allow a chemical reaction to remove the masking moiety from the hapten. (Item 51) 51. The system of either of items 49 or 50, wherein the masked hapten is configured to allow reactive oxygen species to remove the masking moiety from the hapten. (Item 52) 52. The system according to any one of items 49 to 51, wherein the masked hapten comprises a hydroxyphenyl group. (Item 53) 53. The system of any one of items 49 to 52, wherein the masking moiety comprises a 2,4-dinitrophenol (DNP) group. (Item 54) 54. The system according to any one of items 42 to 53, wherein the hapten comprises fluorescein. (Item 55) 55. The system of claim 54, wherein the hapten is a masked hapten comprising hydroxyphenylfluorescein (HPF). (Item 56) 55. The system of claim 54, wherein the hapten is a masked hapten comprising fluorescein-DNP. (Item 57) 57. The system of any one of items 42 to 56, wherein the targeting moiety comprises a phospholipid ether (PLE). (Item 58) 57. The system of any one of items 42 to 56, wherein the targeting moiety comprises folic acid. (Item 59) 58. The system of claim 57, wherein the adapter molecule comprises a HPF conjugated to a PLE. (Item 60) The adapter molecule is HPF-FITC-PEG3-C 18 - The system according to item 57, comprising an alkylphospholipid. (Item 61) The adapter molecule has Formula I: [ka] Item 49. The system according to item 49, comprising a molecule of (Item 62) 58. The system of claim 57, wherein the adapter molecule comprises HPF-{linker}-erufosine. (Item 63) 63. The system of any one of items 42 to 62, wherein the viral envelope comprises a viral fusion glycoprotein derived from the Kokal strain or a functional variant thereof. (Item 64) 64. The system of any one of items 42 to 63, wherein the viral envelope comprises a viral fusion glycoprotein comprising an amino acid sequence at least 95% identical to SEQ ID NO: 1 (Cocal G protein). (Item 65) 65. The system of any one of items 42 to 64, wherein the one or more transduction enhancers comprise one or more of anti-CD3 scFv, CD86, and CD137L. (Item 66) 66. The system of any one of items 42 to 65, wherein the transduction enhancer comprises all of anti-CD3 scFv, CD86, and CD137L. (Item 67) 67. The system of any one of items 42 to 66, wherein the polynucleotide comprises a sequence encoding at least one T cell activation protein. (Item 68) 68. The system of item 67, wherein the at least one T cell activation protein is a dimeric T cell activation receptor. (Item 69) 69. The system of claim 67 or 68, wherein the at least one T cell activation protein comprises a first receptor protein comprising a first dimerization domain and a second receptor protein comprising a second dimerization domain, wherein the first dimerization domain and the second dimerization domain specifically bind to each other in response to a molecule. (Item 70) 70. The system of item 69, wherein the molecule is selected from the list consisting of FK1012, tacrolimus (FK506), FKCsA, rapamycin, coumermycin, gibberellin, HaXS, TMP-HTag, ABT-737, and functional derivatives thereof. (Item 71) 71. The system of any one of items 42 to 70, wherein the receptor that specifically binds to the hapten comprises a hapten-specific antigen-binding fragment of an antibody. (Item 72) 72. The system of claim 71, wherein the antigen-binding fragment comprises a Fab fragment, a single-chain Fv fragment (scFv), or a single-chain heavy-chain antibody. (Item 73) 73. The system according to any one of items 42 to 72, wherein the receptor that specifically binds to the hapten comprises a hapten-specific chimeric antigen receptor. (Item 74) 74. The system of claim 73, wherein the hapten-specific chimeric antigen receptor comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 53. (Item 75) 75. The system of any one of items 42 to 74, wherein T cells in a subject are transduced with the retroviral particles. (Item 76) 76. The system according to any one of items 42 to 75, wherein T cells in the subject express a receptor that specifically binds to the hapten. (Item 77) 77. The system according to any one of items 42 to 76, wherein the adapter molecule specifically binds to and / or labels cancer cells in the subject. (Item 78) 78. The system according to any one of items 42 to 77, wherein the masked hapten is removed by a chemical reaction in the subject. (Item 79) The system according to any one of Items 42 to 78, wherein T cells transduced by the retroviral particles specifically kill cancer cells containing the unmasked hapten. (Item 80) 80. The system according to any one of items 42 to 79, wherein the subject is suffering from cancer and the system treats the cancer. (Item 81) 81. The system of any one of items 42 to 80, wherein the system kills tumor cells. (Item 82) A kit comprising the system according to any one of items 42 to 81 and instructions for use of the system. (Item 83) (a) In the order 5' to 3', (i) a 5'LTR or UTR; (ii) a promoter; and (iii) a sequence encoding a receptor that specifically binds to a hapten; (iv) a polynucleotide comprising a 3'LTR or UTR; (b) (i) a cell surface receptor; (ii) a viral envelope comprising one or more transduction enhancers, The retroviral particle, wherein each of said transduction enhancers is optionally a T cell activating receptor, an NK cell activating receptor, or a costimulatory molecule. (Item 84) 84. A therapeutic composition comprising the retroviral particle of item 83 in an amount sufficient to cause cancer cell death in a subject to which an adapter molecule comprising a targeting moiety and a masked hapten has been, is being, or will be administered. (Item 85) 84. A method of treating cancer and / or killing cancer cells in a subject, comprising administering to the subject a therapeutically effective amount of the retroviral particle of claim 83, wherein before, during, or after the administering step, the subject has been administered or will be administered an adapter molecule comprising a targeting moiety and a masked hapten in a dose effective to label cancer cells with the hapten. (Item 86) 1. A method of treating a tumor and / or killing tumor cells in a subject, comprising administering to said subject an effective amount of an adapter molecule; labeling tumor cells with the adaptor molecule-masked hapten; the masked hapten is activated by reactive oxygen species, thereby generating a hapten; 84. The method, wherein the subject has been or will be administered the retroviral particle of item 83 before, during, or after the administering step. (Item 87) 84. A method of treating cancer and / or killing cancer cells in a subject, comprising administering to the subject a therapeutically effective amount of the retroviral particle of claim 83, wherein prior to the administering step, the subject has been administered a targeting moiety and a masked hapten in a dose effective to label cancer cells with the hapten. (Item 88) 84. A cell line adapted to produce the retroviral particle of item 83. (Item 89) 82. A method for treating cancer and / or killing cancer cells in a subject, comprising administering to the subject the system described in any one of items 42 to 81.

Claims

[Claim 1] The invention described in the specification.

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