Lentiviral vectors for regulated expression of chimeric antigen receptor molecules

Abstract

The present invention contemplates the regulated expression of a chimeric antigen receptor (CAR) in a lentiviral vector. The CAR comprises a HOOK-binding domain that interacts with HOOK, which is preferably encoded by the same lentiviral vector, which prevents the CAR from being properly processed and released to the cell membrane. The present invention includes vectors, methods for preparing vectors, and methods for using vectors, including medical uses. The vector can be used to administer to humans to induce an immune response and treat cancer and tumors.

Description

Technical Field

[0001] The present invention belongs to the field of recombinant vaccine technology and relates to the improvement of lentiviral vectors, which can be used as therapeutic and prophylactic vaccines. The vectors provide improved induction of immune responses over other vectors. Background Art

[0002] Recombinant vaccines have been developed with the advancement of recombinant DNA technology, allowing modification of viral genomes to produce modified viruses. In this way, it has been possible to introduce genetic sequences into non-pathogenic viruses so that they encode immunogenic proteins that need to be expressed in target cells after infection or transduction in order to develop a specific immune response in its host.

[0003] Such vectors constitute a major advance in vaccine technology (Kutzler et al., Nat Rev Genet, 9(10):776-788, 2008). In particular, they have advantages over traditional vaccines: avoidance of live (attenuated) viruses and elimination of the risks associated with inactivated vaccine manufacturing.

[0004] Gene delivery using modified retroviruses (retroviral vectors) was introduced by Mann et al. in the early 1980s (Cell, 33(1): 153-9, 1983). The most commonly used oncogenic retroviruses are based on Moloney murine leukemia virus (MLV). They have a simple genome from which the polyproteins Gag, Pol and Env are produced and are required to be in trans for viral replication (Breckpot et al., 2007, Gene Ther, 14(11): 847-62; He et al., 2007, Expert Rev vaccines, 6(6): 913-24). The cis sequences generally required are the long terminal repeats (LTR) and their surrounding sequences: inverted repeats (IR or att sites) for integration, packaging sequence Ψ, transfer RNA binding site (primer binding site, PBS), and some other sequences involved in reverse transcription (repeated R within the LTR necessary for positive strand initiation and polypurine sequence (PPT)). To generate a replication-defective retroviral vector, the gag, pol and env genes are typically deleted in their entirety and replaced with an expression cassette.

[0005] Retroviral vectors derived from lentiviral genomes (i.e., lentiviral vectors) have emerged as promising tools for gene therapy and immunotherapy purposes because they exhibit several advantages over other viral systems. Specifically, lentiviruses themselves are not toxic, and unlike other retroviruses, lentiviruses are able to transduce non-dividing cells, especially dendritic cells (He et al., 2007, Expert Rev vaccines, 6(6):913-24), allowing antigen presentation through endogenous pathways.

[0006] Slow viruses are linked by similarity in genetic composition, molecular machinery of replication, and biological interactions with their hosts. They are most often referred to as agents of a slow disease syndrome that begins and develops slowly after a prolonged subclinical infection; therefore, they are referred to as "slow" viruses (Narayan et al., 1989, J Gen Virol, 70(7): 1617-39). Its basic structure is the same as that of the retrovirus used but is more complex due to the presence of accessory genes (e.g., vif, vpr, vpu, nef, tat, and rev), which play a key role in the in vivo replication of slow viruses.

[0007] Lentivirus represents a genus of slow viruses of the Retroviridae family, which includes human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious encephalitis virus (EIAV), caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV), and feline immunodeficiency virus (FIV). Lentiviruses can persist indefinitely in their hosts and replicate continuously at varying rates during a lifetime of infection. The continued replication of the virus in its host depends on its ability to evade host defenses.

[0008] The design of recombinant integrative lentiviral vectors is based on the separation of cis- and trans-acting sequences of the lentivirus. Efficient transduction in non-dividing cells requires the presence of two cis-acting sequences in the lentiviral genome: a central polypurine sequence (cPPT) and a central termination sequence (CTS). They lead to the formation of a three-stranded DNA structure, called a central DNA "flap", which maximizes the efficiency of gene entry into the nucleus of non-dividing cells, including dendritic cells (DCs) (Zennou et al., 2000, Cell, 101 (2) 173-85; Arhel et al., 2007, EMBO J, 26 (12): 3025-37).

[0009] Dendritic cells play an important role in antigen presentation as they constitute the major type of antigen presenting cells (APCs) whose primary function is to present antigens and initiate immune responses.

[0010] To generate an immune response, cells must process antigenic proteins into peptides, which are displayed on the cell surface via major histocompatibility complex proteins (MHC). Circulating APCs present the peptide-MHC complex to T cells in the draining lymph nodes, where they interact with T cell receptors and activate T cells in conjunction with co-stimulatory signals.

[0011] Several studies have shown that vaccination using lentiviral vectors results in antigen presentation by DCs and antigen-specific cytotoxic T lymphocytes (CTLs; CD8 + Therefore, in the past 10 years, lentiviral vectors have been engineered for gene transfer and immunotherapy applications.

[0012] The vector usually contains a strong constitutive promoter with an enhancer, such as the CMV promoter. Michelini et al., Vaccine 27(34):4622-29 (2009); Karwacz et al., J. Virol. 83(7):30943103 (2009); Negri et al., Molecular Therapy 15(9):1716-23 (2007); and Buffa et al., J. General Virology 87:1625-1634 (2006).

[0013] The safety of lentiviral vectors has been improved by removing the LTR U3 sequence, creating an "auto-inactivating" vector that is completely free of the viral promoter and enhancer sequences originally present in the LTR.

[0014] Lentiviral particles containing lentiviral vectors can be produced by recombinant technology after transient transfection of cells, such as HEK 293T human cultured cells, with different DNA plasmids:

[0015] (i) a packaging plasmid, which expresses at least Gag, Pol, Rev, Tat and in some cases structural and enzymatic proteins necessary for packaging of the transfer construct;

[0016] (ii) a proviral transfer plasmid containing the expression cassette and HIV cis-acting factors necessary for packaging, reverse transcription, and integration; and

[0017] (iii) A plasmid encoding an envelope, in most cases a glycoprotein of the vesicular stomatitis virus (VSV.G), which allows the formation of mixed particles (pseudotypes) capable of targeting a variety of cells, especially major histocompatibility (MHC) antigen presenting cells (APCs), including DCs.

[0018] This method enables the transfected cells to produce lentiviral particle vectors transiently. However, lentiviral particle vectors can also be continuously produced by stably inserting packaging genes, proviral encoding DNA and envelope genes into the cell genome. This enables the continuous production of lentiviral particle vectors by cells without the need for transient transfection. Of course, a combination of these methods can also be used, in which some DNA / plasmids are integrated into the cell genome and others are provided by transient transfection.

[0019] Non-integrating lentiviral vectors have been designed to reduce the risk of potential carcinogenesis associated with insertional mutagenesis events, particularly for vaccination purposes. Examples of non-integrating lentiviral vectors are provided in Coutant et al., PLOS ONE 7(11): e48644(2102), Karwacz et al., J. Virol. 83(7): 3094-3103(2009), Negri et al., Molecular Therapy 15(9): 1716-1723(2007); Hu et al., Vaccine 28: 6675-6683(2010). Thus, it has been reported that non-integrating lentiviral vector systems can reduce the potential risk of insertional mutagenesis compared to integrating systems. Hu et al., Vaccine 28: 6675-6683(2010).

[0020] In addition, the deletion of the U3 region of the 3′ LTR of the viral promoter and enhancer sequences in the auto-inactivating lentiviral vector limits the possibility of activation of the endogenous promoter. These safety concerns directly reflect the experience gained from the SCID-X1 gene therapy trial conducted in 1998-1999, which used a retroviral vector based on the Moloney virus for children with a rare type of X-linked (SCID-X1 gene) severe immunodeficiency disease (Cavazzana-Calvo et al., 2000, Science., 288(5466): 669-72). In this trial, 4 of 9 children developed leukemia due to the integration of the Moloney-derived lentiviral vector in close proximity to the human LM02 oncogene (Hacein-Bey-Abina et al., 2008, J. Clin. Invest., 118(9): 3132-3142). This suggests that the malignancy is the result of the proximity of the viral U3 promoter / enhancer to the LM02 oncogene. As a result, safety is a major concern for administration of lentiviral vectors to humans.

[0021] Enhancers are cis-acting sequences that can function as transcriptional activators at a distance. They have been widely adopted in virus-derived vectors because they appear to be the most efficient for obtaining strong transgene expression in a variety of cell types, especially DCs (Chinnasamy et al., 2000, Hum Gene Ther 11(13):1901-9; Rouas et al., 2008, Cancer Gene Ther 9(9):715-24; Kimura et al., 2007, Mol Ther 15(7):1390-9; Gruh et al., 2008, J Gene Med 10(1)21-32). However, considering the safety issues of insertional mutagenesis, such transcriptional enhancer sequences should be deleted from lentiviral vector constructs to eliminate the risk of insertional mutagenesis caused by enhancer proximity effects. To date, this enhancer proximity effect is the most common mechanism of insertional mutagenesis and is the only effect described in cases of human or animal tumorigenesis events after gene transfer.

[0022] Therefore, there is a need to develop retroviral, particularly lentiviral, vectors that do not include viral enhancers but still allow for adequate expression of the immunogenic peptides encoded by the transgene, if possible at levels comparable to those observed when using the CMV promoter.

[0023] Recent studies have reported the use of DC-specific promoters derived from major histocompatibility complex class II genes (MHC class II) (Kimura et al., 2007, Mol Ther 15(7): 1390-9) and dectin-2 genes (Lopes et al., 2008, J Virol 82(1): 86-95) instead of viral promoters. The dectin-2 gene promoter used by Lopes et al. contains a putative enhancer and adenovirus conserved sequences (inverted terminal repeats in adenovirus promoters) (Bonkabara et al., 2001, J. Immunology, 167: 6893-6900). The MHC class II gene promoter used by Kimura et al. does not contain any known enhancer.

[0024] However, when administered intravenously, in the absence of an enhancer, the MHC class II promoter was found to be unable to provide adequate transgene expression in DCs. Specifically, lentiviral vectors containing an MHC class II promoter failed to elicit an immune response in immunocompetent C57BL / 6 mice, in contrast to the immune responses observed using the CMV promoter / enhancer. Although integration and sustained transgene expression were observed after injection in mice, lentiviral vectors transcribed by the MHC class II promoter were unable to stimulate antigen-specific CD8+ cytotoxic T lymphocyte responses, even after a vaccination boost. Therefore, the authors of these studies concluded that the use of the MHC class II promoter is only of interest for applications seeking sustained expression in gene replacement therapy, but not in immunotherapy. It should be noted that the MHC class II promoter is weakly expressed in most cell types.

[0025] Therefore, the MHC class II promoter is not an appropriate lentiviral vector promoter for inducing immune responses against antigens by IV injection. In addition, the dectin-2 promoter is weakly expressed in most cell types and appears to contain an enhancer. Therefore, the dectin-2 promoter is not a good lentiviral vector promoter for safety reasons.

[0026] Preferably, in immunotherapy, lentiviral vectors provide efficient transgene expression that elicits the desired specific immune response. This requires high levels of expression in APCs such as dendritic cells.

[0027] It is also preferred that cells transduced by the lentiviral vector be eliminated by an immune response to provide a higher level of safety. That is, the immune response generated against the transgene can induce an immune response in the host sufficient to eliminate the cells transduced by the lentiviral vector. Elimination of transduced cells eliminates the persistence of the lentiviral vector in the host, as well as possible secondary effects of the vector. In order to eliminate transduced cells, a certain level of expression is required in non-dendritic cells to be eliminated by the immune response. Therefore, the antigen needs to be appropriately expressed.

[0028] At the same time, the promoter should maximize immune stimulation by key cells involved in the activation of primary and memory T cells (i.e., dendritic cells) and should minimize the risk of insertional mutagenesis and genotoxicity in stem cells that lead to malignancies. Therefore, the promoter should have sufficiently high activity in dendritic and other cells, but not contain enhancers. Based on these criteria, viral promoters (such as CMV promoters) are not ideal due to the presence of strong enhancers. These criteria are summarized as follows:

[0029] Highly expressed in antigen-presenting cells (including dendritic cells) to induce maximal immune response;

[0030] is expressed in other transduced cell types at levels sufficient to permit elimination by the induced immune response; and

[0031] Enhancer elements are absent to avoid insertion effects.

[0032] Chimeric antigen receptors (CARs) are recombinant receptors for antigens. Sadelain et al., Cancer Discov. 2013 Apr; 3(4): 388-398. They are recombinant receptors that generally target native cell surface antigens. Same as above. CARs involve molecules that do not require recognition of HLA expression or peptide processing. Same as above. CARs have been shown to have T cell-proliferation and activation potential themselves, including chimeric molecules between CD3-ζ or Fc receptor γ and CD8, CD4, CD25 or CD16. The antigen binding portion of CAR is generally an scFv derived from an antibody, a Fab selected from a library, or a natural receptor ligand. Han EQ et al., J Hematol Oncol. 6: 47, 2013.

[0033] CARs have been generated against many different cell surface molecules, including CD19, HER2, GD2, PSMA, and many are in clinical trials. Davila et al., OncoImmunology 1:9, 1577-1583; 2012. The most promising clinical results for CARs have been seen in patients treated with autologous CAR-modified T cells targeting CD19. Maus MV et al., 123(17):2625-35, 2014.

[0034] In vivo elimination of CAR-expressing T cells has been studied recently. Human T cells have been genetically modified with a lentiviral vector to express a CD20-CAR containing a suicide gene that relies on inducible activation of caspase 9. Budde et al., PLoS ONE 8(12): e82742 (2013). This lentiviral vector uses the human EF1α promoter to obtain expression in T cells. Activation of the suicide fusion results in efficient removal of transduced T cells both in vitro and in vivo. Same as above.

[0035] It would be better if the CAR expression on the cell surface could be turned off quickly to prevent adverse events and the system could be reactivated after it has been turned off. Therefore, there is a need in the art for improved methods and vectors for human treatment. The present invention meets these needs in the art. Summary of the invention

[0036] The present invention includes nucleic acid molecules and vectors encoding CAR and methods for preparing and using the nucleic acid molecules and vectors. In one embodiment, the present invention includes a chimeric antigen receptor comprising a binding domain; a transmembrane domain; a HOOK-binding domain, preferably comprising a streptavidin-binding peptide; and an activation domain comprising a T cell activation fragment of at least 100 amino acids of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, SEQ ID NO: 33, or SEQ ID NO: 34.

[0037] In one embodiment, the invention includes a lentiviral vector expressing a chimeric antigen receptor comprising a binding domain; a transmembrane domain; a HOOK-binding domain comprising a streptavidin-binding peptide; and an activation domain comprising a T cell activation fragment of at least 100 amino acids of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, SEQ ID NO: 33, or SEQ ID NO: 34.

[0038] In one embodiment, the present invention includes a nucleic acid vector encoding a chimeric antigen receptor, which comprises a binding domain; a transmembrane domain; a HOOK-binding domain comprising a streptavidin-binding peptide; and an activation domain comprising a T cell activation fragment of at least 100 amino acids of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, SEQ ID NO: 33, or SEQ ID NO: 34.

[0039] In one embodiment, the invention includes a lentiviral vector particle encoding a chimeric antigen receptor, which comprises a binding domain; a transmembrane domain; a HOOK-binding domain comprising a streptavidin-binding peptide; and an activation domain comprising a T cell activation fragment of at least 100 amino acids of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, SEQ ID NO: 33, or SEQ ID NO: 34.

[0040] In one embodiment, the chimeric antigen receptor or lentiviral vector or nucleic acid vector or lentiviral vector particle comprises a single chain Fv antibody or a single domain antibody (ie, a nanobody).

[0041] In one embodiment, the vector encodes a HOOK comprising the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:32.

[0042] In one embodiment, the vector comprises a β2-microglobulin, Ubi, EF1α, MHC I, or MHC II promoter. In one embodiment, the vector is DNA.

[0043] In one embodiment, the lentiviral vector particle comprises vesicular stomatitis virus glycoprotein.In one embodiment, the lentiviral vector particle comprises HIV-1 subtype D Gag and Pol proteins.

[0044] In one embodiment, the invention includes an isolated cell comprising a vector of the invention.

[0045] The present invention includes the use of any chimeric antigen receptor or lentiviral vector or nucleic acid vector or lentiviral vector particle of the present invention to induce an immune response in a human.

[0046] The invention includes methods of inducing an immune response in a human comprising administering to the human a cell transduced with a lentiviral vector particle encoding a chimeric antigen receptor or said lentiviral vector particle and, optionally, subsequently administering to the human biotin.

[0047] In some embodiments, the chimeric antigen receptor or lentiviral vector or nucleic acid vector comprises a nucleotide sequence of any one of SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49. In some embodiments, the chimeric antigen receptor comprises an amino acid sequence of any one of SEQ ID NO: 46, SEQ ID NO: 48, or SEQ ID NO: 50.

[0048] In some embodiments, the lentiviral vector or nucleic acid vector further encodes the amino acid sequence of SEQ ID NO: 42 or comprises a nucleic acid sequence of SEQ ID NO: 43 and / or SEQ ID NO: 44.

[0049] BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Shown is GFP expression in HEK 293T and BDCM cells from lentiviral vectors containing the indicated promoters.

[0051] Figure 2 Cell-specific expression of various promoters analyzed using data from biogps.org is shown. Expression levels were normalized to expression in skeletal muscle (=1.0).

[0052] Figure 3AC shows the expression of CAR in T cells from various lentiviral vectors with human ubiquitin, MHC class I, MHC class II, and β2 microglobulin (β2m) promoters.

[0053] Figure 4 Shown are the titers of pseudotyped vectors encoding codon-optimized VSV-G protein fused to HOOK-streptavidin with a dose gradient of + / - biotin and HOOK-streptavidin encoding vectors.

[0054] Figure 5 AB shows the structure and expression of CAR-RUSH. A) Three CAR-RUSH construction schemes designed CAR-RUSH is a second-generation CAR showing the intracellular domain of 4-1BB and CD3ζ. The specificity of CAR-RUSH is that it allows interaction with HOOK proteins coupled to streptavidin in the presence of SBP (streptavidin binding protein) region, and therefore stays in the endoplasmic reticulum. The differences between SBP1, SBP2 and SBP3 include the position of the SBP region, which has been located between the transmembrane domain and 4-1BB, or between 4-1BB and CD3ζ, or at the C-terminus. B) MFI of the display median fluorescence intensity of CAR-positive T lymphocytes analyzed by FACS. Cells were transduced at an MOI of 10 and analyzed on the third day after transduction. The MFI values ​​reported in the histogram are standardized by subtracting the mfi corresponding to the background from the cells that were not transduced. All CAR-positive T cells showed comparable MFI to the second generation CAR-CD19 (positive control), which showed a 3-fold higher MFI than other tested constructs. All three CAR-RUSH (SBP1, SBP2 and SBP3) proteins showed very similar MFI and were therefore expressed at similar levels on the cell surface.

[0055] Figure 6AB shows the expression and performance of CAR-RUSH constructs after co-transduction and biotin treatment with lentiviral vectors encoding HOOK-streptavidin.Percentage (A) of CAR-positive T lymphocytes analyzed by FACS and MFI (B) showing median fluorescence intensity.Cells were co-transduced with lentiviral vectors encoding HOOK-streptavidin (MOI 5) and lentiviral vectors encoding CAR-SBP (MOI 5), corresponding to a final total MOI of 10.Biotin was added during co-transduction and cells were analyzed on the 3rd day after transduction.The percentages and MFI values ​​reported in the histogram were obtained by subtracting the values ​​from non-transduced cells corresponding to the background.CAR-RUSH constructs are specifically expressed at low percentages after adding biotin to the culture medium, indicating that in the case of co-transduction, CAR-RUSH is efficiently retained by HOOK protein and it can be presented on the cell membrane only after biotin-mediated induction.

[0056] Figure 7 AB shows the expression and performance of CAR-RUSH bicistronic constructs. MFI and percentage of median fluorescence intensity of CAR-positive T lymphocytes analyzed by FACS. Cells were transduced at an MOI of 10 (A) or 30 (B) and analyzed on the 3rd day after transduction. Biotin was added at the time of transduction. The percentages and MFI values ​​reported in the histogram were obtained by subtracting the values ​​from the non-transduced cells corresponding to the background. The SBP1-CD19 and SBP3-CD19 CAR-RUSH constructs showed expression on the cell surface only after adding biotin to the culture medium, indicating that in the absence of biotin, CAR-RUSH was specifically retained by HOOK-streptavidin protein, and after delivering biotin, the RUSH regulatory system induced their transport to the plasma membrane and their expression on the plasma membrane. After adding biotin to the culture medium, SBP1-CD19 and SBP3-CD19 CAR-RUSH constructs showed expression on the cell surface. At high MOI (MOI = 30) and even in the absence of biotin, SBP1-CD19 showed slight cell surface expression. Given these results, CAR-RUSH is believed to be largely retained by HOOK streptavidin protein in the absence of biotin. In addition, as expected, biotin delivery induces their transport to the plasma membrane and their expression on the plasma membrane.

[0057] Figure 8AB shows CAR-RUSH system switching evaluation (off / on). Purified T cells were transduced with lentivirus encoding CAR-RUSH system at day 0 and biotin was added or not added at the same time. On the 3rd day, CAR expression on the surface of T cells was analyzed by flow cytometry. The percentage (A) of CAR-positive T lymphocytes from a representative donor analyzed by FACS and the MFI (B) showing median fluorescence intensity. CAR-RUSH constructs were transduced at an MOI of 10 or 20, while biotin was added and T cells were analyzed for CAR expression on the cell surface on the 3rd day after transduction. This experiment was conducted to study the induction kinetics of CAR-RUSH constructs after biotin delivery and the efficiency of CAR-RUSH retention in the absence of biotin inducers. As shown in the figure above, a low percentage of CAR-positive T cells was detected, and depending on the CAR-RUSH construct, the effect depended on or was independent of the addition of biotin. According to the analyzed CAR-RUSH constructs, a low percentage of CAR-positive T cells was detected. The ability of biotin to induce CAR-RUSH expression at the cell surface was tested for CAR-SBP1 and CAR-SBP2. CAR-SBP3 showed slight CAR expression in the presence or absence of biotin.

[0058] Fig. 9AD shows CAR-RUSH system switching evaluation (off / on / off). Purified T cells were transduced with lentivirus encoding the CAR-RUSH system on day 0 and biotin was added or not added at the same time. On the 3rd day, CAR expression in the presence or absence of biotin was analyzed in a group of wells by flow cytometry. The wells of other groups were washed and put back into the culture containing or not containing biotin to monitor the ability of CAR-positive cells to turn off the expression of CAR. Then the CAR expression on the T cell surface of T cells was analyzed on the 7th day. The percentage (A) of CAR-positive T lymphocytes derived from healthy donors 32 and the MFI (B) showing the median fluorescence intensity were analyzed by FACS. CAR-RUSH constructs were transduced at an MOI of 10 and analyzed on the cell surface on the 7th day after transduction after adding or not adding biotin on the 3rd day. As shown in A), a low percentage of CAR-positive T cells were detected. For CAR-SBP2 and CAR-SBP3, biotin induces CAR-RUSH expression on the cell surface, and especially for CAR-SBP3, CAR expression is still re-induced after washing and re-addition of biotin on the 3rd day. The percentage (C) of CAR-positive T lymphocytes derived from healthy donor 31 by FACS analysis and MFI (D) showing median fluorescence intensity. The CAR-RUSH construct was transduced at an MOI of 20 and analyzed for CAR expression on the cell surface on the 7th day after transduction after adding or not adding biotin on the 3rd day. All three tested CAR-RUSHs showed a low percentage of CAR-positive cells. In addition, except for CAR-SBP1, which showed CAR expression in the presence or absence of biotin, CAR-RUSH was expressed only in conditions where biotin was added on day 0 and / or day 7.

[0059] Fig.10 AC shows the amino acid and nucleotide sequences of the second generation of CAR-CD19 and its components. A) Nucleotide sequence (SEQ ID NO: 39), B) amino acid sequence (SEQ ID NO: 51), and C) key points indicate signal sequence, CD19 ScFv, CD8 hinge, transmembrane domain, 4-1BB, and CD3z sequence.

[0060] Fig.11 The structure of the bicistronic HOOK Streptavidin-IRES-CAR-SBP-CD19 generation 2 construct is shown. DETAILED DESCRIPTION OF THE INVENTION

[0061] A variety of lentiviral constructs encoding chimeric antigen receptors (CARs) were generated for expression of CARs in T cells. It is also unknown which promoter will be most suitable for expression in human T cells. To compare promoters, renal cell lines and dendritic cell lines were transduced with lentiviral vectors expressing green fluorescent protein (GFP) from various promoters. The human EF1α promoter was found to be the strongest promoter in the dendritic cell line BDCM ( Figure 1 ). This promoter is also very strong in the kidney cell line 293T. The cell-specific expression of various promoters was analyzed using the dataset from biogps.org ( Figure 2 ). From this dataset, expression of the EF1α promoter in T cells was similar to that in BDCA+ cells. In fact, the human EF1α promoter was previously used to express CAR in activated human T cells. Budde et al., PLoS ONE 8(12): e82742 (2013).

[0062] Based on the differences in expression in different cell types, it is not obvious which promoter can be successfully used to express CAR in human T cells. The human ubiquitin MHC I, MHC II, and β2 microglobulin (β2m) promoters were evaluated for their suitability for expressing CAR in human T cells. Surprisingly, all of these promoters were found to function in human T cells ( Figure 3 AC).

[0063] To find the optimal conditions for generating CAR T-cells, several parameters that may affect T-cell transduction and CAR expression were tested (e.g., MOI, incubation time, promoter, T-cell activation, and purification strategy).

[0064] After subtracting the background (% of CAR+ cells in untransduced (UTD) controls), CAR+ cells were detected at 24 hours (UBC vectors of MOI=10 and 30), 96 hours (β2M vectors of MOI=3 and UBC vectors of MOI=3 and 10) and day 8 (β2M vectors of MOI=30 and UBC vectors of each tested MOI). At 96 hours and day 8, a high percentage (about 30%) of CAR-CD19 positive cells were found compared to untransduced lymphocytes. On day 7, when the live CD3+ population was analyzed, a high percentage (about 70%) of CAR-CD19 positive cells were found compared to untransduced lymphocytes.

[0065] Strong expression of CAR-CD19 was found in several donors (up to 70% of CD3 positive lymphocytes). CAR expression was found even at MOI=3; however, a high percentage of CAR-positive cells was achieved at a later time point (day 7 in this experiment). Interestingly, CAR expression between donors is variable. For CAR-CD19, expression is limited to the CD3 positive population, because CD3 negative cells cannot express CAR-CD19.

[0066] The HOOK-streptavidin sequence was cloned into a pseudo vector encoding a codon-optimized VSG-G protein as a fusion with VSV-G. The vector was evaluated by co-transfection with a lentivirus encoding GFP and increasing doses of the HOOK-streptavidin encoding vector. No reduction in titer due to retention of VSV protein in the ER was observed ( Figure 4 ). Immunofluorescence studies showed no retention of VSV protein in the ER. This is in contrast to the results shown for VSV protein alone. Nature Methods 9(5): 493-500(2102). Therefore, it is unclear whether the HOOK-streptavidin system can regulate CAR expression.

[0067] Evaluation of various positions between the intracellular domains with streptavidin binding protein (SBP) on the surface of T cells to examine whether the presence of the SBP sequence modifies CAR expression. CAR-CD19 + with various positions of SBP was cloned into a lentiviral vector under the control of the β2m promoter.

[0068] Human T cells were transduced with lentiviral vectors and the expression of CD19 on the surface of the cells was evaluated. CAR-CD19-SBP was expressed on the surface of T cells, with a slightly lower MFI compared to CAR-CD19 ( Figure 5 ).

[0069] In the presence of HOOK-streptavidin, CAR-CD19 lentiviral vectors with SBP at different positions between intracellular domains were evaluated. Human T- cells were co-transduced with a lentiviral vector expressing CAR-CD19-SBP and a lentiviral vector expressing HOOK-streptavidin, and the percentage and MFI of transduced cells were evaluated before or after the addition of biotin. It was found that HOOK-streptavidin and CAR-CD19-SBP can be co-expressed in human T- cells. The presence of HOOK-streptavidin can retain CAR-CD19-SBP in the endoplasmic reticulum of T- cells and add biotin in the culture medium to induce CAR-CD19 release and its expression on the cell surface ( Figure 6 ).

[0070] Then, various lentiviral vectors were constructed containing HOOK and CAR containing SBP at various positions ( Fig.11 ). The construct contains the β2m promoter linked to HOOK streptavidin, followed by an IRES linked to CAR-CD19 with SBPs at 3 different locations.

[0071] Human T-cells from various donors were transduced with lentiviral vectors and the expression of CAR-CD19-SBP was evaluated on the cell surface in the presence and absence of biotin. HOOK-IRES-CAR-CD19-SBP was expressed in human T-cells. Expression of HOOK-streptavidin retained the CAR-CD19-SBP in the endoplasmic reticulum of T-cells regardless of their location. Addition of biotin to the culture medium induced the release of CAR-CD19 and its expression on the cell surface, regardless of its location ( Figure 7 ).

[0072] The present invention includes lentiviral vectors encoding CAR and their use in inducing an immune response in a host, especially a human.

[0073] Before disclosing and describing the proteins, compositions, methods and other embodiments of the present invention, it should be understood that the terminology herein is only used for the purpose of describing specific embodiments and is not intended to be limiting. It must be noted that the singular forms "a", "an", and "the" used in this specification and the appended claims include plural referents unless the context clearly indicates otherwise.

[0074] The term "comprising" as used herein is synonymous with "including" or "containing", and is a closed or open expression that does not exclude other unrecited elements or method steps.

[0075] The full names of amino acids herein may be used interchangeably with their respective standard three-letter and single-letter abbreviations. For the avoidance of doubt, those are: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Val, V).

[0076] The term "in vitro" as used herein refers to events that occur in an artificial environment (eg, in a test tube or reaction vessel, in cell culture, in a petri dish, etc.), rather than within an organism (eg, an animal, plant, or microorganism).

[0077] As used herein, the term "in vivo" refers to events that occur within an organism (eg, an animal, plant, or microorganism).

[0078] As used herein, the term "isolated" refers to: (1) a substance or entity that has been separated from at least some of the components with which it was originally associated (in a natural or experimental setting), and (2) a substance or entity that has been artificially generated, prepared, and / or manufactured. An isolated substance and / or entity may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the other components with which it was originally associated. In some embodiments, the isolated substance is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is considered "pure" if it is substantially free of other components.

[0079] The "isolated" products of the present invention, including isolated nucleic acids, proteins, polypeptides and antibodies, are not natural products (i.e., "non-naturally occurring"). In contrast, the "isolated" nucleic acids, proteins, polypeptides and antibodies of the present invention are "artificial" products. The "isolated" products of the present invention may be "significantly different" or "clearly different" from natural products. As non-limiting examples, isolated nucleic acids may be purified, recombined, synthesized, labeled, and / or attached to a solid substrate. Such nucleic acids may be significantly different or clearly different from naturally occurring nucleic acids. As other non-limiting examples, the "isolated" proteins, polypeptides and antibodies of the present invention may be purified, recombined, synthesized, labeled, and / or attached to a solid substrate. Such proteins, polypeptides and antibodies may be significantly different or clearly different from naturally occurring proteins, polypeptides and antibodies.

[0080] As used herein, the term "peptide" refers to a short polypeptide, for example, which typically contains less than about 50 amino acids and more typically contains less than about 30 amino acids. The term herein includes analogs and mimetics that mimic structure and biological function.

[0081] The term "polypeptide" includes naturally occurring and non-naturally occurring proteins, fragments, mutants, derivatives and analogs thereof. A polypeptide may be a monomer or a multimer. In addition, a polypeptide may contain a plurality of different domains, wherein each domain has one or more different activities. For the avoidance of doubt, a "polypeptide" may be any length exceeding two amino acids.

[0082] The term "isolated protein" or "isolated polypeptide" refers to a particular protein or polypeptide, in the original or derived sense, which (1) is not associated with naturally associated components that accompany it in its natural state, (2) exists in the form of a pure material not found in nature, where purity can be determined by the presence of other cellular materials (e.g., the absence of other proteins from the same species), (3) is expressed by cells from other species, or (4) does not exist in nature (e.g., it is a fragment of a polypeptide found in nature or it contains amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it is naturally derived is "isolated" from its naturally associated components. A polypeptide or protein can also be rendered substantially free of naturally associated components by separation, using protein purification techniques known in the art. As defined herein, "isolated" does not require the physical removal of the protein, polypeptide, peptide or oligopeptide from the cell in which it is synthesized.

[0083] The protein or polypeptide may be purified. Preferably, the purified protein or polypeptide has a purity exceeding 50%, 75%, 85%, 90%, 95%, 97%, 98%, or 99%. In the context of the present invention, a purified protein having a purity exceeding 50% (etc.) means that the purified protein sample contains less than 50% (etc.) other proteins. For example, if a sample containing a protein contains less than 1% contaminating host cell proteins, its purity may be 99%.

[0084] As used herein, the term "polypeptide fragment" refers to a polypeptide having a deletion, for example, a deletion of the amino terminus and / or the carboxyl terminus compared to a full-length polypeptide (e.g., a naturally occurring protein). In one embodiment, the polypeptide fragment is a continuous sequence, wherein the amino acid sequence of the fragment is identical to the corresponding positions in the naturally occurring sequence. The fragment length is typically at least 5, 6, 7, 8, 9, or 10 amino acids, or at least 12, 14, 16, or 18 amino acids, or at least 20 amino acids, or at least 25, 30, 35, 40, or 45 amino acids, or at least 50 or 60 amino acids, or at least 70 amino acids, or at least 100 amino acids.

[0085] The term "fusion protein" refers to a polypeptide comprising a polypeptide or fragment coupled to a heterologous amino acid sequence. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements, which can be from two or more different proteins. The fusion protein contains at least 10 consecutive amino acids from the polypeptide of interest, or at least 20 or 30 amino acids, or at least 40, 50 or 60 amino acids, or at least 75, 100 or 125 amino acids. The heterologous polypeptide length contained in the fusion protein is generally at least 6 amino acids, or at least 8 amino acids, or at least 15, 20 or 25 amino acids. Particularly useful are fusions comprising larger polypeptides, such as IgG Fc regions, or even entire proteins, such as proteins containing green fluorescent protein ("GFP") chromophores. Fusion proteins can be recombinantly generated by constructing a nucleic acid sequence encoding a polypeptide or fragment thereof fused in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be chemically generated by cross-linking a polypeptide or fragment thereof with another protein.

[0086] As used herein, "recombinant" may refer to a biomolecule, e.g., a gene or protein, or to an organism. The term "recombinant" may be applied to cloned DNA isolates, chemically synthesized polynucleotides, or polynucleotides biologically synthesized by heterologous systems, as well as proteins or polypeptides and / or RNA encoded by such nucleic acids. A "recombinant" nucleic acid is a nucleic acid linked to nucleotides or polynucleotides to which it is not linked in nature. A "recombinant" protein or polypeptide can be (1) a protein or polypeptide linked to amino acids or polypeptides to which it is not linked in nature; and / or (2) a protein or polypeptide produced by transcription and / or translation of a recombinant nucleic acid. Thus, a protein synthesized by a microorganism is recombinant, e.g., if it is synthesized from mRNA synthesized by a recombinant nucleic acid present in the cell. A "recombinant" cell is a cell that contains a "recombinant" biomolecule. For example, a T cell containing a "recombinant" nucleic acid is a "recombinant" cell.

[0087] The term "polynucleotide", "nucleic acid molecule", "nucleic acid", or "nucleic acid sequence" refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-natural internucleoside bonds, or both. Nucleic acids can be in any topological configuration. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, tetramer, partially double-stranded, branched, hairpin-shaped, circular or locked configurations. The nucleic acids (also referred to as polynucleotides) can include sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They can be chemically or biologically modified or can contain non-natural or derived nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged connections (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged connections (e.g., phosphorothioates, phosphorodithioates, etc.), side parts (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylating agents, and modified connections (e.g., α-anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides that bind to a specified sequence through hydrogen bonds and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide connections replace phosphate connections in the main chain of the molecule. Other modifications may include, for example, analogs in which the ribose ring contains a bridging moiety or other structure, such as modifications found in "locked" nucleic acids.

[0088] "Synthetic" RNA, DNA or mixed polymers are produced outside the cell, for example, by chemical synthesis.

[0089] As used herein, the term "nucleic acid fragment" refers to a nucleic acid sequence that has a deletion compared to a full-length reference nucleotide sequence, for example, a 5'-terminal or 3'-terminal deletion. In one embodiment, the nucleic acid fragment is a continuous sequence, wherein the nucleotide sequence of the fragment is identical to the corresponding position in the naturally occurring sequence. In some embodiments, the fragment length is at least 10, 15, 20, or 25 nucleotides, or at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 nucleotides. In some embodiments, the nucleic acid sequence fragment is an open reading frame sequence fragment. In some embodiments, the fragment encodes a polypeptide fragment (as defined herein) of a protein encoded by an open reading frame nucleotide sequence.

[0090] The nucleic acid may be purified. Preferably, the purity of the purified nucleic acid exceeds 50%, 75%, 85%, 90%, 95%, 97%, 98%, or 99%. Within the scope of the present invention, a purified nucleic acid having a purity of at least 50% means that the purified nucleic acid contains less than 50% of other nucleic acids. For example, if a plasmid sample contains less than 1% contaminating bacterial DNA, its purity may be at least 99%.

[0091] As used herein, an endogenous nucleic acid sequence (or the encoded protein product of the sequence) in the genome of an organism is considered to be "recombinant" herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence. In this article, a heterologous sequence is a sequence that is not naturally adjacent to an endogenous nucleic acid sequence, whether the heterologous sequence is endogenous (from the same host cell or its progeny) or exogenous (from a different host cell or its progeny). For example, the natural promoter of a gene in the genome of a host cell can be replaced by a promoter sequence (e.g., by homologous recombination) so that the gene has an altered expression pattern. The gene will now become "recombinant" because it is separated from at least some of the sequences that are naturally flanked by it.

[0092] A nucleic acid is also considered "recombinant" if it contains any modification that does not occur naturally with the corresponding nucleic acid in the genome. For example, an endogenous coding sequence is considered "recombinant" if it contains an insertion, deletion, or point mutation introduced artificially (e.g., by human intervention). "Recombinant nucleic acid" also includes nucleic acids that are integrated into the chromosome of a host cell at a heterologous site and nucleic acid constructs that exist as protoplasts.

[0093] As used herein, the term "degenerate variant" of a reference nucleic acid sequence includes nucleic acid sequences that can be translated according to the standard genetic code to provide the same amino acid sequence as translated from the reference nucleic acid sequence. The terms "degenerate oligonucleotide" or "degenerate primer" are used to refer to oligonucleotides that are capable of hybridizing to a target nucleic acid sequence that are not necessarily identical in sequence but are homologous to each other in one or more specific segments.

[0094] When used in reference to nucleic acid sequences, the terms "percent sequence identity" or "identity" refer to the residues in two sequences that are identical when aligned for maximum correspondence. The length of the sequence identity comparison can be over a stretch of at least about 9 nucleotides, typically at least about 20 nucleotides, typically at least about 24 nucleotides, generally at least about 28 nucleotides, typically at least about 32, and even more typically at least about 36 or more nucleotides. There are a variety of different algorithms known in the art that can be used to measure nucleotide sequence identity. For example, FASTA, Gap or Bestfit are used to compare polynucleotide sequences, which are programs in the Wisconsin package version 10.0 of the Genetic Computer Group (GCG) in Madison, Wisconsin. FASTA can provide an alignment and a percentage of sequence identity for the best overlap region between a query sequence and a search sequence. Pearson, Methods Enzymol. 183: 63-98 (1990). For example, percent sequence identity between nucleic acid sequences can be determined using FASTA with default parameters (wordlength 6 and NOPAM coefficients for the scoring matrix) as provided in GCG version 6.1 (incorporated herein by reference) or Gap with default parameters. Alternatively, sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), particularly blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0095] As used herein, "expression control sequence" refers to a polynucleotide sequence that affects the expression of the coding sequence to which they are operably linked. Expression control sequences are sequences that control the transcription, post-transcriptional events, and translation of nucleic acid sequences. Expression control sequences include suitable transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and sequences that enhance protein secretion when necessary. The nature of such control sequences varies depending on the host biotin; in prokaryotes, such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences. The term "control sequence" is intended to include at a minimum all components whose presence is necessary for expression, and may also include other components whose presence is beneficial, such as leader sequences and fusion partner sequences.

[0096] As used herein, "operably linked" or "operably linked" expression control sequences refers to linkages in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, and in which the expression control sequence functions in trans or at a distance to control the gene of interest.

[0097] As used herein, "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected. One type of vector is a "plasmid", which generally refers to a circular double-stranded DNA ring, in which other DNA segments can be connected, but also includes linear double-stranded molecules, such as those obtained from amplification by polymerase chain reaction (PCR) or treatment of circular plasmids with restriction enzymes. Other vectors include cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). Another type of vector is a viral vector, in which additional DNA segments can be connected to enter the viral genome (described in detail below). Some vectors can replicate autonomously in the host cell transduced therein (for example, a vector with a replication origin that functions in the host cell). Other vectors can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome. In addition, some vectors can guide the expression of genes operatively connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or "expression vectors" for short). The integration cosmid vector pYUB412 is an example of a "vector".

[0098] The term "recombinant host cell" (or "recombinant cell" or "host cell" for short) as used herein is intended to refer to a cell into which a recombinant nucleic acid, such as a recombinant vector, has been introduced. In some cases, the word "cell" is replaced by a name that describes the cell type. For example, a "recombinant microorganism" is a recombinant host cell, which is a microbial host cell. It should be understood that the term refers not only to the specific object cell, but also to the offspring of the cell. Due to certain changes that may occur during continuous passage due to mutations or environmental influences, such offspring may not in fact be identical to the parent cell, but still fall within the scope of the terms "recombinant host cell", "recombinant cell" and "host cell" as used herein. The recombinant host cell can be an isolated cell or cell line grown in culture or can be a cell located in a living tissue or organism.

[0099] As used herein, the term "mammal" refers to any member of the class Mammalia, including placental mammals and marsupial mammals. Thus, "mammals" include humans, primates, livestock, and laboratory mammals. Exemplary mammals include rodents, mice, rats, rabbits, dogs, cats, sheep, horses, goats, llamas, cattle, primates, pigs, and any other mammal. In some embodiments, the mammal is at least one of a transgenic mammal, a genetically engineered mammal, and a cloned mammal.

[0100] Chimeric Antigen Receptor

[0101] The present invention includes a chimeric antigen receptor (CAR), which includes an extracellular antigen binding domain (binding domain), a hinge and a transmembrane domain (transmembrane domain), a HOOK-binding domain, and an intracellular signal transduction domain (activation domain). CAR may contain 1, 2, 3 or more of these domains.

[0102] The present invention individually encompasses all possible combinations of the specific polypeptides used herein and fragments thereof.

[0103] The present invention includes CARs comprising a HOOK-binding domain. A "HOOK-binding domain" is a domain that reversibly binds directly or indirectly to a "HOOK" protein in a cell, and its binding prevents CAR from leaving the endoplasmic reticulum (ER) or the Golgi apparatus under appropriate conditions.

[0104] In one embodiment, the HOOK-binding domain includes a streptavidin-binding peptide (SBP) that can bind to a HOOK protein containing core streptavidin. Biotin releases the CAR containing the hook-binding domain from HOOK by competing with SBP. The CAR can then move to the cell membrane.

[0105] Preferably, a system called RUSH (Resting Using Selective HOOK) system may be employed, Boncompain et al., Nat. Methods 9:493-498, 2012, which is incorporated herein by reference.

[0106] Preferably, the HOOK-binding domain comprises the following amino acid sequence:

[0107] MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO: 1) or encoded by the following nucleic acid sequence:

[0108]

[0109] Shorter SBP fragments deleted at their N- and C-termini can be used with the same efficacy. See Barrette-Ng, IH, SCWu, WMTjia, SLWong, and KKNg. 2013, The structure of the SBP-Tag-streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits, Acta crystallographica. Part D, Biologicalcrystallography 69: 879-887.

[0110] One embodiment of the HOOK-binding domain is shown in WO2010 / 142785, which is incorporated herein by reference. FKBPF-K506 binding domain 12 (FKBPI2) can be used in conjunction with FKBP-rapamycin associated protein (FRAP) used as a HOOK. In this embodiment, the interaction occurs only in the presence of rapamycin or its analogs, which can mediate the interaction between FKBP12 and FRAP, and can be specifically selected from FKI012, FK-CsA and rapamycin.

[0111] In one embodiment, HOOK-binding domains are located in the intracytoplasmic region of CAR. In other embodiments, HOOK-binding domains are located in other positions, that is, completely between different intracytoplasmic elements (e.g., transmembrane region and the first costimulatory element) or different connections between the same stimulatory elements.

[0112] The present invention includes CARs containing a binding domain comprising an antibody that specifically binds to a human polypeptide. The term "antibody" includes polyclonal antibodies, monoclonal antibodies, fragments thereof such as F(ab')2 and Fab fragments, single-chain variable fragments (scFv), single-domain antibody fragments (VHH or nanobodies, preferably camelids), and divalent and trivalent antibody fragments (diabodies and triantibodies).

[0113] Preferably, the antibody is a single chain Fv antibody or a nanobody.

[0114] In one embodiment, the antibody is monospecific. In one embodiment, the antibody is multispecific for 2, 3, or 4 polypeptides. Preferably, the antibody is bispecific.

[0115] Antibodies can be synthetic, monoclonal or polyclonal and can be prepared by techniques well known in the art. Such antibodies specifically bind to human proteins through the antibody's antigen binding site (as opposed to non-specific binding). Human proteins, polypeptide fragments and peptides can be used as immunogens in generating antibodies immunoreactive therewith. Human proteins, polypeptides and peptides contain antigenic determinants or epitopes that trigger antibody formation.

[0116] These antigenic determinants or epitopes can be linear or conformational (discontinuous). Linear epitopes contain a single segment of amino acids of a polypeptide, while conformational or discontinuous epitopes contain segments of amino acids from different regions of a polypeptide that are closely adjacent after protein folding (CA Janeway, Jr. and P. Travers, Immuno Biology 3: 9 (Garland Publishing Inc., 2nd edition, 1996)). Since folded proteins have complex surfaces, the number of accessible epitopes is very large; however, due to protein conformation and steric hindrance, the number of antibodies that actually bind to epitopes is less than the number of accessible epitopes (CA Janeway, Jr. and P. Travers, Immuno Biology 2: 14 (Garland Publishing Inc., 2nd edition, 1996)). Epitopes can be identified by any method known in the art.

[0117] Therefore, one aspect of the present invention relates to antigenic epitopes of human proteins. Such epitopes can be used to generate antibodies, especially monoclonal antibodies, as described in detail below.

[0118] If the antibody is greater than or equal to about 10 7 M -1If the Ka of the binding partners or antibodies is greater than 1:1, then they are defined as specifically binding. The affinity of a binding partner or antibody can be readily determined using conventional techniques, such as those described in Scatchard et al., Ann. NY Acad. Sci., 51:660 (1949).

[0119] Polyclonal antibodies can be easily generated from a variety of sources, such as horses, cattle, goats, sheep, dogs, chickens, rabbits, mice, or rats, using methods well known in the art. In general, a properly coupled purified human protein or polypeptide is generally administered to a host animal by parenteral injection. Immunogenicity can be enhanced by the use of an adjuvant, for example, complete or incomplete Freund's adjuvant. After booster immunization, a small serum sample is collected and tested for reactivity against a human protein or polypeptide. Examples of various tests that can be used for such determination include those described in Antibodies: A Laboratory Manual, compiled by Harlow and Lane, Cold Spring Harbor Laboratory Press, 1988; and processes such as countercurrent immunoelectrophoresis (CIEP), radioimmunoassay, radio-immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot assay, and sandwich assay. See U.S. Pat. Nos. 4,376,110 and 4,486,530.

[0120] Monoclonal antibodies can be readily prepared using well-known methods. See, e.g., the procedures described in U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Planu Publishing Company, Kennett, McKeam and Bechtol, ed., 1980.

[0121] For example, a host animal, such as a mouse, can be injected intraperitoneally at least once, and at least twice at intervals of about 3 weeks with isolated and purified human proteins or coupled human polypeptides, for example, peptides comprising or consisting of the above-mentioned specific amino acids. Mouse serum is then tested by conventional dot blotting techniques or antibody capture (ABC) to determine which animal is best fused. After about 2-3 weeks, intravenous boosting of human proteins or polypeptides is given to mice. Afterwards, mice are killed, and spleen cells are fused with commercially available myeloma cells such as Ag8.653 (ATCC) according to a maturation protocol. In brief, myeloma cells are washed several times in culture medium and fused to mouse spleen cells at a ratio of about 3 spleen cells: 1 myeloma cell. Fusion agents can be any suitable reagent used in the art, for example, polyethylene glycol (PEG). The fusion is inoculated onto a plate containing culture medium to allow selective growth of fused cells. The fused cells can then be grown for about 8 days. The supernatant from the resulting hybridoma is collected and added to a plate coated with goat anti-mouse Ig for the first time. After washing, a marker, such as a labeled human protein or polypeptide, is added to each well and incubated. Positive wells can then be tested. Positive clones are grown in large cultures and the supernatant is then purified on a protein A column (Pharmacia).

[0122] Other techniques may be used to generate the monoclonal antibodies of the invention, such as those described in Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas," Strategies in Molecular Biology 3: 1-9 (1990), which is incorporated herein by reference. Similarly, binding partners may be constructed using recombinant DNA technology to incorporate the variable regions of genes encoding specific binding antibodies. Such techniques are described in Larrick et al., Biotechnology, 7: 394 (1989).

[0123] The present invention also includes binding fragments of such antibodies that can be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fab and F(ab')2 fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.

[0124] The monoclonal antibodies of the present invention include chimeric antibodies, for example, humanized forms of mouse monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and provide the advantage of reduced immunogenicity when the antibody is administered to a human. In one embodiment, a humanized monoclonal antibody comprises a variable region (or its antigen binding site) of a mouse antibody and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment may comprise an antigen binding site of a mouse monoclonal antibody and a variable region fragment (lacking an antigen binding site) derived from a human antibody. The processes for producing chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (Bio / Technology 7:934, 1989), and Winter and Harris (TIPS 14:139, May 1993). Procedures for producing antibodies by transgenic means can be found in GB 2,272,440, US Pat. Nos. 5,569,825 and 5,545,806.

[0125] Antibodies produced by genetic engineering methods can be used, such as chimeric and humanized monoclonal antibodies, which contain both human and non-human portions, which can be prepared using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by genetic engineering using standard DNA techniques known in the art, for example, using Robinson et al., International Publication No. WO 87 / 02671; Akira et al., European Patent Application 0184187; Taniguchi, M., European Patent Application 0171496; Morrison et al., European Patent Application 0173494; Neuberger et al., PCT International Publication No. WO 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 0125023; Better et al., Science 240:1041-1043, 1988; Liu et al., PNAS 84:3439-3443, 1987; Liu et al., J. Immunol. 139:3521-3526, 1987; Sun et al., PNAS 84:214 218, 1987; Nishimura et al., Canc. Res. 47:9991005, 1987; Wood et al., Nature 314:446-449, 1985; and Shaw et al., J. Natl. Cancer Inst. 80:1553-1559, 1988); Morrison, SL, Science 229:1202-1207, 1985; Oi et al., BioTechniques 4:214, 1986; Winter, U.S. Pat. No. 5,225,539; Jones et al., Nature 321:552-525, 1986; Verhoeyan et al., Science 239:1534, 1988; and Beidler et al., J. Immunol. 141:4053-4060, 1988.

[0126] A library of immunoglobulins can be expressed from a population of display packages (preferably derived from filamentous bacteriophage) to form an antibody display library. Examples of methods and reagents particularly suitable for generating antibody display libraries can be found in, e.g., Ladner et al., U.S. Pat. No. 5,223,409; Kang et al., PCT Publication No. WO 92 / 18619; Dower et al., PCT Publication No. WO 91 / 17271; Winter et al., PCT Publication No. WO 92 / 20791; Markland et al., PCT Publication No. WO 92 / 15679; Breitling et al., PCT Publication No. WO 93 / 01288; McCafferty et al., PCT Publication No. WO 92 / 01047; Garrard et al., PCT Publication No. WO 92 / 09690; Ladner et al., PCT Publication No. WO 90 / 02809; Fuchs et al., (1991) Bio / Technology 9:1370-1372; Hay et al., (1992) Hum Antibod Hybridomas 3:81-85; Huse et al., (1989) Science 246:1275-1281; Griffths et al., (1993) supra; Hawkins et al., (1992) J Mol Biol 226:889-896; Clackson et al., (1991) Nature 352:624-628; Gram et al., (1992) PNAS 89:3576-3580; Garrad et al., (1991) Bio / Technology 9:1373-1377; Hoogenboom et al., (1991) Nuc Acid Res 19:4133-4137; and Barbas et al., (1991) PNAS 88:7978-7982. Once displayed on the surface of a display package (e.g., a filamentous phage), the antibody library is secreted to identify and isolate packages expressing antibodies that bind to a human protein or polypeptide. In a preferred embodiment, the initial screening of the library comprises panning with an immobilized human protein or polypeptide, and selecting display packages that express and display antibodies that bind to an immobilized human protein or polypeptide.

[0127] When used in conjunction with synthetic and semisynthetic antibodies, such terms are intended to encompass, but are not limited to, antibody fragments, isotype-switched antibodies, humanized antibodies (e.g., mouse-human, human-mouse), hybrids, antibodies with multiple specificities, and fully synthetic antibody-like molecules.

[0128] The present invention includes CARs comprising an activation domain of a CD3-ζ or Fc receptor γ amino acid sequence. Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398, which is incorporated herein by reference. The present invention also includes CARs comprising an activation domain of a CD3-ζ chain and a co-stimulatory receptor such as CD28, 4-1BB (CD137), DAP10, OX40 (CD134), ICOS, CD27, or a cytoplasmic domain of CD40L. Same as above.

[0129] Preferably, the CAR comprises a fragment of at least 50, 60, 70, 80, 90, 100, 110, 120, 150, or 200 amino acids of at least one of the following amino acid sequences having T-cell activation activity.

[0130] CD3-ζ:

[0131]

[0132] CD28:

[0133]

[0134] 4-1BB(CD137):

[0135]

[0136] DAP10:

[0137]

[0138] OX40 (CD134):

[0139]

[0140] ICOS:

[0141]

[0142] CD27:

[0143]

[0144] CD40L (CD154):

[0145]

[0146] In various embodiments, the CAR comprises a fragment of at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, or 200 amino acids that is at least 90%, preferably greater than 95%, and more preferably greater than 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34.

[0147] In various embodiments, the activation domain of CAR comprises 1, 2, 3, or 4 fragments of at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, or 200 amino acids, which have at least 90%, preferably more than 95%, and more preferably more than 99% identity with the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34.

[0148] The present invention includes CARs comprising a transmembrane (TM) domain, preferably a fragment of at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, or 200 amino acids, most preferably a fragment of at least one of the CD28, CD3z, CD8, CD4, FcRγ, TM regions.

[0149] CAR can be purified. Preferably, the purity of the purified CAR exceeds 50%, 75%, 85%, 90%, 95%, 97%, 98%, or 99%. In the context of the present invention, a purified CAR with a purity exceeding 50% (etc.) means that the purified CAR sample contains less than 50% (etc.) of other proteins. For example, if a sample of a recombinant CAR purified from a host cell contains less than 1% of contaminating host cell proteins, its purity can be 99%.

[0150] In a preferred embodiment, CAR encodes the amino acid sequence of any one of SEQ ID NO:46, SEQ ID NO:48, or SEQ ID NO:50.

[0151] Particularly preferred CARs include those encoding Fig.11Those of any component of the CAR shown in , including signal sequence, CD19 ScFv, CD8 hinge, transmembrane domain, 4-1BB, and / or CD3z sequence. Other preferred CARs include those encoding any component of the CAR of SEQ ID NO: 44-50. Particularly preferred CARs have a CD19ScFv domain replaced by another binding region.

[0152] Nucleic Acids

[0153] The present invention includes nucleic acids encoding CAR. The nucleic acid can be single-stranded or double-stranded. The nucleic acid can be an RNA or DNA molecule. The preferred nucleic acid encodes an amino acid sequence of at least one of the SEQ ID NOs detailed herein. The present invention includes isolated nucleic acids of the present invention inserted into a vector.

[0154] In one embodiment, the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 2. In one embodiment, the nucleic acid sequence comprises one or more of the following nucleic acid sequences:

[0155] HOOK: (nucleus + HA tag + other structures)

[0156]

[0157] Core Streptavidin:

[0158]

[0159] HA tag: TATCCGTACGACGTACCAGACTACGCA (SEQ ID NO: 37).

[0160] Preferred nucleic acids are at least 50, 60, 70, 80, 90, 100, 110, 120, 150, 200, 300, 400, 500, or 600 nucleotides.

[0161] The nucleic acid may be purified. Preferably, the purity of the purified nucleic acid exceeds 50%, 75%, 85%, 90%, 95%, 97%, 98%, or 99%. Within the scope of the present invention, a purified nucleic acid having a purity exceeding 50% means that the purified nucleic acid contains less than 50% other nucleic acids. For example, if a plasmid sample purified from a host bacterium contains less than 1% contaminating bacterial DNA, its purity may be at least 99%.

[0162] Particularly preferred nucleic acids include the following:

[0163] CAR_CD19 second generation: 1518bp

[0164]

[0165] CAR_CD19 third generation: 1641bp

[0166]

[0167] Particularly preferred nucleic acids include those comprising or encoding Fig. 9 Those of any component of the CAR shown in , including signal sequence, CD19 ScFv, CD8 hinge, transmembrane domain, 4-1BB, and / or CD3z sequence. Other preferred nucleic acids include those comprising or encoding any component of the CAR of SEQ ID NO: 45-50.

[0168] In some embodiments, the nucleic acid comprises a HOOK operably linked to a promoter, preferably UBC or β2M, and a CAR comprising a HOOK-binding protein operably linked to an IRES. In a preferred embodiment, the HOOK is a streptavidin protein, preferably core streptavidin, and the HOOK-binding protein is a streptavidin-binding protein.

[0169] Carrier

[0170] The present invention includes vectors encoding CAR and HOOK-binding domains. Preferred vectors include nucleic acid sequences or amino acid sequences encoding at least one of the SEQ ID NOs described in detail herein.

[0171] The vector may also encode a "HOOK". A "HOOK" is a protein that prevents a CAR containing a HOOK-binding domain from leaving the endoplasmic reticulum (ER) or the Golgi apparatus by directly or indirectly reversibly binding to the HOOK-binding domain within the CAR.

[0172] Retention can occur in the lumen of the ER or at its cytoplasmic face, depending on the topoisomerism of the protein and the orientation triggered by the interacting domains. Boncompain et al., Current Protocols in Cell Biology 15.19.1-15.19.16, December 2012, which is incorporated herein by reference.

[0173] In some embodiments, the HOOK for the ER comprises a mutant of the matrix interaction molecule 1 (STIM1-NN; type I protein), which is located in the ER but may not bind to microtubules, an isoform of the human non-variant chain of the major inhibitory compatibility complex (Ii; type II protein) with an N-terminal arginine-based motif; or a C-terminal ER retention signal (Lys-Asp-Glu-Leu; KDEL). Boncompain et al., Nat. Methods 9:493-498, 2012, which is incorporated herein by reference. The HOOK can be fused to the core streptavidin in its lumen or cytoplasmic domain, depending on the HOOK-binding protein, supra, Figure 1 .

[0174] In an alternative embodiment, the HOOK for the Golgi apparatus may be Golgin-84, which is used as a cytoplasmic Golgi apparatus HOOK.

[0175] Preferably, the HOOK comprises a streptavidin protein sequence, most preferably core streptavidin. US Patent No. 5,672,691, which is incorporated herein by reference.

[0176] Preferably, the HOOK comprises one of the following streptavidin protein sequences:

[0177] or

[0178] or

[0179]

[0180] Preferably, HOOK is encoded by the following nucleotide sequence:

[0181]

[0182] In some embodiments, the glycine at amino acid 49 of SEQ ID NO: 31 or SEQ ID NO: 32 is substituted with a bulky residue (e.g., threonine) to reduce biotin binding affinity without affecting SBP binding affinity. Wu et al., PLoS ONE 8(7): e69530 (2013), which is incorporated herein by reference. Another mutation can also be introduced to further favor SBP binding to biotin (mutation S27A).

[0183] In some embodiments, the vector comprises a HOOK operably linked to a promoter, preferably UBC or β2M, and a CAR comprising a HOOK-binding protein operably linked to an IRES.

[0184] The preferred IRES nucleotide sequence is:

[0185]

[0186] In a preferred embodiment, HOOK is a streptavidin protein, preferably core streptavidin, and the HOOK-binding protein is a streptavidin-binding protein. A preferred vector is a lentiviral vector comprising any one of the following nucleotide or amino acid sequences:

[0187] CAR-CD19 second generation-SBP1(nt):

[0188]

[0189]

[0190] CAR-CD19 second generation - SBP1 (aa):

[0191]

[0192] CAR-CD19 second generation - SBP2 (nt):

[0193]

[0194] CAR-CD19 second generation - SBP2 (aa):

[0195]

[0196] CAR-CD19 second generation - SBP3 (nt):

[0197]

[0198] CAR-CD19 second generation - SBP3 (aa):

[0199]

[0200] The vector may be an expression vector. The vector may be a plasmid vector. Preferably, the vector is a lentiviral vector.

[0201] In the context of the present invention, a "lentiviral vector" refers to a non-replicating vector that is used to transduce host cells with a transgene comprising cis-acting lentiviral RNA or DNA sequences and requires lentiviral proteins (e.g. Gag, Pol and / or Env) provided in trans. Lentiviral vectors lack expression of functional Gag, Pol and Env proteins. Lentiviral vectors can exist in the form of RNA or DNA molecules, depending on the generation or development stage of the retroviral vector.

[0202] The lentiviral vector can be in the form of a recombinant DNA molecule, such as a plasmid. The lentiviral vector can be in the form of a lentiviral vector particle, such as an RNA molecule in a complex of a lentivirus and other proteins. Typically, the lentiviral particle vector corresponding to the modified or recombinant lentiviral particle contains a genome consisting of two single-stranded RNA copies. These RNA sequences can be obtained by transcription from a double-stranded DNA sequence (proviral vector DNA) inserted into the host cell genome, or can be obtained by transient expression of plasmid DNA (plasmid vector DNA) in a transformed host cell.

[0203] Preferably, the lentiviral vector particles are integration-competent. Thus, they contain a functional integrase protein. Non-integrating vector particles have one or more mutations that eliminate most or all of the integration capacity of the lentiviral vector particles. For example, non-integrating vector particles may contain mutations in the integrase encoded by the lentiviral pol gene that result in reduced integration capacity. In contrast, integrating vector particles contain a functional integrase protein that does not contain any mutations that eliminate most or all of the integration capacity of the lentiviral vector particles.

[0204] Lentiviral vectors are derived from lentiviruses, particularly human immunodeficiency virus (HIV-1 or HIV-2), simian immunodeficiency virus (SIV), equine infectious encephalitis virus (EIAV), caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV), and feline immunodeficiency virus (FIV), which have been modified to remove genetic determinants involved in pathogenicity and to introduce new determinants useful for obtaining a therapeutic effect.

[0205] This type of vector is based on the separation of cis- and trans-acting sequences. To make a replication-defective vector, the trans-acting sequences (such as the gag, pol, tat, rev and env genes) can be deleted and replaced with an expression cassette encoding the transgene.

[0206] Efficient integration and replication in non-dividing cells generally require the presence of two cis-acting sequences in the center of the lentiviral genome: a central polypurine sequence (cPPT) and a central termination sequence (CTS). This results in the formation of a three-stranded DNA structure, called a central DNA "flap," which serves as a signal for uncoating of the pre-integration complex at the nuclear pore and for efficient introduction of the expression cassette into the nucleus of non-dividing cells such as dendritic cells.

[0207] In one embodiment, the present invention comprises a central polypurine sequence and a central termination sequence referred to as a cPPT / CTS sequence, in particular in European patent application EP 2169073.

[0208] Other sequences are usually present in cis, such as the long terminal repeats (LTR) involved in the integration of the vector proviral DNA sequence into the host cell genome. The vector can be obtained by mutating the LTR sequence, for example, by mutation in domain U3 of the LTR (ΔU3) (Miyoshi H et al., 1998, J Virol. 72 (10): 8150-7; Zufferey et al., 1998, J Virol 72 (12): 9873-80).

[0209] Preferably, the vector does not contain an enhancer. In one embodiment, the present invention comprises a lentiviral vector comprising an LTR sequence, preferably having a mutated U3 region (ΔU3) that removes the promoter and enhancer sequences in the 3' LTR.

[0210] A packaging sequence Ψ (psi) may also be incorporated to assist in encapsidation of the polynucleotide sequence into vector particles (Kessler et al., 2007, Leukemia, 21(9): 1859-74; Paschen et al., 2004, Cancer Immunol Immunother 12(6): 196-203).

[0211] In one embodiment, the invention includes a lentiviral vector comprising a lentiviral packaging sequence Ψ (psi).

[0212] Other additional functional sequences, such as transfer RNA-binding site or primer binding site (PBS) or woodchuck post regulatory element (WPRE) may also be preferably included in the lentiviral vector polynucleotide sequence of the present invention to obtain more stable expression of the transgene in vivo.

[0213] In one embodiment, the present invention includes a lentiviral vector comprising a PBS. In one embodiment, the present invention includes a lentiviral vector comprising a WPRE and / or an IRES.

[0214] Thus, in a preferred embodiment, the lentiviral vector comprises at least one cPPT / CTS sequence, one Ψ sequence, one (preferably two) LTR sequences and an expression cassette comprising a transgene under the transcriptional control of a β2m or MHC class I promoter.

[0215] Promoter

[0216] The present invention includes the use of a promoter to drive high expression of CAR from a lentiviral vector in T cells (preferably human T cells). Preferred promoters are human ubiquitin, MHC class I, MHC class II, and β2 microglobulin (β2m) promoters.

[0217] In various embodiments, the promoter drives high expression in antigen presenting cells, including dendritic cells. Preferably, the promoter lacks enhancer elements to avoid insertion effects.

[0218] Most preferably, the promoter is not the CMV promoter / enhancer.Preferably, the promoter is not the dectin-2 or MHCII promoter.

[0219] The sequences of various mammalian (human) MHC class I promoters are shown below:

[0220] HLA-A2 (MHC I):

[0221]

[0222] HLA-B7 (MHC I):

[0223]

[0224] HLA-Cw5(MHC I):

[0225]

[0226] HLA-E(MHC I):

[0227]

[0228] HLA-F(MHC I):

[0229]

[0230] The sequence of the human β2-microglobulin promoter is shown below:

[0231]

[0232] The sequence of the human ubiquitin (Ubi) promoter is shown below:

[0233]

[0234] The sequence of the human HLA-DRα promoter is shown below:

[0235]

[0236] In various embodiments, the lentiviral vector comprises a β2m, Ubi, MHC II or MHC class I promoter. Preferably, the MHC class I promoter is an HLA-A2 promoter, an HLA-B7 promoter, an HLA-Cw5 promoter, an HLA-F or an HLA-E promoter. In various embodiments, the promoter sequence comprises a polynucleotide sequence having greater than 90%, preferably greater than 95%, and more preferably greater than 99% identity to the promoter sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 38, or SEQ ID NO: 41.

[0237] In some embodiments, expression of the promoter in BDCA+ dendritic cells is at least 10, 12, 15, 20, 25, 30, 35, 40, 50, or 60 times greater than expression of the promoter in skeletal muscle cells.

[0238] In one embodiment, the present invention includes a lentiviral vector particle comprising a lentiviral vector comprising a dendritic cell-specific promoter directing expression of a microbial or tumor antigen, wherein the lentiviral vector particle exhibits higher antigen expression in BDCM cells than in HEK293T cells.

[0239] The present invention includes lentiviral vectors comprising a promoter without an enhancer.

[0240] The present invention includes inserting MHC class I (MHCI), Ubi, EF1α or β2 microglobulin promoter (β2m) promoter into a lentiviral vector. "MHC class I (MHCI) promoter" or "β2 microglobulin promoter" or "MHC class II (MHCII)" or "human ubiquitin promoter" used herein include naturally occurring or synthetic MHC class I promoters or β2 microglobulin promoters or MHC class II promoters or human ubiquitin promoters. The term "MHC class I promoter" does not include β2m promoter.

[0241] In one embodiment, the lentiviral vector particles comprising the promoter show higher expression in BDCM cells than in HEK293T cells.

[0242] The promoter may be a naturally occurring promoter. Examples of naturally occurring promoters are human β2m, HLA-A2, HLA-B7, HLA-Cw5, HLA-E, HLA-F, HLA-DRα, and ubiquitin gene promoters.

[0243] These naturally occurring MHC I promoters are generally cloned or reproduced from the promoter region of genes encoding MHC class I proteins, and are subsequently referred to as putatively encoding, such proteins in genome databases (according to: NCBI Polynucleotide Database http: / / www.ncbi.nlm.nih.gov / guide / dna-rna). Both β2m and MHC class I proteins enter the major histocompatibility complex (MHC).

[0244] The proteins encoded by these genes are present in almost all cell types. MHC I proteins are generally present on the surface of leukocyte membranes, where they bind to β2-microglobulin (β2m). The role of these associated proteins is to present peptides of endogenous origin to CD8+T cells. Therefore, it plays a central role in the generation of antigen-specific immune responses. Because MHC I class proteins have been widely studied and described over the years, their genes have been maturely characterized and can be well detected using sequence alignment tools (such as BLAST method) (Altschul, SF et al. (1990). Basic local alignment search tool. (Basic local alignment search tool) J. Mol. Biol. 215 (3): 403-410).

[0245] MHC class I promoters also share the ability to be strongly activated in antigen presenting cells, including dendritic cells, and to a reduced degree in most other human tissues.

[0246] The promoter of the present invention may also contain regulatory elements, such as one or more Sp1 and ETs binding sites. In a preferred embodiment, the MHC class I promoter contains two Sp1 binding sites and one Ets binding site. In other embodiments, Ap1 and / or Ap2 sites are also included in the promoter.

[0247] Preferred promoters are the naturally occurring human β2m, HLA-A2, HLA-B7, HLA-Cw5, HLA-E and HLA-F promoters.

[0248] Promoters can also be synthetic. Synthetic promoters include promoters that are synthesized using molecular biology techniques to assemble the individual components of the promoter or promoters that are derived from naturally occurring promoters using molecular biology techniques.

[0249] In various embodiments, the synthetic promoter comprises a polynucleotide sequence having greater than 90%, preferably greater than 95%, more preferably greater than 99%, or 100% identity to the promoter sequence of β2m, Ubi, MHC II or MHC class I gene promoter (SEQ ID NO: 9-14, 38, or 41).

[0250] Transcription of MHC-type genes is usually mediated by two major regulatory elements: interferon-stimulated response elements (ISRE) and SXY modules (including W / S, X1X2 / site α and Y / enhancer B regulatory elements). See Van den Elsen, Immunogenetics (1998) 48: 208-211.

[0251] These regulatory promoter elements are located in a region extending from approximately nucleotides -220 to -95 upstream of the transcription start site and mediate tissue-specific and cytokine-induced transcription of MHC class I genes.

[0252] The ISRE of the MHC class I gene promoter typically contains a binding site for a member of the interferon regulatory factor (IRF) family. Thus, one property of the MHC class I promoter is binding to members of the interferon regulatory factor (IRF) family. This can be verified, for example, by a gel shift assay.

[0253] Another regulatory element, enhancer A, which contains a binding site for nuclear transcription factor kappa B (NF-κB), is present in most cases. Thus, one property of MHC class I promoters is the binding of nuclear transcription factor kappa B (NF-κB). This can be verified, for example, by gel shift assays.

[0254] In addition to the ISRE, MHC class I promoters typically share another set of conserved upstream sequence motifs consisting of three regulatory elements: the S or W box, the X1 / CREX2 box or site α, and the Y box or enhancer B, which are collectively referred to as the SXY module. The SXY module typically binds cooperatively to a multiprotein complex containing regulatory factor X (RFX; consisting of RFX5, RFXB / ANK, and RFXAP), cAMP response element binding protein (CREB) / activating transcription factor (ATF), and nuclear factor Y (NFY) that acts as an enhanceosome to drive transactivation of these genes. Therefore, one property of MHC class I promoters is binding to these factors. This can be verified, for example, by gel shift assays.

[0255] In contrast, the MHC class II promoter does not display enhancer A or ISRE elements (Van den Elsen, PJ et al., 1998, Immunogenetics. 48: 208-221). In addition, RFX and CIITA were found to be critical in the regulation of class II MHC genes, as shown in studies using cell lines established from patients with bare lymphocyte syndrome (BLS; a severe combined immunodeficiency caused by mutations in one of the RFX subunits or CIITA). Furthermore, the absence of CIITA or one of the RFX subunits affects the function and assembly of the MHC enhanceome, respectively, resulting in a lack of class II MHC and decreased levels of class I MHC transcription (Van den Elsen, PJ et al., 2004, Current Opinion in Immunology, 16: 67-75).

[0256] In one embodiment, the present invention includes a method comprising inserting a promoter of the present invention, particularly a β2m, Ubi, MHC type II, or MHC I type promoter, into a lentiviral vector to direct the expression of the CAR of the present invention. The method may also include inserting any other nucleic acid element described herein, such as a DNA flap sequence.

[0257] Isolated cells

[0258] The present invention includes cells, especially cells of the human immune system, which contain lentiviral vector particles and vectors encoding the CAR of the present invention. Preferably, the cells are T cells, including Tαβ and Tγδ cells, or NK cells.

[0259] In one embodiment, the cell contains a vector integrated into the cell genome. In one embodiment, the cell contains a vector that transiently expresses CAR. In one embodiment, the cell produces a lentiviral vector particle encoding CAR.

[0260] In various embodiments, the present invention includes a cell line, a cell population, or a cell culture comprising a vector or a lentiviral vector particle encoding a CAR.

[0261] Lentiviral vector particles

[0262] The present invention provides a method for producing a lentiviral vector particle. The lentiviral vector particle (or lentiviral particle vector) includes a lentiviral vector associated with a viral protein. The vector is preferably an integrating vector.

[0263] In one embodiment, the lentiviral vector particle encodes the CAR of the present invention.

[0264] In one embodiment, the lentiviral vector particles contain HIV-1 Gag and Pol proteins. Preferably, the lentiviral vector particles contain subtype D, especially HIV-1 NDK , Gag and Pol proteins.

[0265] According to one embodiment of the method, lentiviral vector particles are obtained in host cells transformed with a DNA plasmid.

[0266] Such a DNA plasmid may contain:

[0267] - Bacterial origin of replication (e.g. pUC ori);

[0268] - an antibiotic resistance gene for selection (such as KanR); and more specifically:

[0269] - A lentiviral vector comprising at least one nucleic acid encoding a CAR transcriptionally linked to a β2m, Ubi, MHC class II, or MHC class I promoter.

[0270] The method allows the production of recombinant vector particles according to the invention, comprising the following steps:

[0271] i) transfecting suitable host cells with a lentiviral vector;

[0272] ii) transfecting the host cell with a packaging plasmid vector containing viral DNA sequences encoding at least the structural and polymerase (+ / - integrase) activities of a retrovirus, preferably a lentivirus; such packaging plasmids have been described in the art (Dull et al., 1998, J Virol, 72(11):8463-71; Zufferey et al., 1998, J Virol 72(12):9873-80).

[0273] iii) culturing the transfected host cells to allow the lentiviral vector to be expressed and packaged into lentiviral vector particles; and

[0274] iv) harvesting the lentiviral vector particles obtained by expression and packaging in step iii) in the cultured host cells.

[0275] For different reasons, pseudotyping (i.e., adding or replacing specific particle envelope proteins) of the prepared retroviral particles may be helpful. For example, it may be advantageous to have different envelope proteins to distinguish recombinant particles from natural particles or other recombinant particles. In vaccination strategies, pseudotyped particle vectors are more likely to escape the immune system when the latter have developed immunity to the lentivirus. This is particularly helpful if continuous injections of similar particle vectors are necessary to immunize the patient against the disease.

[0276] To prepare pseudotypes of the retroviral particles of the invention, host cells can also be transfected with one or several envelope DNA plasmids encoding viral envelope proteins, preferably VSV-G envelope proteins.

[0277] Suitable host cells are preferably cultured human cell lines, such as HEK cell lines.

[0278] Alternatively, the method for producing vector particles can be carried out in a host cell whose genome has been stably transformed with one or more of the following components: a lentiviral vector DNA sequence, a packaging gene, and an envelope gene. Such DNA sequences can be considered similar to the proviral vectors of the present invention, containing additional promoters to allow transcription of the vector sequence and increase particle productivity.

[0279] In a preferred embodiment, the host cell is also modified to produce viral particles in a continuous manner in the culture medium without swelling or death of the entire cell. For such techniques for producing viral particles, see Strang et al., 2005, J Virol 79(3): 1165-71; Relander et al., 2005, Mol Ther 11(3): 452-9; Stewart et al., 2009, Gene Ther, 16(6): 805-14; and Stuart et al., 2011, Hum gene Ther.

[0280] The object of the present invention consists of a host cell transformed with a lentiviral particle vector.

[0281] The lentiviral particle vector may include the following elements, as defined above:

[0282] -cPPT / CTS polynucleotide sequence; and

[0283] - A nucleic acid encoding a CAR under the control of a β2m, Ubi or MHC class I promoter, and optionally one of the other elements described above.

[0284] Preferably, the dose of lentiviral vector particles is 10 6 , 2x10 6 , 5x10 6 , 10 7 , 2x10 7 , 5x10 7 , 10 8 , 2x10 8 , 5x10 8 , or 10 9 TU.

[0285] Methods for expressing CAR in cells

[0286] The present invention includes a method for expressing CAR in cells, especially T cells, and preferably proliferating T cells. The method includes transducing cells using a lentiviral vector or lentiviral particle vector of the present invention under conditions that allow CAR expression, and preferably proliferating the T cells.

[0287] The cell is preferably a mammalian cell, in particular a human cell. Particularly preferred are human non-dividing cells.

[0288] Preferably, the cells are primary T cells or NK cells.

[0289] The method may further comprise harvesting or isolating the CAR.

[0290] The lentiviral vector or lentiviral particle vector preferably comprises the promoter of the present invention.

[0291] In one embodiment, the invention includes treating cells with biotin to release CAR from HOOK. Preferably, cells are treated with biotin at an initial concentration of at least 0.2, 0.4, 0.8, 1.6, 2.5, 5, 10, 20, 40, or 80 μM.

[0292] In one embodiment, the present invention includes a method of expressing a CAR, comprising inserting a β2m, Ubi, or MHCI promoter into a lentiviral vector so that it directs the expression of a nucleic acid encoding a CAR and transducing cells, preferably T or NK cells, with a vector containing the promoter, and optionally treating the cells with an initial concentration of biotin of at least 0.2, 0.4, 0.8, 1.6, 2.5, 5, 10, 20, 40, or 80 μM.

[0293] Therapeutic uses of lentiviral vectors

[0294] The present invention also relates to the use of the lentiviral vector of the present invention, especially in the form of lentiviral vector particles, for preparing a therapeutic composition or vaccine, wherein the therapeutic composition or vaccine can induce or cause an immune response to the CAR encoded by the vector or enhance the response.

[0295] The present invention includes methods of administering a lentiviral vector (or "lentiviral vector") to a human. Preferably, the lentiviral particle is an integrated lentiviral particle comprising a functional integrase protein.

[0296] Preferred modes of administration include reperfusion of the modified T cells, preferably intravenous or intraarticular administration, most preferably intratumoral administration.

[0297] In one embodiment, the present invention includes a method of inducing an immune response in a human, comprising administering a lentiviral vector comprising a functional integrase protein to T or NK cells and administering the modified cells to the human; wherein the integrated lentiviral vector includes a promoter that directs CAR expression; and generating an immune response to the CAR.

[0298] The present invention can also be used in treatment regimens against tumors and cancers, and in particular can be used in immunotherapy or vaccination treatment regimens against cancers and tumors.

[0299] The present invention also relates to an immunogenic composition comprising a lentiviral vector as defined above.

[0300] The immunogenic compositions of the present invention preferably comprise cPPT and CTS sequences in vectors and vector particles to induce or stimulate nuclear import of the vector genome in target cells.

[0301] During reverse transcription, the cPPT and CTS sequences induce the formation of a three-stranded DNA structure called a DNA triplex, which stimulates nuclear import of the DNA vector sequence. Preferably, the vector comprises CAR and regulatory signals for reverse transcription, expression and encapsidation of a retroviral or retroviral-like origin.

[0302] The lentiviral vectors of the present invention have the ability to redirect the specificity and function of T lymphocytes and / or other immune cells. They can quickly generate T cells that target specific tumor antigens or antigens associated with other lesions such as autoimmune diseases.

[0303] The lentiviral vector of the present invention can be used in therapeutic methods and methods for inducing immune responses, comprising administering the lentiviral vector to cells, preferably T or NK cells, administering the cells to a host, and generating a specific immune response that redirects the specificity and function of T lymphocytes and / or other immune cells.

[0304] A particular advantage of the immunogenic compositions of the invention is that they can be used to redirect the specificity and function of T lymphocytes and other immune cells against a variety of antigens to which the CAR in the vector or vector particle is directed.

[0305] Thus, the present invention includes compositions that can be used in therapeutic immunization regimens.

[0306] In particular, it may be used in combination with adjuvants, other immunogenic compositions, chemotherapy or any other therapeutic treatment.

[0307] The present invention includes a composition for administration to a human comprising a lentiviral vector particle comprising a functional integrase protein and a lentiviral vector; wherein the DNA of the lentiviral vector comprises a promoter directing the expression of amino acids comprising or consisting of a CAR.

[0308] In one embodiment, the invention comprises administering to a human, preferably intramuscularly, a lentiviral vector encoding a chimeric antigen receptor, or a cell transduced by the lentiviral vector, the chimeric antigen receptor comprising a binding domain; a transmembrane domain; a HOOK-binding domain, preferably comprising a streptavidin-binding peptide; and an activation domain, comprising a T cell activation fragment of at least 100 amino acids of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, SEQ ID NO: 33, or SEQ ID NO: 34. Preferably, the lentiviral vector further comprises HOOK, preferably comprising a streptavidin protein, most preferably comprising the amino acid sequence of SEQ ID NO: 32, SEQ ID NO: 33, or a mutant thereof, the mutant having a glycine mutation at amino acid 49, preferably to a threonine mutation. Preferably, the HOOK-binding domain comprises the amino acid sequence of SEQ ID NO: 1 or is encoded by the nucleic acid sequence of SEQ ID NO: 2.

[0309] The method may further comprise administering biotin to the human to release the CAR from the ER or Golgi apparatus. Preferably, biotin is administered at an initial concentration of at least 0.2, 0.4, 0.8, 1.6, 3.2, 5, 10, 20, 40, or 80 μM.

[0310] Therefore, different embodiments of the present invention have been described, and those skilled in the art should note that the contents disclosed herein are only exemplary, and that various other substitutions, adjustments or modifications may be included within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments shown herein. Example

[0311] Example 1. Molecular Construction

[0312] PCR amplification of the proviral region of pTRIPΔU3-CMV-GFP (15) was performed using forward (5'-CTTACTAGTTGGAAGGGCTAATTCACTCCCAAC-3'; SEQ ID NO: 15) and reverse (5'-CATTCTAGAACTGCTAGAGATTTTCCACACTG-3'; SEQ ID NO: 16) oligonucleotides including restriction sites for SDeI and XbaI, respectively. The resulting fragment was digested and cloned between the SpeI and XbaI sites of the pVAX-1 plasmid (Invitrogen, Life Technologies), from which the MluI site had been deleted. The resulting plasmid was named pFLAP-CMV-GFP. The SV40 sequence was amplified from the pTRIPΔU3-CMV-GFP plasmid by PCR (using 5'-TACCCCGGGCCATGGCCTCCAAAAAAGCCTCCTCACTACTTC-3' (SEQ ID NO: 17) and 5'-ACTCCCGGGTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC-3' (SEQ ID NO: 18) oligonucleotides) and cloned into the Pml1 site of pFLAP-CMV-GFP, and the resulting plasmid was then named pFLAP-CMV-GFP-SV. The CMV promoter was amplified using forward (5'-TACACGCGTGGAGTTCCGCGTTACATAACTTACGG-3'; SEQ ID NO: 19) and reverse (5'-CGTGGATCCGATCGCGGTGTCTTCTATGGAGGTCAAAAC-3'; SEQ ID NO: 20) oligonucleotides including MluI and BamHI sites, respectively. The resulting fragment was cloned back between the MluI and BamHI sites of pFlap-CMV-GFP-SV to facilitate replacement of the promoter within the lentiviral vector. The promoter was then amplified from HEK 293T cell DNA by PCR using 5'-GCCGGCGCGCCGAGAAACCCTGCAGGGAATTCCC-3' (SEQ ID NO: 21) and 5'-CGTGGATCCGATCGCTCGGCCCGAATGCTGTCAGCTTCAGG-3' (SEQ ID NO: 22) for the β2m promoter and cloned between the MluI and BamH1 sites of pFLAP-CMV-GFP-SV to generate pFlap-β2m-SV. The amplified β2m promoter sequence is as follows:

[0313] CAR can be synthesized and cloned in the position of GFP gene between BamHI and XhoI sites of pFlap-β2m-SV.

[0314] For example, pFlap-β2m-GFP-SV can be digested with BamHI and XhoI and a DNA linker containing a multiple cloning site (MCS, carrying SalI, SacII, NdeI, AscI and NheI restriction sites) can be placed between these sites in the position of the GFP gene to allow insertion of the nucleic acid sequence encoding the CAR.

[0315] The packaging plasmid pTHV-GP-N was constructed by PCR amplification of the HIV-1 NDK genome (using the following oligonucleotides, 5'-atgcatgcgtcgacctcgagttaatcctcatcctgtctacttgccac-3' (SEQ ID NO: 24) and 5'-gcatgcatcggccggggcggcgactgGTgagagGCCACCatgggtgcgagagcgtcagtattaag-3' (SEQ ID NO: 25)). The resulting fragment was digested with EagI and SalI restriction enzymes and inserted into the p8.74 packaging plasmid (15), from which the Eag1-SalI fragment had been previously removed.

[0316] Pseudotyped plasmids were generated by synthesizing codon optimized genes corresponding to the Indiana (GenBank #CAX62728.1), New Jersey (GenBank #CAX62729.1) and Cocal (GenBank #CAX62731.1) strains of vesicular stomatitis virus. These genes were then digested with EcoR1 and BamH1 and cloned between the corresponding restriction sites of the pVAX1 plasmid (Invitrogen, Life Technologies).

[0317] Plasmids can be generated using Nucleobond Xtra Maxi EF columns according to the manufacturer's instructions (MN Corporation).

[0318] Example 2. Lentivirus production

[0319] R&D Production: Vectors can be produced by transient calcium phosphate transfection of HEK 293T as described previously (25). HEK293T (human embryonic kidney cell line, ATCC CRL-11268, (Graham et al., 1977)) cells were maintained in Dulbecco's modified Eagle's medium (DMEM / High Modified, Hyclone) supplemented with 10% fetal bovine serum (FBS, PAA), 1% L-glutamine (Eurobio), 1% penicillin-streptomycin (Gibco, Life Technologies), and 1% sodium pyruvate (Gibco, Life Technologies). The cell line was maintained in an incubator at 37°C in a humidified atmosphere with 5% CO2. Lentiviral vectors were produced by transient transfection of HEK 293T cells using a standard calcium phosphate precipitation protocol. HEK 293T cells were grown at 7x10 6 10 cells were seeded in 10 mL of complete medium in a 10 cm 2 Tissue culture dish (BD Falcon) and kept in an incubator in a humid atmosphere of 5% CO2 at 37°C for 24 hours to adhere. For each generated vector, the tissue culture dish was transfected as follows: lentiviral backbone plasmid pFlap-promoter-CAR_CD19 (10 μg), pThV-Env1 encoding envelope plasmid (2 μg) and pThV-GP packaging plasmid (10 μg) were mixed with 353 μL of sterile distilled water (Gibco of Life Technologies) and 125 μL of CaCl2 (Fluka). Then 500 μL of 37°C preheated HBS 2X pH=7.3 was added dropwise to the DNA mixture, and 1 mL of the resulting precipitate was added to the culture medium of the cells. The transfected cells were then incubated at 37°C, 5% CO2. The culture medium was replaced with 7 mL of serum-free harvesting culture medium 24 hours after transfection and the viral supernatant was harvested by centrifugation at 2500 rpm for 5 minutes after another 24 hours. Then, the harvested clarified culture (210 mL) was treated with DNase (Roche) in the presence of MgCl2 (Sigma-Aldrich) for 30 minutes to avoid residual transfection DNA, and ultra-concentrated by centrifugation at 22000 rpm for 1 hour at 4°C. Each vector pellet was resuspended in 70 μl PBS-lactose (40 mg / L), pooled, aliquoted in 30 μL and stored at -70°C ± 10°C.

[0320] For product characterization and drug release, quality tests may be performed as follows: Regulatory texts for vaccines: quality controls required for vaccines are as per the European Pharmacopoeia (section 6.16), “Guideline on quality, non-clinical and clinical aspects of vaccines derived from live recombinant viral vectors” (EMA / CHMP / 141697 / 2009), “Guideline for the development and manufacture of lentiviral vectors” (CHMP / BWP / 2458 / 03); Regulatory texts for gene therapy medicinal products: quality controls required for transgenic medicinal products for human use are as per the European Pharmacopoeia (section 5.14), quality controls specific to gene therapy products as defined in the “Guideline Note on quality, preclinical and clinical aspects of transgene removal free products” (CHMP / BWP / 3088 / 99); Regulatory texts for biotechnological products (ICH Q5A to ICH Q5E); Regulatory texts for labeling (ICH Q6A and ICH Q6B) and quality controls required for parenteral preparations as per the European Pharmacopoeia (section 7.0).

[0321] Example 3. Lentiviral vector titer

[0322] qPCR reaction: HEK 293T cells were seeded in 6-well plates (BD Falcon) in culture medium and incubated for 4 hours at 37°C in a humidified atmosphere of 5% CO2. Cells were transduced with 3 serial dilutions of lentiviral vector. After 72 hours of incubation, cells were harvested and transduced HEK 293T cell pellets were generated. Whole genomic DNA was extracted from transduced cell pellets using a method based on the QIAGEN QIAamp DNA mini kit manual. Proviral quantification was performed using Taqman qPCR. Amplification was performed using a master mix (Fermentas Thermo Scientific), Flap A primer (CCCAAGAACCCAAGGAACA; SEQ ID NO: 26) and Flap S primer (AGACAA GATAGAGGAAGAGCAAAAC; SEQ ID NO: 27) primers, and Lenti TM probe (6FAM-AACCATTAGGAGTAGCACCCACCAAGG-BBQ; SEQ ID NO: 52). Normalization was performed using quantification of the actin gene (same mix, actin A-CGGTGAGGATCTTCATGAGGTAGT-(SEQ ID NO: 28), actin S-AACACCCCAGCCATGTACGT-(SEQ ID NO: 29) primers and HUMURA ​​ACT™ probe-6FAM-CCAGCCAGGTCCAGACGCAGGA-BBQ-(SEQ ID NO: 30). Both reactions were performed on a MasterCycler Ep Realplex S (Eppendorf, 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 sec at 95°C and 1 min at 63°C). Analysis was performed on the MasterCycler Ep Realplex software.

[0323] Example 4. Regulated CAR

[0324] The lentiviral vector encoding CAR was generated. CAR_CD19 second and third generation sequences (SEQ ID NO: 39 and SEQ ID NO: 40) were purchased from GeneArt (Life Technologies) and cloned to replace the GFP gene between the BamHI and XhoI restriction sites of pFlap-ΔU3-β2m-GFP, pFlap-ΔU3-HLA-A2-GFP, pFlap-ΔU3-HLA-DRα-GFP or pFlap-ΔU3-UBC-GFP, depending on the desired promoter.

[0325] CAR was generated as a fusion protein with SBP at 3 different positions. The lentiviral vector was further modified to contain the HOOK of ER fused to the core streptavidin protein. The promoter was the β2m promoter.

[0326] These lentiviral vectors will be used in vitro and animal models of cancer. Biotin will be given at an initial concentration of 40 μM (titration of higher and lower concentrations) to test release from the ER. First, the migration of CAR to the cell surface (GFP at the binding domain position or between the signal sequence and the binding domain) will be analyzed in vitro. Next, the same analysis will be performed in animal models (mice and rats) by injecting CAR-T cells and evaluating migration (GFP) on the surface of cells from animals.

[0327] Will generate simultaneously contain HOOK-binding domain (SBP) " second generation " CAR (2 endocytoplasmic activation domains) and " third generation " CAR (3 endocytoplasmic activation domains).Initially, the binding domains evaluated will be anti-CD19, anti-PDL-1, anti-PD1, anti-hedgehog, anti-CD123, and anti-CD123 / CD33.

[0328] CARMIN 1.0: A second generation (containing CD3_ and 4-1BB co-signaling domains) and a third generation (containing CD3_, CD28 and 4-1BB domains) lentiviral vector for CD19 (CD19+ leukemia and lymphoma), LMP-1 and -2 (EBV-induced leukemia). The lentiviral vector allows for optimized expression of CAR in T cells and studies on the effects on the efficacy of CAR-T cells. Hematological malignancies can be used as a benchmark.

[0329] CARMIN 2.0; Development of a switchable on / off system based on a W protein anchored to the membrane of the endoplasmic reticulum (ER) by HOOK, and its binding partner Y introduced into the CAR structure. The interaction between X-HOOK (e.g., streptavidin) and Y (e.g., streptavidin-binding protein)-CAR causes CAR to be retained in the ER. The addition of Z protein (e.g., biotin) replaces the balance of binding of X to Z instead of Y, thereby causing CAR to be released from the ER and expressed in the cytoplasmic membrane. The release of CAR will be stopped by Z consumption (or antagonists), and the remaining cells can be easily reactivated by the reintroduction of Z inducers.

[0330] HOOK and CAR can be vectorized in a lentiviral vector and can be used in the clinic (b2m-HOOK-IRES-CAR). It can be evaluated in vitro on fixed cells (HEK293T, Jurkat, HeLa) and on primary cells (T-cells). This system will increase the safety of CAR-T cells. The switchable CD19-CAR system can evaluate expression in vitro and evaluate efficacy in vivo.

[0331] Most of the scFv used so far is mouse-derived. Neutralizing antibodies for these mouse scFv can show the efficacy of CAR. Alternatively, we will develop camelid nano antibodies to be used as binding domains because they are highly homologous to human antibody VH domains and they show high antigen binding ability. A second-generation CAR containing nano antibodies for Her2 as binding domains will be used for concept verification. These technology platforms allow CAR design, production and evaluation of flexibility and responsiveness, thus generating optimized CAR-T cells. This differential induction and reversible (on / off) CAR T- cell technology is intended to be delivered (automated process) at the patient's bedside.

[0332] Example 5. Expression in human T cells

[0333] Peripheral blood mononuclear cells (PBMCs) were purified from peripheral blood by gradient density centrifugation on Ficoll. After PBMC washing, CD3+ T cells were purified by negative magnetic selection (i.e., unwanted cells were magnetically labeled while T cells remained untouched) using a pan-T cell isolation kit (Miltenyi Biotec). This step is required to obtain a highly purified population of T cells. Based on the yield obtained after this separation step, 10 7 Up to 10 8 T cells at 2,5x10 6 / ml in an optimized serum-free cell culture medium developed for the culture and proliferation of human T cells (TexMACS culture medium, Miltenyi Biotec). These T cells are activated by T cell activation / proliferation from Miltenyi Biotec. The kit consists of anti-biotin MACSiBead particles and biotinylated antibodies for human CD2, CD3 and CD28. Anti-biotin MACSiBead particles loaded with biotinylated antibodies are used to simulate antigen presenting cells and activate T cells. The optimal activation of T cells is completed by using 1 loaded anti-biotin MACSiBead particle for every 2 cells. T cells are activated for 3 days. Then the transduction of activated T cells is performed at an MOI of 4, which means that 1 T cell is incubated with 4 transduction units of lentiviral particles. The expression of CAR added by lentiviral particles for 48 or 72 hours is evaluated by flow cytometry, by using biotinylated goat anti-mouse IgG, followed by staining the mouse CD19-binding domain with streptavidin coupled to phycoerythrin. T cell subsets were characterized by CD3, CD4 and CD8 staining. This enabled specific detection of CAR on the surface of T cells. The entire process was performed with TexMACS medium, allowing the survival and proliferation of T cells.

[0334] Example 6. Expression and structure of CAR-RUSH

[0335] Qualified blood was obtained from the National Blood Service (Rungis, France). Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll (lymphocyte separation medium, Eurobio) gradient density separation. T cells were then purified from PBMCs by magnetic separation using a pan-T cell isolation kit (Miltenyi Biotec). T cells were isolated according to the manufacturer's instructions. 2,5x10 6 Cells were placed in culture medium at a concentration of 10 cells / ml and activated for 3 days with a 1:2 bead:T cell ratio using a T cell activation / proliferation kit from Miltenyi Biotec (anti-CD2 / -CD3 / -CD28 nanoparticles prepared according to the manufacturer's instructions). After activation, T cells were harvested, counted, and cultured in TexMACS medium in 24-well plates at 37°C / 5% CO2. Transduction was performed by adding lentiviral vectors at different MOIs directly to the wells. The different lentiviral vectors tested were: (i) a second-generation anti-CD19 CAR containing 4-1BB and CD3ζ intracellular domains; (ii) the same vector containing streptavidin binding protein at 3 different positions (CAR-SBP1, CAR-SBP2, CAR-SBP3).

[0336] The volume of vector added to each well according to the MOI was calculated as follows: Volume to be added (ul) = (MOI x number of cells (in millions) / vector concentration (transducing units / ml)) x 1000.

[0337] Use 3.10 9 TU / ml vector titer, 200,000 cells transduced at an MOI of 10->vector volume to be added (μl) = (10x0.2x10 6 / 3x10 9 )x1000=0.51μl. Human recombinant IL-2 (Miltenyi Biotec) was added at 50IU / ml on the day of transduction. On the 3rd day, the transduced T cells were harvested, fully washed with DPBS1X and immunostained for the molecules of interest in 96-well plates. T cells were first incubated with a viability dye coupled to eFluor 780 (fixable viability dye, eBiosciences) and incubated at 4°C for 30 minutes. Incubation was carried out in DPBS1X without azide and protein. The cells were then washed with DPBS 1X and incubated at 4°C for 30 minutes with biotinylated goat anti-mouse IgG (Fab') 2 (Jackson ImmunoResearch). After incubation, cells were washed with autoMACS running buffer (Miltenyi Biotec) and incubated for the third time using a mixture of streptavidin coupled to phycoerythrin (Jackson ImmunoResearch) and mouse anti-human CD3 coupled to PE-Cy7 (BD Biosciences). Incubation was performed for 30 minutes at 4°C. After the third incubation, cells were washed in autoMACS running buffer and fixed in CellFIX (BD Biosciences) before data acquisition on a MACSQuant analyzer (Miltenyi Biotec). Flow cytometry data were analyzed using FlowJo software.

[0338] Example 7. Expression and performance of CAR-RUSH constructs after co-transduction with lentiviral vector encoding HOOK-streptavidin and biotin treatment.

[0339] Qualified blood was obtained from the National Blood Service (Rungis, France). Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll (lymphocyte separation medium, Eurobio) gradient density separation. T cells were then purified from PBMCs by magnetic separation using a pan-T cell isolation kit (Miltenyi Biotec). T cells were isolated according to the manufacturer's instructions. 2,5x10 6Cells were placed in culture medium at a concentration of 10 cells / ml and activated for 3 days by the T cell activation / proliferation kit from Miltenyi Biotec (anti-CD2 / -CD3 / -CD28 nanoparticles prepared according to the manufacturer's instructions) at a beads:T cell ratio of 1:2.

[0340] After activation, T cells were harvested, counted and cultured in TexMACS medium in 24-well plates at 37°C / 5% CO2. Co-transduction was performed by adding different MOIs of lentiviral vectors directly to the wells. The two lentiviral vectors tested were: (i) HOOK-streptavidin vector and (ii) second-generation anti-CD19 CAR (CAR-SBP1, CAR-SBP2 and CAR-SBP3) containing 4-1BB and CD3ζ intracellular domains of streptavidin binding protein at 3 different positions.

[0341] The volume of each vector added to each well according to the MOI was calculated as follows: Volume to be added (μl) = (MOI x number of cells (in millions) / vector concentration (transducing units / ml)) x 1000.

[0342] Human recombinant IL-2 (50 IU / ml; Miltenyi Biotec) and biotin (40 μM; Sigma-Aldrich) were added on the day of transduction.

[0343] On the 3rd day, the transduced T cells were harvested, washed thoroughly with DPBS 1X and immunostained for molecules of interest in 96-well plates. T cells were first incubated with a viability dye (fixable viability dye, eBiosciences) coupled to eFluor 780 and incubated at 4°C for 30 minutes. Incubation was carried out in DPBS 1X without azide and protein. The cells were then washed with DPBS 1X and incubated at 4°C for 30 minutes with biotinylated goat anti-mouse IgG (Fab') 2 (Jackson ImmunoResearch). After incubation, cells were washed with autoMACS running buffer (Miltenyi Biotech) and incubated for the third time using a mixture of streptavidin coupled to phycoerythrin (Jackson ImmunoResearch) and mouse anti-human CD3 coupled to PE-Cy7 (BD Biosciences). Incubation was carried out at 4°C for 30 minutes. After the third incubation, cells were washed in autoMACS running buffer and fixed in CellFIX (BD Biosciences) prior to data acquisition on a MACSQuant analyzer (Miltenyi Biotec). Flow cytometry data were analyzed using FlowJo software.

[0344] Example 8. Expression and performance of CAR-RUSH bicistronic constructs

[0345] Qualified blood was obtained from the National Blood Service (Rungis, France). Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll (lymphocyte separation medium, Eurobio) gradient density separation. T cells were then purified from PBMCs by magnetic separation using a pan-T cell isolation kit (Miltenyi Biotec). T cells were isolated according to the manufacturer's instructions. 2,5x10 6 Cells were placed in culture medium at a concentration of 10 cells / ml and activated for 3 days by the T cell activation / proliferation kit from Miltenyi Biotec (anti-CD2 / -CD3 / -CD28 nanoparticles prepared according to the manufacturer's instructions) at a beads:T cell ratio of 1:2.

[0346] After activation, T cells were harvested, counted and cultured in TexMACS medium in 24-well plates at 37°C / 5% CO2. Transduction was performed by adding different MOIs of lentiviral vectors directly to the wells. The different lentiviral vectors tested were: (i) "classic" second-generation anti-CD19 CAR; (ii) three constructs containing HOOK-streptavidin, IRES, and a second-generation anti-CD19 CAR containing 4-1BB and CD3ζ intracellular domains with streptavidin binding proteins at three different locations (HOOK-IRES-CAR-SBP1, HoOK-IRES-CAR-SBP2, HOOK-IRES-CAR-SBP3).

[0347] The volume of vector added to each well according to the MOI was calculated as follows: Volume to be added (ul) = (MOI x number of cells (in millions) / vector concentration (transducing units / ml)) x 1000.

[0348] The different MOIs tested were 10, 20 or 30, depending on the experiment.

[0349] Human recombinant IL-2 (50 IU / ml; Miltenyi Biotec) and biotin (40 μM; Sigma-Aldrich) were added on the day of transduction.

[0350] On the 3rd and 7th days, the transduced T cells were harvested, washed thoroughly with DPBS 1X and immunostained for molecules of interest in 96-well plates. T cells were first incubated with a viability dye coupled to eFluor 780 (fixable viability dye, eBiosciences) and incubated at 4°C for 30 minutes. Incubation was carried out in DPBS 1X without azide and protein. The cells were then washed with DPBS 1X and incubated at 4°C for 30 minutes with biotinylated goat anti-mouse IgG (Fab') 2 (Jackson ImmunoResearch). After incubation, cells were washed with autoMACS running buffer (Miltenyi Biotech) and incubated for the third time using a mixture of streptavidin coupled to phycoerythrin (Jackson ImmunoResearch) and mouse anti-human CD3 coupled to PE-Cy7 (BD Biosciences). Incubation was carried out at 4°C for 30 minutes. After the third incubation, cells were washed in autoMACS running buffer and fixed in CellFIX (BD Biosciences) prior to data acquisition on a MACSQuant analyzer (Miltenyi Biotec). Flow cytometry data were analyzed using FlowJo software.

[0351] Example 9. CAR-RUSH system switching evaluation (off / on / off)

[0352] Qualified blood was obtained from the National Blood Service (Rungis, France). Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll (lymphocyte separation medium, Eurobio) gradient density separation. T cells were then purified from PBMCs by magnetic separation using a pan-T cell isolation kit (Miltenyi Biotec). T cells were isolated according to the manufacturer's instructions. 2,5x10 6 Cells were placed in culture medium at a concentration of 10 cells / ml and activated for 3 days by the T cell activation / proliferation kit from Miltenyi Biotec (anti-CD2 / -CD3 / -CD28 nanoparticles prepared according to the manufacturer's instructions) at a beads:T cell ratio of 1:2.

[0353] After activation, T cells were harvested, counted and cultured in TexMACS medium in 24-well plates at 37°C / 5% CO2. Transduction was performed by adding different MOIs of lentiviral vectors directly to the wells. The different lentiviral vectors tested were: (i) "classic" second-generation anti-CD19 CAR; (ii) three constructs containing HOOK-streptavidin, IRES, and a second-generation anti-CD19 CAR containing 4-1BB and CD3ζ intracellular domains with streptavidin binding proteins at three different locations (HOOK-IRES-CAR-SBP1, HOOK-IRES-CAR-SBP2, HOOK-IRES-CAR-SBP3).

[0354] The volume of vector added to each well according to the MOI was calculated as follows: Volume to be added (μl) = (MOI x number of cells (in millions) / vector concentration (transducing units / ml)) x 1000.

[0355] The different MOIs tested were 10 and 20.

[0356] Human recombinant IL-2 (50 IU / ml; Miltenyi Biotec) and biotin (40 μM; Sigma-Aldrich) were added on the day of transduction.

[0357] On day 3, cells were washed and biotin was added at 40 μM or not to reinduce CAR-SBP expression.

[0358] On the 7th day, the transduced T cells were harvested, washed thoroughly with DPBS 1X and immunostained for molecules of interest in 96-well plates. T cells were first incubated with a viability dye coupled to eFluor 780 (fixable viability dye, eBiosciences) and incubated at 4°C for 30 minutes. Incubation was carried out in DPBS 1X without azide and protein. The cells were then washed with DPBS 1X and incubated at 4°C for 30 minutes with biotinylated goat anti-mouse IgG (Fab') 2 (Jackson ImmunoResearch). After incubation, cells were washed with autoMACS running buffer (Miltenyi Biotech) and incubated for the third time using a mixture of streptavidin coupled to phycoerythrin (Jackson ImmunoResearch) and mouse anti-human CD3 coupled to PE-Cy7 (BD Biosciences). Incubation was carried out at 4°C for 30 minutes. After the third incubation, cells were washed in autoMACS running buffer and fixed in CellFIX (BD Biosciences) prior to data acquisition on a MACSQuant analyzer (Miltenyi Biotec). Sequence Listing <110> THERAVECTYS Institut Curie Central Scientific Research Center Sorbonne University National Institute of Health and Medical Research (Institut National de la Sante et de la Recherche Medicalale) <120> Lentiviral vectors for regulated expression of chimeric antigen receptor molecules <130> THERA14.16-PCT <150> 62 / 028,889 <151> 2014-07-25 <150> 62 / 155,811 <151> 2015-05-01 <160> 52 <170> PatentIn version 3.5 <210> 1 <211> 38 <212> PRT <213> Artificial sequence <220> <223> Hook-binding domain <400> 1 Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly 1 5 10 15 Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro 20 25 30 Gln Gly Gln Arg Glu Pro 35 <210> 2 <211> 114 <212> DNA <213> Artificial sequence <220> <223> Hook-binding domain <400> 2 atggacgaga aaaccaccgg ctggcgggga ggccacgtgg tggaaggact ggccggcgag 60 ctggaacagc tgcgggccag actggaacac cacccccagg gccagagaga gccc 114 <210> 3 <211> 163 <212> PRT <213> Homo sapiens <400> 3 Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu Pro 1 5 10 15 Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys Tyr 20 25 30 Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala Leu 35 40 45 Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 50 55 60 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 65 70 75 80 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 85 90 95 Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 100 105 110 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 115 120 125 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 130 135 140 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 145 150 155 160 Pro Pro Arg <210> 4 <211> 220 <212> PRT <213> Homo sapiens <400> 4 Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val 1 5 10 15 Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr 20 25 30 Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser 35 40 45 Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu 50 55 60 Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser 65 70 75 80 Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr 85 90 95 Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys 100 105 110 Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser 115 120 125 Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro 130 135 140 Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly 145 150 155 160 Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile 165 170 175 Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 180 185 190 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 195 200 205 Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 210 215 220 <210> 5 <211> 255 <212> PRT <213> Homo sapiens <400> 5 Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu 1 5 10 15 Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro 20 25 30 Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys 35 40 45 Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile 50 55 60 Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser 65 70 75 80 Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly 85 90 95 Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu 100 105 110 Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln 115 120 125 Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys 130 135 140 Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro 145 150 155 160 Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala 165 170 175 Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu 180 185 190 Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200 205 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255 <210> 6 <211> 93 <212> PRT <213> Homo sapiens <400> 6 Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala 1 5 10 15 Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr 20 25 30 Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro 35 40 45 Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile 50 55 60 Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln 65 70 75 80 Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly 85 90 <210> 7 <211> 277 <212> PRT <213> Homo sapiens <400> 7 Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val 20 25 30 Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro 35 40 45 Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys 50 55 60 Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro 65 70 75 80 Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys 85 90 95 Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly 100 105 110 Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys 115 120 125 Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp 130 135 140 Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn 145 150 155 160 Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro 165 170 175 Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr 180 185 190 Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu 195 200 205 Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val 210 215 220 Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu 225 230 235 240 Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 245 250 255 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 260 265 270 Thr Leu Ala Lys Ile 275 <210> 8 <211> 199 <212> PRT <213> Homo sapiens <400> 8 Met Lys Ser Gly Leu Trp Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys 1 5 10 15 Val Leu Thr Gly Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile 20 25 30 Phe His Asn Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val 35 40 45 Gln Gln Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp 50 55 60 Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu 65 70 75 80 Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu 85 90 95 Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser 100 105 110 Ile Phe Asp Pro Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu 115 120 125 His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Phe Trp Leu Pro 130 135 140 Gly Cys Ala Ala Phe Val Val Cys Ile Leu Gly Cys Ile Leu 145 150 155 160 Ile Cys Trp Leu Thr Lys Lys Tyr Ser Ser Ser Val His Asp Pro 165 170 175 Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser 180 185 190 Arg Leu Thr Asp Val Thr Leu 195 <210> 9 <211> 220 <212> DNA <213> Humans (Homo sapiens) <400> 9 attggggagt cccagccttg gggattcccc aactccgcag ttctttct ccctccca 60 acctatgtag ggtccttctt cctggatact cacgacgcgg acccagttct cactcccatt 120 gggtgtcggg tttccagaga agccaatcag tgtcgtcgcg gtcgcggttc taaagtccgc 180 acgcacccac cgggactcag attctcccca gacgccgagg 220 <210> 10 <211> 197 <212> DNA <213> Homo sapiens <400> 10 ggggaggcgc agcgttgggg attccccact cccctgagtt tcacttcttc tcccaacttg 60 tgtcgggtcc ttcttccagg atactcgtga cgcgtcccca cttcccactc ccattgggta 120 ttggatatct agagaagcca atcagcgtcg ccgcggtccc agttctaaag tccccacgca 180 cccacccgga ctcagag 197 <210> 11 <211> 216 <212> DNA <213> Homo sapiens <400> 11 cactggggag gcgccgcgtt gaggattctc cactcccctc agtttcactt cttctcccaa 60 cctgcgtcgg gtccttcttc ctgaatactc atgacgcgtc cccaattccc actcccattg 120 ggtgtcgggt tctagagaag ccaatcagcg tctccgcagt cccggtctaa agtccccagt 180 cacccacccg gactcagatt ctccccagac gccgag 216 <210> 12 <211> 205 <212> DNA <213> Homo sapiens <400> 12 taagaactgc tgattgctgg gaaactctgc agtttcccgt tcctctcgta acctggtcat 60 gtgtccttct tcctggatac tcatgacgca gactcagttc tcattcccaa tgggtgtcgg 120 gtttctagag aagccaatca gcgtcgccac gactcccgac tataaagtcc ccatccggac 180 tcaagaagtt ctcaggactc agagg 205 <210> 13 <211> 252 <212> DNA <213> Homo sapiens <400> 13 aggccccgag gcggtgtctg gggttggaag gctcagtatt gagaattccc catctcccca 60 gagtttctct ttctctccca acccgtgtca ggtccttcat cctggatact cataacgcgg 120 ccccatttct cactcccatt gggcgtcgcg tttctagaga agccaatcag tgtcgccgca 180 gttcccaggt tctaaagtcc cacgcacccc gcgggactca tatttttccc agacgcggag 240 gttggggtca tg 252 <210> 14 <211> 192 <212> DNA <213> Homo sapiens <400> 14 aacatcacga gactctaaga aaaggaaact gaaaacggga aagtccctct ctctaacctg 60 gcactgcgtc gctggcttgg agacaggtga cggtccctgc gggccttgtc ctgattggct 120 gggcacgcgt ttaatataag tggaggcgtc gcgctggcgg gcattcctga agctgacagc 180 attcgggccg ag 192 <210> 15 <211> 33 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 15 cttactagtt ggaagggcta attcactccc aac 33 <210> 16 <211> 32 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 16 cattctagaa ctgctagaga ttttccacac tg 32 <210> 17 <211> 42 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 17 taccccgggc catggcctcc aaaaaagcct cctcactact tc 42 <210> 18 <211> 44 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 18 actcccgggt aatttttttt atttatgcag aggccgaggc cgcc 44 <210> 19 <211> 35 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 19 tacacgcgtg gagttccgcg ttacataact tacgg 35 <210> 20 <211> 39 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 20 cgtggatccg atcgcggtgt cttctatgga ggtcaaaac 39 <210> twenty one <211> 34 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> twenty one gccggcgcgc cgagaaaccc tgcagggaat tccc 34 <210> twenty two <211> 41 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> twenty two cgtggatccg atcgctcggc ccgaatgctg tcagcttcag g 41 <210> twenty three <211> 517 <212> DNA <213> Artificial sequence <220> <223> Promoter <400> 23 gagaaaccct gcagggaatt ccccagctgt agttataaac agaagttctc cttctgctag 60 gtagcattca aagatcttaa tcttctgggt ttccgttttc tcgaatgaaa aatgcaggtc 120 cgagcagtta actggcgggg gcaccattag caagtcactt agcatctctg gggccagtct 180 gcaaagcgag ggggcagcct taatgtgcct ccagcctgaa gtcctagaat gagcgcccgg 240 tgtcccaagc tggggcgcgc accccagatc ggagggcgcc gatgtacaga cagcaaactc 300 acccagtcta gtgcatgcct tcttaaacat cacgagactc taagaaaagg aaactgaaaa 360 cgggaaagtc cctctctcta acctggcact gcgtcgctgg cttggagaca ggtgacggtc 420 cctgcgggcc ttgtcctgat tggctgggca cgcgtttaat ataagtggag gcgtcgcgct 480 ggcgggcatt cctgaagctg acagcattcg ggccgag 517 <210> 24 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer <400> 24 atgcatgcgt cgacctcgag ttaatcctca tcctgtctac ttgccac 47 <210> 25 <211> 65 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 25 gcatgcatcg gccggggcgg cgactggtga gaggccacca tgggtgcgag agcgtcagta 60 ttaag 65 <210> 26 <211> 19 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 26 cccaagaacc caaggaaca 19 <210> 27 <211> 25 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 27 agacaagata gaggaagagc aaaac 25 <210> 28 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 28 cggtgaggat cttcatgagg tagt 24 <210> 29 <211> 20 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 29 aacaccccag ccatgtacgt 20 <210> 30 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 30 ccagccaggt ccagacgcag ga 22 <210> 31 <211> 160 <212> PRT <213> Artificial sequence <220> <223> Hook peptide <400> 31 Met Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala 1 5 10 15 Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val 20 25 30 Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala Val 35 40 45 Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala 50 55 60 Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp 65 70 75 80 Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln 85 90 95 Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr 100 105 110 Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val Gly His 115 120 125 Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala 130 135 140 Lys Lys Ala Gly Val Asn Asn Gly Asn Pro Leu Asp Ala Val Gln Gln 145 150 155 160 <210> 32 <211> 160 <212> PRT <213> Artificial Sequence <220> <223> Hook Peptide <400> 32 Met Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala 1 5 10 15 Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val 20 25 30 Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala Val 35 40 45 Gly Asn Ala Glu Ser Arg Tyr Thr Leu Thr Gly Arg Tyr Asp Ser Ala 50 55 60 Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Arg Val Ala Trp 65 70 75 80 Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln 85 90 95 Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Thr Leu Thr 100 105 110 Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Arg Gly His 115 120 125 Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala 130 135 140 Lys Lys Ala Gly Val Asn Asn Gly Asn Pro Leu Asp Ala Val Gln Gln 145 150 155 160 <210> 33 <211> 260 <212> PRT <213> Homo sapiens <400> 33 Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val 1 5 10 15 Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr 20 25 30 Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe 35 40 45 Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro 50 55 60 Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His 65 70 75 80 Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys 85 90 95 Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys 100 105 110 Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu 115 120 125 Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His 130 135 140 Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met 145 150 155 160 Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr 165 170 175 His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile 180 185 190 Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala 195 200 205 Leu Phe Leu His Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser 210 215 220 Pro Val Glu Pro Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu 225 230 235 240 Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro 245 250 255 Ala Cys Ser Pro 260 <210> 34 <211> 261 <212> PRT <213> Homo sapiens <400> 34 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly Leu Leu Lys Leu 260 <210> 35 <211> 1224 <212> DNA <213> Artificial sequence <220> <223> Hook sequence <400> 35 atgcaccgga ggagatcacg ctcttgtagg gaggaccaga aacctgtcac cggtgaccct 60 agcaaagact caaaagctca ggtgtccgct gccgaggctg gcattactgg aacatggtac 120 aatcagctcg ggagcacctt tattgtgact gctggagccg atggagccct caccggaaca 180 tacgaatctg ctgtgggaaa cgccgaatca cggtacgtcc tcactggccg atacgatagt 240 gcccctgcca ccgacggatc tgggactgcc ctgggatgga ctgtcgcttg gaaaaacaac 300 taccggaatg ctcattctgc cacaacatgg agtggacagt acgtgggagg cgctgaggct 360 agaatcaata cacagtggct gctcacatct ggcacaaccg aggcaaatgc ttggaaatcc 420 accctggtgg gacatgacac attcaccaaa gtgaaaccct ccgccgcttc aatcgatgcc 480 gccaaaaaag ccggagtcaa caacggcaat cctctggatg ccgtccagca ggtcgactat 540 ccgtacgacg taccagacta cgcagtcgga ccgatggacg atcagaggga cctcattagc 600 aacaacgaac agctgcctat gctgggacgg cgacctggag cccctgaatc caaatgctct 660 aggggagcac tgtacactgg cttctccatt ctcgtgacac tgctgctggc cgggcaggct 720 actactgctt acttcctgta ccagcagcag gggcggctgg acaaactcac tgtgacatct 780 cagaacctcc agctggaaaa tctgaggatg aaactgccca aaccccctaa acccgtgtcc 840 aaaatgagga tggccacacc tctgctcatg caggcactgc caatgggagc cctgccccag 900 gggcccatgc agaatgccac caagtatggc aacatgacag aggaccatgt gatgcacctg 960 ctccagaatg ctgaccccct gaaggtgtac ccgccactga aggggagctt cccggagaac 1020 ctgagacacc ttaagaacac catggagacc atagactgga aggtctttga gagctggatg 1080 caccattggc tcctgtttga aatgagcagg cactccttgg agcaaaagcc cactgacgct 1140 ccaccgaaag agtcactgga actggaggac ccgtcttctg ggctgggtgt gaccaagcag 1200 gatctgggcc cagtccccat gtga 1224 <210> 36 <211> 477 <212> DNA <213> Artificial sequence <220> <223> Hook sequence <400> 36 gaccctagca aagactcaaa agctcaggtg tccgctgccg aggctggcat tactggaaca 60 tggtacaatc agctcgggag cacctttatt gtgactgctg gagccgatgg agccctcacc 120 ggaacatacg aatctgctgt gggaaacgcc gaatcacggt acgtcctcac tggccgatac 180 gatagtgccc ctgccaccga cggatctggg actgccctgg gatggactgt cgcttggaaa 240 aacaactacc ggaatgctca ttctgccaca acatggagtg gacagtacgt gggaggcgct 300 gaggctagaa tcaatacaca gtggctgctc acatctggca caaccgaggc aaatgcttgg 360 aaatccaccc tggtgggaca tgacacattc accaaagtga aaccctccgc cgcttcaatc 420 gatgccgcca aaaaagccgg agtcaacaac ggcaatcctc tggatgccgt ccagcag 477 <210> 37 <211> 27 <212> DNA <213> Artificial sequence <220> <223> HA tag <400> 37 tatccgtacg acgtaccaga ctacgca 27 <210> 38 <211> 1216 <212> DNA <213> Homo sapiens <400> 38 ggcctccgcg ccgggttttg ggcctcccgc gggcgccccc ctcctcacgg cgagcgctgc 60 cacgtcagac gaagggcgca gcgagcgtcc tgatccttcc gcccggacgc tcaggacagc 120 ggcccgctgc tcataagact cggccttaga accccagtat cagcagaagg acattttagg 180 acgggacttg ggtgactcta gggcactggt tttctttcca gagagcggaa caggcgagga 240 aaagtagtcc cttctcggcg attctgcgga gggatctccg tggggcggtg aacgccgatg 300 attatataag gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg gatttgggtc 360 gcggttcttg tttgtggatc gctgtgatcg tcacttggtg agtagcgggc tgctgggctg 420 gccggggctt tcgtggccgc cgggccgctc ggtgggacgg aagcgtgtgg agagaccgcc 480 aagggctgta gtctgggtcc gcgagcaagg ttgccctgaa ctgggggttg gggggagcgc 540 agcaaaatgg cggctgttcc cgagtcttga atggaagacg cttgtgaggc gggctgtgag 600 gtcgttgaaa caaggtgggg ggcatggtgg gcggcaagaa cccaaggtct tgaggccttc 660 gctaatgcgg gaaagctctt attcgggtga gatgggctgg ggcaccatct ggggaccctg 720 acgtgaagtt tgtcactgac tggagaactc ggtttgtcgt ctgttgcggg ggcggcagtt 780 atggcggtgc cgttgggcag tgcacccgta cctttgggag cgcgcgccct cgtcgtgtcg 840 tgacgtcacc cgttctgttg gcttataatg cagggtgggg ccacctgccg gtaggtgtgc 900 ggtaggcttt tctccgtcgc aggacgcagg gttcgggcct agggtaggct ctcctgaatc 960 gacaggcgcc ggacctctgg tgaggggagg gataagtgag gcgtcagttt ctttggtcgg 1020 ttttatgtac ctatcttctt aagtagctga agctccggtt ttgaactatg cgctcggggt 1080 tggcgagtgt gttttgtgaa gttttttagg caccttttga aatgtaatca tttgggtcaa 1140 tatgtaattt tcagtgttag actagtaaat tgtccgctaa attctggccg tttttggctt 1200 ttttgttaga ccgatc 1216 <210> 39 <211> 1518 <212> DNA <213> Artificial Sequence <220> <223> CAR <400> 39 atggccctgc ctgtgacagc cctgctgctg cccctggctc tcctgctgca tgccgccaga 60 cccgctagcg acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac 120 agagtgacca tcagctgccg ggccagccag gacatcagca agtacctgaa ctggtatcag 180 cagaaacccg acggcaccgt gaagctgctg atctaccaca ccagccggct ccacagcggc 240 gtgcccagca gattttctgg cagcggcagc ggcaccgact acagcctgac catctccaac 300 ctggaacagg aagatatcgc tacctacttc tgtcagcaag gcaacaccct gccctacacc 360 ttcggcggag gcaccaagct ggaaatcacc ggcggaggcg gaagtggagg tggaggatct 420 ggcggcggag gctccgaagt gaagctgcag gaaagcggcc ctggcctcgt ggcccctagc 480 cagagcctgt ccgtgacctg taccgtgtcc ggcgtgtccc tgcccgacta cggcgtgtcc 540 tggatcagac agcctcccag aaagggcctg gaatggctgg gcgtgatctg gggcagcgag 600 acaacctact acaacagcgc cctgaagtcc cggctgacca tcatcaagga caacagcaag 660 agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc 720 gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 780 gtgaccgtgt ccagccatat ggccctgagc aacagcatca tgtacttcag ccacttcgtg 840 cccgtgtttc tgcccgccaa gcccaccacc acccctgccc ctagacctcc caccccagcc 900 ccaacaatcg ccagccagcc tctgtccctg cggcccgaag cctgtagacc tgctgccggc 960 ggagccgtgc acaccagagg cctggatatc tacatctggg cccctctggc cggcacctgt 1020 ggcgtgctgc tgctgagcct ggtgatcaca aagcggggca gaagaagct gctgtacatc 1080 ttcaagcagc cattcatgcg gcccgtgcag accacccagg aagaggacgg ctgcagctgc 1140 cggttccccg aggagga aggcggctgc gaactgccca agctgtgcta cctgctggac 1200 ggcatcctgt tcatctatgg cgtgatcctg accgccctgt tcctgagagt gaagttcagc 1260 agaagcgccg acgcccctgc ctaccagcag ggccagaacc agctctacaa cgagctgaac 1320 ctgggcagac gggaagagga cgacgtgctggagaagggagaggccggga ccctgagatg 1380 ggcggcaagc cccagcggcg gaagaaccct caggaagcc tgtataacga actgcagaaa 1440 gacaagatgg ccgaggccta cagcgagatc ggcatgaagg gcgagcggcg gagaggcaag 1500 ggccacgatg gcctgtac 1518 <210> 40 <211> 1641 <212> DNA <213> Artificial Sequence <220> <223> CAR <400> 40 atggccctgc ctgtgacagc cctgctgctg cccctcgctc tgctgctgca tgccgccaga 60 cccgctagcg acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac 120 agagtgacca tcagctgccg ggccagccag gacatcagca agtacctgaa ctggtatcag 180 cagaaacccg acggcaccgt gaagctgctg atctaccaca ccagccggct ccacagcggc 240 gtgcccagca gattttctgg cagcggcagc ggcaccgact acagcctgac catctccaac 300 ctggaacagg aagatatcgc tacctacttc tgtcagcaag gcaacaccct gccctacacc 360 ttcggcggag gcaccaagct ggaaatcacc ggcggaggcg gaagtggagg gggaggatct 420 ggcggcggag gctccgaagt gaagctgcag gaaagcggcc ctggcctggt ggcccctagc 480 cagagcctgt ccgtgacctg taccgtgtcc ggcgtgtccc tgcccgacta cggcgtgtcc 540 tggatcagac agccccccag aaagggcctg gaatggctgg gcgtgatctg gggcagcgag 600 acaacctact acaacagcgc cctgaagtcc cggctgacca tcatcaagga caacagcaag 660 agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc 720 gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 780 gtgaccgtgt ccagccatat ggccctgagc aacagcatca tgtacttcag ccacttcgtg 840 cccgtgtttc tgcccgccaa gcccaccacc acccctgccc ctagacctcc caccccagcc 900 ccaacaatcg ccagccagcc tctgtccctg aggcccgaag cctgtagacc tgctgccggc 960 ggagccgtgc acaccagagg cctggatatc tacatctggg cccctctggc cggcacctgt 1020 ggcgtgctgc tgctgagcct ggtgatcacc cggtccaagc ggagcagact gctgcactcc 1080 gactacatga acatgacccc cagacggcct ggccccaccc ggaagcacta ccagccttac 1140 gcccctcccc gggacttcgc cgcctacaga agcaagcggg gcagaaagaa gctgctgtac 1200 atcttcaagc agcccttcat gcggcccgtg cagaccaccc aggaagagga cggctgcagc 1260 tgccggttcc ccgaggaaga ggaaggcggc tgcgaactgc ccaagctgtg ctacctgctg 1320 gacggcatcc tgttcatcta tggcgtgatc ctgaccgccc tgttcctgag agtgaagttc 1380 agcagaagcg ccgacgcccc tgcctaccag cagggccaga accagctgta caacgagctg 1440 aacctgggca gacgggaaga gtacgacgtg ctggacaagc gcagaggccg ggaccctgag 1500 atgggcggca agcctcagcg gcggaagaac cctcaggaag gcctgtataa cgaactgcag 1560 aaagacaaga tggccgaggc ctacagcgag atcggcatga agggcgagcg gcggagaggc 1620 aagggccacg atggcctgta c 1641 <210> 41 <211> 300 <212> DNA <213> Homo sapiens <400> 41 gtctagaagt cagattgggg ttaaagagtc tgtccgtgat tgactaacag tcttaaatac 60 ttgatttgtt gttgttgttg tcctgtttgt ttaagaactt tacttcttta tccaatgaac 120 ggagtatctt gtgtcctgga ccctttgcaa gaacccttcc cctagcaaca gatgcgtcat 180 ctcaaaatat ttttctgatt ggccaaagag taattgattt gcattttaat ggtcagactc 240 tattacaccc cacattctct tttcttttat tcttgtctgt tctgcctcac tcccgagctc 300 <210> 42 <211> 407 <212> PRT <213> Artificial Sequence <220> <223> Hook Peptide <400> 42 Met His Arg Arg Arg Ser Arg Ser Cys Arg Glu Asp Gln Lys Pro Val 1 5 10 15 Thr Gly Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu 20 25 30 Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile 35 40 45 Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala 50 55 60 Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser 65 70 75 80 Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala 85 90 95 Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly 100 105 110 Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu 115 120 125 Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val Gly 130 135 140 His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser Ile Asp Ala 145 150 155 160 Ala Lys Lys Ala Gly Val Asn Asn Gly Asn Pro Leu Asp Ala Val Gln 165 170 175 Gln Val Asp Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Val Gly Pro Met 180 185 190 Asp Asp Gln Arg Asp Leu Ile Ser Asn Asn Glu Gln Leu Pro Met Leu 195 200 205 Gly Arg Arg Pro Gly Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala Leu 210 215 220 Tyr Thr Gly Phe Ser Ile Leu Val Thr Leu Leu Leu Ala Gly Gln Ala 225 230 235 240 Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu 245 250 255 Thr Val Thr Ser Gln Asn Leu Gln Leu Glu Asn Leu Arg Met Lys Leu 260 265 270 Pro Lys Pro Pro Lys Pro Val Ser Lys Met Arg Met Ala Thr Pro Leu 275 280 285 Leu Met Gln Ala Leu Pro Met Gly Ala Leu Pro Gln Gly Pro Met Gln 290 295 300 Asn Ala Thr Lys Tyr Gly Asn Met Thr Glu Asp His Val Met His Leu 305 310 315 320 Leu Gln Asn Ala Asp Pro Leu Lys Val Tyr Pro Pro Leu Lys Gly Ser 325 330 335 Phe Pro Glu Asn Leu Arg His Leu Lys Asn Thr Met Glu Thr Ile Asp 340 345 350 Trp Lys Val Phe Glu Ser Trp Met His His Trp Leu Leu Phe Glu Met 355 360 365 Ser Arg His Ser Leu Glu Gln Lys Pro Thr Asp Ala Pro Pro Lys Glu 370 375 380 Ser Leu Glu Leu Glu Asp Pro Ser Ser Gly Leu Gly Val Thr Lys Gln 385 390 395 400 Asp Leu Gly Pro Val Pro Met 405 <210> 43 <211> 1224 <212> DNA <213> Artificial Sequence <220> <223> Hook Sequence <400> 43 atgcaccgga ggagatcacg ctcttgtagg gaggaccaga aacctgtcac cggtgaccct 60 agcaaagact caaaagctca ggtgtccgct gccgaggctg gcattactgg aacatggtac 120 aatcagctcg ggagcacctt tattgtgact gctggagccg atggagccct caccggaaca 180 tacgaatctg ctgtgggaaa cgccgaatca cggtacgtcc tcactggccg atacgatagt 240 gcccctgcca ccgacggatc tgggactgcc ctgggatgga ctgtcgcttg gaaaaacaac 300 taccggaatg ctcattctgc cacaacatgg agtggacagt acgtgggagg cgctgaggct 360 agaatcaata cacagtggct gctcacatct ggcacaaccg aggcaaatgc ttggaaatcc 420 accctggtgg gacatgacac attcaccaaa gtgaaaccct ccgccgcttc aatcgatgcc 480 gccaaaaaag ccggagtcaa caacggcaat cctctggatg ccgtccagca ggtcgactat 540 ccgtacgacg taccagacta cgcagtcgga ccgatggacg atcagaggga cctcattagc 600 aacaacgaac agctgcctat gctgggacgg cgacctggag cccctgaatc caaatgctct 660 aggggagcac tgtacactgg cttctccatt ctcgtgacac tgctgctggc cgggcaggct 720 actactgctt acttcctgta ccagcagcag gggcggctgg acaaactcac tgtgacatct 780 cagaacctcc agctggaaaa tctgaggatg aaactgccca aaccccctaa acccgtgtcc 840 aaaatgagga tggccacacc tctgctcatg caggcactgc caatgggagc cctgccccag 900 gggcccatgc agaatgccac caagtatggc aacatgacag aggaccatgt gatgcacctg 960 ctccagaatg ctgaccccct gaaggtgtac ccgccactga aggggagctt cccggagaac 1020 ctgagacacc ttaagaacac catggagacc atagactgga aggtctttga gagctggatg 1080 caccattggc tcctgtttga aatgagcagg cactccttgg agcaaaagcc cactgacgct 1140 ccaccgaaag agtcactgga actggaggac ccgtcttctg ggctgggtgt gaccaagcag 1200 gatctgggcc cagtccccat gtga 1224 <210> 44 <211> 574 <212> DNA <213> Artificial Sequence <220> <223> IRES <400> 44 gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60 gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120 ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180 gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240 aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300 tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360 acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca 420 aggggctgaa ggatgcccag aaggtacgcc attgtatggg atctgatctg gggcctcggt 480 gcacatgctt tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc cgaaccacgg 540 ggacgtggtt ttcctttgaa aaacacgatg ataa 574 <210> 45 <211> 1638 <212> DNA <213> Artificial Sequence <220> <223> CAR <400> 45 atggccctgc ctgtgacagc cctgctgctg cccctggctc tcctgctgca tgccgccaga 60 cccgctagcg acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac 120 agagtgacca tcagctgccg ggccagccag gacatcagca agtacctgaa ctggtatcag 180 cagaaacccg acggcaccgt gaagctgctg atctaccaca ccagccggct ccacagcggc 240 gtgcccagca gattttctgg cagcggcagc ggcaccgact acagcctgac catctccaac 300 ctggaacagg aagatatcgc tacctacttc tgtcagcaag gcaacaccct gccctacacc 360 ttcggcggag gcaccaagct ggaaatcacc ggcggaggcg gaagtggagg tggaggatct 420 ggcggcggag gctccgaagt gaagctgcag gaaagcggcc ctggcctcgt ggcccctagc 480 cagagcctgt ccgtgacctg taccgtgtcc ggcgtgtccc tgcccgacta cggcgtgtcc 540 tggatcagac agcctcccag aaagggcctg gaatggctgg gcgtgatctg gggcagcgag 600 acaacctact acaacagcgc cctgaagtcc cggctgacca tcatcaagga caacagcaag 660 agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc 720 gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 780 gtgaccgtgt ccagccatat ggccctgagc aacagcatca tgtacttcag ccacttcgtg 840 cccgtgtttc tgcccgccaa gcccaccacc acccctgccc ctagacctcc caccccagcc 900 ccaacaatcg ccagccagcc tctgtccctg cggcccgaag cctgtagacc tgctgccggc 960 ggagccgtgc acaccagagg cctggatatc tacatctggg cccctctggc cggcacctgt 1020 ggcgtgctgc tgctgagcct ggtgatcacc accggtatgg acgagaaaac caccggctgg 1080 cgggaggcc acgtggtgga aggactggcc ggcgagctgg aacagctgcg ggccagactg 1140 gaacaccacc cccagggcca gagagagccc aagcggggca gaagaagct gctgtacatc 1200 ttcaagcagc ccttcatgcg gcccgtgcag accacccagg aagaggacgg ctgcagctgc 1260 cggttccccg aggagga aggcggctgc gaactgccca agctgtgcta cctgctggac 1320 ggcatcctgt tcatctacgg cgtgatcctg accgccctgt tcctgagagt gaagttcagc 1380 agaagcgccg acgcccctgc ctaccagcag ggccagaacc agctgtacaa cgagctgaac 1440 ctgggcagac gggaagagat cgacgtgctggagaagggagaggccggga ccctgagatg 1500 ggcggcaagc cccagcggcg gaagacccc caggaaggcc tgtataacga actgcagaaa 1560 gacaagatgg ccgaggccta cagcgagatc ggcatgaagg gcgagcggcg gagaggcaag 1620 ggccacgatg gcctgtac 1638 <210> 46 <211> 546 <212> PRT <213> Artificial Sequence <220> <223> CAR <400> 46 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ala Ser Asp Ile Gln Met Thr Gln Thr Thr Ser 20 25 30 Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala 35 40 45 Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp 50 55 60 Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu 85 90 95 Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln 100 105 110 Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 115 120 125 Ile Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser 145 150 155 160 Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp 165 170 175 Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp 180 185 190 Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu 195 200 205 Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe 210 215 220 Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys 225 230 235 240 Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly 245 250 255 Gln Gly Thr Ser Val Thr Val Ser Ser His Met Ala Leu Ser Asn Ser 260 265 270 Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu Pro Ala Lys Pro 275 280 285 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 290 295 300 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 305 310 315 320 Gly Ala Val His Thr Arg Gly Leu Asp Ile Tyr Ile Trp Ala Pro Leu 325 330 335 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Thr Gly 340 345 350 Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly 355 360 365 Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro 370 375 380 Gln Gly Gln Arg Glu Pro Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 385 390 395 400 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 405 410 415 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 420 425 430 Pro Lys Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val 435 440 445 Ile Leu Thr Ala Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 450 455 460 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 465 470 475 480 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 485 490 495 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 500 505 510 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 515 520 525 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 530 535 540 Leu Tyr 545 <210> 47 <211> 1638 <212> DNA <213> Artificial Sequence <220> <223> CAR <400> 47 atggccctgc ctgtgacagc cctgctgctg cccctggctc tcctgctgca tgccgccaga 60 cccgctagcg acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac 120 agagtgacca tcagctgccg ggccagccag gacatcagca agtacctgaa ctggtatcag 180 cagaaacccg acggcaccgt gaagctgctg atctaccaca ccagccggct ccacagcggc 240 gtgcccagca gattttctgg cagcggcagc ggcaccgact acagcctgac catctccaac 300 ctggaacagg aagatatcgc tacctacttc tgtcagcaag gcaacaccct gccctacacc 360 ttcggcggag gcaccaagct ggaaatcacc ggcggaggcg gaagtggagg tggaggatct 420 ggcggcggag gctccgaagt gaagctgcag gaaagcggcc ctggcctcgt ggcccctagc 480 cagagcctgt ccgtgacctg taccgtgtcc ggcgtgtccc tgcccgacta cggcgtgtcc 540 tggatcagac agcctcccag aaagggcctg gaatggctgg gcgtgatctg gggcagcgag 600 acaacctact acaacagcgc cctgaagtcc cggctgacca tcatcaagga caacagcaag 660 agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc 720 gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 780 gtgaccgtgt ccagccatat ggccctgagc aacagcatca tgtacttcag ccacttcgtg 840 cccgtgtttc tgcccgccaa gcccaccacc acccctgccc ctagacctcc caccccagcc 900 ccaacaatcg ccagccagcc tctgtccctg cggcccgaag cctgtagacc tgctgccggc 960 ggagccgtgc acaccagagg cctggatatc tacatctggg cccctctggc cggcacctgt 1020 ggcgtgctgc tgctgagcct ggtgatcaca aagcggggca gaagaagct gctgtacatc 1080 ttcaagcagc ccttcatgcg gcccgtgcag accacccagg aagaggacgg ctgcagctgc 1140 cggttccccg agggaagga aggcggctgc gagctgaccg gtatggacga gaaaaccacc 1200 ggctggcggg gaggccacgt ggtggagga ctggccggcg agctggaaca gctgcgggcc 1260 agatggaac accaccccca gggccagagg gaaccccca agctgtgcta cctgctggac 1320 ggcatcctgt tcatctacgg cgtgatcctg accgccctgt tcctgagagt gaagttcagc 1380 agaagcgccg acgcccctgc ctaccagcag ggccagaacc agctgtacaa cgagctgaac 1440 ctgggcagac gggaagagat cgacgtgctggagaagggagaggccggga ccctgagatg 1500 ggcggcaagc cccagcggcg gaagacccc caggaaggcc tgtataacga actgcagaaa 1560 gacaagatgg ccgaggccta cagcgagatc ggcatgaagg gcgagcggcg gagaggcaag 1620 ggccacgatg gcctgtac 1638 <210> 48 <211> 546 <212> PRT <213> Artificial Sequence <220> <223> CAR <400> 48 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ala Ser Asp Ile Gln Met Thr Gln Thr Thr Ser 20 25 30 Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala 35 40 45 Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp 50 55 60 Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu 85 90 95 Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln 100 105 110 Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 115 120 125 Ile Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser 145 150 155 160 Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp 165 170 175 Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp 180 185 190 Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu 195 200 205 Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe 210 215 220 Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys 225 230 235 240 Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly 245 250 255 Gln Gly Thr Ser Val Thr Val Ser Ser His Met Ala Leu Ser Asn Ser 260 265 270 Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu Pro Ala Lys Pro 275 280 285 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 290 295 300 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 305 310 315 320 Gly Ala Val His Thr Arg Gly Leu Asp Ile Tyr Ile Trp Ala Pro Leu 325 330 335 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Lys Arg 340 345 350 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 355 360 365 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 370 375 380 Glu Glu Glu Gly Gly Cys Glu Leu Thr Gly Met Asp Glu Lys Thr Thr 385 390 395 400 Gly Trp Arg Gly Gly His Val Val Glu Gly Leu Ala Gly Glu Leu Glu 405 410 415 Gln Leu Arg Ala Arg Leu Glu His His Pro Gln Gly Gln Arg Glu Pro 420 425 430 Pro Lys Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val 435 440 445 Ile Leu Thr Ala Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 450 455 460 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 465 470 475 480 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 485 490 495 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 500 505 510 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 515 520 525 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 530 535 540 Leu Tyr 545 <210> 49 <211> 1638 <212> DNA <213> Artificial Sequence <220> <223> CAR <400> 49 atggccctgc ctgtgacagc cctgctgctg cccctggctc tcctgctgca tgccgccaga 60 cccgctagcg acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac 120 agagtgacca tcagctgccg ggccagccag gacatcagca agtacctgaa ctggtatcag 180 cagaaacccg acggcaccgt gaagctgctg atctaccaca ccagccggct ccacagcggc 240 gtgcccagca gattttctgg cagcggcagc ggcaccgact acagcctgac catctccaac 300 ctggaacagg aagatatcgc tacctacttc tgtcagcaag gcaacaccct gccctacacc 360 ttcggcggag gcaccaagct ggaaatcacc ggcggaggcg gaagtggagg tggaggatct 420 ggcggcggag gctccgaagt gaagctgcag gaaagcggcc ctggcctcgt ggcccctagc 480 cagagcctgt ccgtgacctg taccgtgtcc ggcgtgtccc tgcccgacta cggcgtgtcc 540 tggatcagac agcctcccag aaagggcctg gaatggctgg gcgtgatctg gggcagcgag 600 acaacctact acaacagcgc cctgaagtcc cggctgacca tcatcaagga caacagcaag 660 agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc 720 gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 780 gtgaccgtgt ccagccatat ggccctgagc aacagcatca tgtacttcag ccacttcgtg 840 cccgtgtttc tgcccgccaa gcccaccacc acccctgccc ctagacctcc caccccagcc 900 ccaacaatcg ccagccagcc tctgtccctg cggcccgaag cctgtagacc tgctgccggc 960 ggagccgtgc acaccagagg cctggatatc tacatctggg cccctctggc cggcacctgt 1020 ggcgtgctgc tgctgagcct ggtgatcaca aagcggggca gaagaagct gctgtacatc 1080 ttcaagcagc ccttcatgcg gcccgtgcag accacccagg aagaggacgg ctgcagctgc 1140 cggttccccg aggagga aggcggctgc gaactgccca agctgtgcta cctgctggac 1200 ggcatcctgt tcatctacgg cgtgatcctg accgccctgt tcctgagagt gaagttcagc 1260 agaagcgccg acgcccctgc ctaccagcag ggccagaacc agctctacaa cgagctgaac 1320 ctgggcagac gggaagagga cgacgtgctggagaagggagaggccggga ccctgagatg 1380 ggcggcaagc cccagcggcg gaagacccc caggaaggcc tgtataacga actgcagaaa 1440 gacaagatgg ccgaggccta cagcgagatc ggcatgaagg gcgagcggcg gagaggcaag 1500 ggccacgatg gcctgtacac cggtatggac gagaaaacca ccggctggcg gggaggccac 1560 gtggtggaag gactggccgg cgagctggaa cagctgcggg ccagactgga acaccacccc 1620 cagggccaga gggaaccc 1638 <210> 50 <211> 546 <212> PRT <213> Artificial Sequence <220> <223> CAR <400> 50 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ala Ser Asp Ile Gln Met Thr Gln Thr Thr Ser 20 25 30 Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala 35 40 45 Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp 50 55 60 Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu 85 90 95 Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln 100 105 110 Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 115 120 125 Ile Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser 145 150 155 160 Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp 165 170 175 Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp 180 185 190 Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu 195 200 205 Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe 210 215 220 Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys 225 230 235 240 Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly 245 250 255 Gln Gly Thr Ser Val Thr Val Ser Ser His Met Ala Leu Ser Asn Ser 260 265 270 Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu Pro Ala Lys Pro 275 280 285 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 290 295 300 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 305 310 315 320 Gly Ala Val His Thr Arg Gly Leu Asp Ile Tyr Ile Trp Ala Pro Leu 325 330 335 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Lys Arg 340 345 350 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 355 360 365 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 370 375 380 Glu Glu Glu Gly Gly Cys Glu Leu Pro Lys Leu Cys Tyr Leu Leu Asp 385 390 395 400 Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala Leu Phe Leu Arg 405 410 415 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 420 425 430 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 435 440 445 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 450 455 460 Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 465 470 475 480 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 485 490 495 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Thr Gly Met Asp Glu Lys 500 505 510 Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly Leu Ala Gly Glu 515 520 525 Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro Gln Gly Gln Arg 530 535 540 Glu Pro 545 <210> 51 <211> 506 <212> PRT <213> Artificial Sequence <220> <223> CAR <400> 51 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ala Ser Asp Ile Gln Met Thr Gln Thr Thr Ser 20 25 30 Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala 35 40 45 Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp 50 55 60 Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu 85 90 95 Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln 100 105 110 Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 115 120 125 Ile Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser 145 150 155 160 Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp 165 170 175 Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp 180 185 190 Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu 195 200 205 Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe 210 215 220 Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys 225 230 235 240 Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly 245 250 255 Gln Gly Thr Ser Val Thr Val Ser Ser His Met Ala Leu Ser Asn Ser 260 265 270 Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu Pro Ala Lys Pro 275 280 285 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 290 295 300 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 305 310 315 320 Gly Ala Val His Thr Arg Gly Leu Asp Ile Tyr Ile Trp Ala Pro Leu 325 330 335 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Lys Arg 340 345 350 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 355 360 365 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 370 375 380 Glu Glu Glu Gly Gly Cys Glu Leu Pro Lys Leu Cys Tyr Leu Leu Asp 385 390 395 400 Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala Leu Phe Leu Arg 405 410 415 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 420 425 430 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 435 440 445 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 450 455 460 Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 465 470 475 480 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 485 490 495 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 500 505 <210> 52 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 52 aaccattagg agtagcaccc accaagg 27

Claims

1. An isolated cell comprising a nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: - Binding domain; - transmembrane domain; - a HOOK-binding domain comprising a streptavidin-binding peptide; and - an activation domain comprising: - a T cell activating fragment of at least 100 amino acids of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34, or - a T cell activation fragment of at least 90 consecutive amino acids of SEQ ID NO: 6, The HOOK-binding domain is located in the intracytoplasmic region of the chimeric antigen receptor, The HOOK-binding domain is encoded by the nucleic acid sequence SEQ ID NO:

2.

2. An isolated cell comprising a nucleic acid vector encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: - Binding domain; - transmembrane domain; - a HOOK-binding domain comprising a streptavidin-binding peptide; and - an activation domain comprising: - a T cell activating fragment of at least 100 amino acids of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34, or - a T cell activation fragment of at least 90 consecutive amino acids of SEQ ID NO: 6, The HOOK-binding domain is located in the intracytoplasmic region of the chimeric antigen receptor, The HOOK-binding domain is encoded by the nucleic acid sequence SEQ ID NO:

2.

3. An isolated cell comprising a lentiviral vector encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: - Binding domain; - transmembrane domain; - a HOOK-binding domain comprising a streptavidin-binding peptide; and - an activation domain comprising: - a T cell activating fragment of at least 100 amino acids of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34, or - a T cell activation fragment of at least 90 consecutive amino acids of SEQ ID NO: 6, The HOOK-binding domain is located in the intracytoplasmic region of the chimeric antigen receptor, The HOOK-binding domain is encoded by the nucleic acid sequence SEQ ID NO:

2.

4. An isolated cell comprising a vector expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: - Binding domain; - transmembrane domain; - a HOOK-binding domain comprising a streptavidin-binding peptide; and - an activation domain comprising: - a T cell activating fragment of at least 100 amino acids of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34, or - a T cell activation fragment of at least 90 consecutive amino acids of SEQ ID NO: 6, The HOOK-binding domain is located in the intracytoplasmic region of the chimeric antigen receptor, The HOOK-binding domain is encoded by the nucleic acid sequence SEQ ID NO:

2.

5. An isolated cell comprising a lentiviral vector particle expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: - Binding domain; - transmembrane domain; - a HOOK-binding domain comprising a streptavidin-binding peptide; and - an activation domain comprising: - a T cell activating fragment of at least 100 amino acids of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34, or - a T cell activation fragment of at least 90 consecutive amino acids of SEQ ID NO: 6, The HOOK-binding domain is located in the intracytoplasmic region of the chimeric antigen receptor, The HOOK-binding domain is encoded by the nucleic acid sequence SEQ ID NO:

2.

6. The isolated cell of any one of claims 1-5, wherein the isolated cell is an immune cell.

7. The isolated cell of any one of claims 1-5, wherein the isolated cell is (i) T cells, or (ii) NK cells.

8. The isolated cell of claim 7, wherein the T cell is a primary T cell.

9. The isolated cell of any one of claims 1-5, wherein the isolated cell is a mammalian cell.

10. The isolated cell of any one of claims 1-5, wherein the isolated cell is a human cell.

11. The isolated cell of any one of claims 1-5, wherein the binding domain comprises a single-chain Fv antibody or a nanobody.

12. The isolated cell of any one of claims 1-5, wherein the HOOK comprises the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:

32.

13. The isolated cell of any one of claims 2-5, wherein the vector, the lentiviral vector or the lentiviral vector particle comprises a β2-microglobulin, ubiquitin, MHC I or MHC II promoter.

14. The isolated cell of any one of claims 1-5, wherein the chimeric antigen receptor comprises the amino acid sequence of any one of SEQ ID NO:46, SEQ ID NO:48, or SEQ ID NO:

50.

15. Use of the isolated cell according to any one of claims 1 to 5 in the preparation of a medicament for inducing an immune response in a human, wherein the isolated cell is an immune cell.

16. Use of the isolated cells according to any one of claims 1 to 5 in the preparation of a medicament for treating cancer and tumors, wherein the isolated cells are immune cells.

17. Use of the isolated cell according to any one of claims 1 to 5 in the preparation of a medicament for treating an autoimmune disease, wherein the isolated cell is an immune cell.

18. The use of claim 15, wherein the isolated cells are administered to a human intramuscularly, intravenously, intraarticularly or intratumorally.

19. The use of claim 16, wherein the isolated cells are administered to a human intramuscularly, intravenously, intraarticularly or intratumorally.

20. The use of claim 17, wherein the isolated cells are administered to a human intramuscularly, intravenously, intraarticularly or intratumorally.

21. The use of claim 18, 19 or 20, wherein biotin is administered to the human after administration of the cells.

22. A non-therapeutic method for expressing a chimeric antigen receptor in a cell, comprising: Under conditions that allow expression of the chimeric antigen receptor, Expressing a nucleic acid molecule encoding a chimeric antigen receptor, or a vector expressing a chimeric antigen receptor, in a cell, or cells were transduced with lentiviral vector particles expressing chimeric antigen receptors; The chimeric antigen receptor comprises: - Binding domain; - transmembrane domain; - a HOOK-binding domain comprising a streptavidin-binding peptide; and - an activation domain comprising: - a T cell activating fragment of at least 100 amino acids of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34, or - a T cell activation fragment of at least 90 consecutive amino acids of SEQ ID NO: 6, The HOOK-binding domain is located in the intracytoplasmic region of the chimeric antigen receptor, The HOOK-binding domain is encoded by the nucleic acid sequence SEQ ID NO:

2.

23. The method of claim 22, wherein the vector comprises a nucleic acid vector.

24. The method of claim 22, wherein the vector comprises a lentiviral vector.

25. The method of claim 22, further comprising harvesting or isolating the chimeric antigen receptor.

26. The method of any one of claims 22-25, wherein the cells are treated with biotin.

27. The method of claim 26, wherein the cells are treated with biotin at an initial concentration of at least 0.2, 0.4, 0.8, 1.6, 2.5, 5, 10, 20, 40 or 80 μM.

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