Intein-based sorting system and modular chimeric polypeptides

Abstract

The present application relates to intein-based multiplex methods and compositions for generating engineered cells expressing modular polypeptides, such as CAR and CCR.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 381,921, filed on November 1, 2022, the entire contents of which are incorporated herein by reference. 2. Background Technology

[0003] Cell-based immunotherapy has been shown to have the potential to treat a variety of cancers. In many methods of this type of method, T cells and / or other immune cells are modified to target specific antigens via expressing polypeptides (e.g., chimeric antigen receptors (CARs) and chimeric costimulatory receptors (CCRs)) that are specific to the target antigen. For example, the targeted T cell therapy using the CAR for cancer-associated antigen CD19 has been shown to be clinically successful in treating hematological malignancies. However, in order to ensure that cancer is effectively eradicated with extremely low toxicity, cell-based immunotherapy may need to target multiple antigens. Targeting multiple antigens generally requires that large recombinant constructs be delivered and incorporated into appropriate T cells and / or other immune cells. However, it is difficult to stably incorporate a large amount of genetic information into primary T cells with the required efficiency. For example, when the viral vector insert approaches and exceeds the viral packaging limit (retrovirus is about 6kb to 8kb, and slow virus is about 10kb to 12kb), both retrovirus and slow virus show a significantly decreased viral titer. Low viral titers result in low transduction efficiency and low copy number incorporation per cell, resulting in low expression levels of gene constructs. Therefore, there is a need to develop novel genetic engineering strategies to express the desired polypeptides and therapeutic strategies that can induce effective cancer eradication with minimal toxicity and off-target activity. 3. Summary of the Invention

[0004] In certain embodiments, the present disclosure relates to cells, systems, and methods related to the production of modular polypeptides (e.g., chimeric antigen receptors (CARs) and other cell surface transmembrane polypeptides), wherein orthogonal split intein tags are used to perform post-translational trans-splicing of polypeptide subunits to generate functional mature CARs or other transmembrane constructs. In certain embodiments, trans-splicing peptide subunits include, but are not limited to, antigen binding domains (which may include one or more affinity tags); surface-presented membrane-bound cytokines and chemokines; spacers and transmembrane domains (which may include one or more affinity tags); positive and / or negative co-stimulatory molecule extracellular domains; intracellular signaling domains; and intracellular non-signaling domains. In certain embodiments, antigen binding and signaling intein-tagged subunits can be multiplexed to produce a mixed population of mature trans-splicing inteins inside a single cell. In certain embodiments, the antigen binding domain polypeptide interacts with the spacer-transmembrane domain polypeptide.

[0005] In certain embodiments, the present disclosure relates to a modular CAR system of affinity tags or a system containing a polypeptide of an extracellular and transmembrane domain, which utilizes orthogonal intein and endoplasmic reticulum (ER) retention motif to constitute a cell sorting system. In certain embodiments, the ER retention motif retains the construct containing the transmembrane domain in a non-spliced ​​state within the cell. In certain embodiments, the ER retention motif is excised during trans-splicing with a homologous orthogonal intein pair, thereby enabling mature spliced ​​intein-tagged transmembrane polypeptides to be transported to the cell surface. In certain embodiments, the ER retention motif can be attached to a plurality of different intracellular polypeptides, and by utilizing orthogonal inteins, multiple rounds of trans-splicing and ER retention motif cutting can be engineered to retain trans-splicing transmembrane polypeptides in the ER or transport them to the cell membrane. In certain embodiments, multiple vectors are used to perform multiple genetic modifications on cells, wherein each vector encodes a single subunit of a modular intein-tagged polypeptide, which, when combined together, reconstructs the mature affinity-tagged transmembrane polypeptides expressed on the cell surface. In certain embodiments, magnetic bead-based sorting (MACS) targeting specific affinity tags can selectively enrich for cells that have correctly trans-spliced ​​mature affinity-tagged polypeptide products, thereby selectively isolating cells that have incorporated at least one copy of each vector encoding an intein-tagged polypeptide subunit (molecular coincidence detector). For example, this method can achieve selective sorting of cells that have incorporated at least four unique vectors. In certain embodiments, additional transgenes can be incorporated into each vector to enhance the ability to transfer genetic information into the cell. In addition, in certain embodiments, trans-splicing of degenerate modular peptides can generate a greater number of mature trans-spliced ​​polypeptides from a given set of available precursor polypeptides.

[0006] In certain embodiments, the present disclosure relates to a system for producing affinity-tagged modular CARs using a polypeptide degradation system of a drug-regulated intracellular intein-tagged polypeptide. In certain embodiments, an unstable degradation determinant is attached to an intracellular intein-tagged polypeptide so that the intracellular intein undergoes selective drug-regulated degradation, thereby performing drug-regulated trans-splicing with an intein-tagged transmembrane polypeptide. In certain embodiments, the transmembrane polypeptide of the intein tag contains an ER retention motif, and when the intracellular intein-tagged polypeptide containing a degradation determinant is degraded, the polypeptide remains in the cell. In certain embodiments, when there is a small molecule, the transmembrane polypeptide of the intein tag is trans-splicing with the intracellular intein-tagged polypeptide containing a degradation determinant, which promotes the increase in the concentration of the intracellular intein-tagged polypeptide, the ER retention motif cutting, and the cell surface transport (drug system) of the mature trans-splicing polypeptide. In certain embodiments, in the absence of a small molecule, an intein-tagged transmembrane polypeptide undergoes trans-splicing with an intein-tagged intracellular polypeptide containing a degron, which promotes elevated intracellular intein-tagged polypeptide concentrations, cleavage of the ER retention motif, and cell surface trafficking of the mature trans-spliced ​​polypeptide (drug withdrawal system). In certain embodiments, multiplexing of orthogonal drug destabilization motifs can enable selective splicing of multiple different intein-tagged intracellular domain polypeptides by inducing selective orthogonal degradation or stabilization. In certain embodiments, MACS sorting can selectively enrich for cells that have correctly spliced ​​mature, surface-expressed, affinity-tagged trans-splicing products, thereby selectively isolating cells that have incorporated at least one copy of each vector encoding an intein-tagged polypeptide subunit.

[0007] In certain embodiments, the present disclosure relates to a system for producing modular CARs of affinity tags using low-affinity orthogonal inteins, which require small molecules to regulate the dimerization of additional heterodimerization polypeptide substituents. In certain embodiments, the polypeptide subunit dimerization of the intein tag induced by small molecules promotes trans-splicing to form a mature polypeptide. In certain embodiments of this system, trans-splicing causes the cleavage of the ER retention motif, thereby causing the mature trans-splicing polypeptide to migrate from the ER to the cell surface. In certain embodiments of this system, the receptor does not contain the ER retention motif and is not dependent on trans-splicing for transport to the cell surface. In certain embodiments, the orthogonal principle of intein trans-splicing can be used to generate a combination of the above embodiments to implement the reorganization of specific molecules. For example, a specific antigen binding domain can be spliced ​​with an intracellular domain intein containing CD3z, while other antigen binding domains are spliced ​​with costimulatory domain intein (e.g., 4-1BB and CD28). Additionally, subsets of orthogonal intein and polypeptide subunits can be arranged into sorting systems, while other orthogonal inteins can be used to achieve small-molecule-induced splicing of CAR subunits.

[0008] In certain non-limiting embodiments, the subject matter disclosed herein relates to methods for modifying cells, comprising delivering the disclosed system to the cells. In certain of these methods, the cells are mammalian cells. In certain of these methods, the mammalian cells are immune cells. In certain of these methods, the immune cells are T cells.

[0009] In certain non-limiting embodiments, the subject matter disclosed herein relates to methods for enriching a modified cell population, wherein the enrichment comprises: modifying the cell population as disclosed herein; culturing the cell population; and enriching the modified cell population by selecting for surface expression of an extein. In certain of these methods, the cells are mammalian cells. In certain of these methods, the mammalian cells are immune cells. In certain of these methods, the immune cells are T cells.

[0010] In certain non-limiting embodiments, the subject matter disclosed herein relates to methods for treating a disease, comprising providing a modified cell population comprising a system as described herein, or cells modified according to the modification methods disclosed herein, or an enriched cell population enriched according to the methods disclosed herein to a subject in need thereof. In certain embodiments of such embodiments, the subject is a human subject. In certain embodiments of such embodiments, the disease is cancer, an autoimmune disease, an inflammatory disease, or a graft-versus-host disease. In certain embodiments of such embodiments, the cancer is leukemia, lymphoma, myeloma, ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, testicular cancer, anal cancer, skin cancer, gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, or soft tissue sarcoma. In certain embodiments of such embodiments, the leukemia is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, or B-cell prolymphocytic leukemia. In certain embodiments of such embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. In certain embodiments of such embodiments, the non-Hodgkin's lymphoma is B-cell non-Hodgkin's lymphoma or T-cell non-Hodgkin's lymphoma. In certain embodiments of such embodiments, the cancer comprises cells expressing CD19 or CD20. In certain of such embodiments, the cancer comprises cells expressing at least one antigen selected from the group consisting of: CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, an antigen of cytomegalovirus (CMV) infected cells, ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, C CDC155, CCR1, CD10, CD117, CD123, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD30, CD300LF, CD32, CD321, CD33, C D34, CD36, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3,CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, COLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-a, FOLR2, FRMD5, GABRB2, GAS2, GD 2, GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B, kappa-light chain, L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MY ADML2, NGFR, NKCS1, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, PRAME, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), peptidase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine peptide kinase Erb-B2,RHBDL3, RNF173, RNF183, ROR1, RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5 , SLC25A36, SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STO N2, SUN3, survivin, SUSD2, SYNC, TACSTD2, TAS1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycopolypeptide 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms' tumor polypeptide (WT-1), WNT4, WT1, and ZDHHC11. 4. Description of the Figures

[0011] Figures 1A to 1D An exemplary split-intein-mediated trans-splicing reaction is depicted. Figure 1A An exemplary intein trans-splicing reaction is shown, wherein a first polypeptide comprises a split intein fused to the N-terminus of an intein, and a second polypeptide comprises a complementary split intein fused to the C-terminus of the intein. Following association of the split intein, trans-splicing of the first and second polypeptides (extein) is catalyzed, resulting in the covalent attachment of the first and second polypeptides via a peptide bond and the release of the non-covalently associated intein. Figure 1B The degeneracy of intein-based trans-splicing reactions is shown, which can generate a combination of mature trans-spliced ​​polypeptides from a given set of available precursor polypeptides. Figure 1C Exemplary illustrations of intein degeneracy are provided using anti-CD19 scFv or anti-CD20 scFv fused to the N-terminus of a split intein and the costimulatory molecules CD28z or 4-1BB fused to the C-terminus of a complementary split intein. Figure 1D A comparison of the coding required to transduce an exogenous gene by conventional gene expression and intein-based gene expression (e.g., CAR) is shown. The degeneracy of intein-based trans-splicing reactions enables the expression of large constructs with higher efficiency than when using conventional methods and enables the encoding of a greater number of receptors using the same amount of DNA, providing a clear advantage.

[0012] Figures 2A to 2B An exemplary two-vector intein sorting system is depicted. Figure 2A As shown, in this exemplary method, vector 1 encodes a secreted affinity-tagged molecule fused to an intein N-terminal domain. Vector 2 encodes a C-terminal intein capture domain fused to a spacer, a transmembrane domain, a linker, and a non-signaling cytoplasmic domain. Trans-splicing of the two intein-tagged polypeptides results in cell-selective surface display of the affinity tag on cells transduced with the dual vectors, due to selective intracellular splicing. This enables selective MACS sorting based on the affinity tag for cells that have incorporated both vectors and completed the trans-splicing reaction. Each vector can carry additional transgenes to enhance the functionality of the engineered cells. Figure 2B Depicted are the results of exemplary experiments using a dual-vector intein sorting system utilizing a secreted affinity tag with an intein tag and a transmembrane intein capture handle.

[0013] Figures 3A to 3I An exemplary method for sorting and post-translational CAR assembly based on endoplasmic reticulum retention of dual-carrier inteins is depicted. Figure 3A It is shown that in this exemplary method, vector 1 encodes an intein-tagged antigen-binding construct consisting of an scFv fused to an affinity tag, a spacer, a transmembrane domain, an intein N-terminus, and an endoplasmic reticulum (ER) retention motif; and vector 2 encodes a cytoplasmic signaling domain shuttle construct consisting of an intein C-terminus fused to a costimulatory domain and a T cell receptor signaling domain (e.g., CD3ζ). Trans-splicing of two intein-tagged polypeptides results in an intracellular splicing junction and recapitulates a fully functional chimeric antigen receptor (CAR). Due to ER retention, the surface expression of the affinity-tagged antigen-binding domain is significantly reduced until the two inteins undergo trans-splicing, which removes the ER retention motif and results in cell surface transport. This enables selective MACS sorting based on affinity tags for cells that have incorporated two vectors and completed the trans-splicing reaction. Each vector can carry additional transgenes to enhance engineered cell functionality. Figure 3B Depicted is an exemplary two-vector intein modular CAR sorting system in which an extracellular scFv with a spacer is fused to a transmembrane intein capture handle with an inwardly rectifier potassium (Kir6.2) RXR ER retention motif. Figure 3C Depicted is an exemplary two-vector intein modular CAR sorting system in which an extracellular scFv with a spacer is fused to a transmembrane intein capture handle with an E319K ER retention motif. Figure 3D Shown is the efficacy of CD19-CAR T cells in killing CD19-expressing BM185 cells. Figure 3E An exemplary three-vector based intein retention is shown, using either the RXR or E319K (KKXX) retention motif. Figures 3F to 3I demonstrated the ability of the RXR-type ER retention motif to function within the interior of a polypeptide chain.

[0014] Figures 4A to 4G . Figure 4A An exemplary dual-vector intein sorting system is depicted for generating cells expressing modular dual CARs with dual antigen specificity. In this exemplary method, vector 1 encodes a dual-affinity-tagged intein scFv in which each scFv is fused to an affinity tag and the N-terminus of a degenerate intein; and vector 2 encodes a transmembrane signaling domain shuttle consisting of an intein C-terminus fused to a spacer domain, a transmembrane domain, a co-stimulatory domain, and a T cell receptor signaling domain. After capture and splicing of the intein domains, both antigen-binding domains are displayed on the cell surface, reproducing the full-length CAR molecule. Due to selective intracellular splicing, trans-splicing of the two intein-tagged polypeptides results in cell-selective surface display of the affinity tag in cells transduced with the dual vectors. This enables selective MACS sorting based on the affinity tag for cells that have incorporated both vectors and completed the trans-splicing reaction. Each vector can carry additional transgenes to enhance the functionality of the engineered cells. Figure 4B Depicted are the results of MACS sorting enrichment of an exemplary modular scFv intein-CAR with dual antigen specificity, which possesses a dual secretory intein-tagged affinity-tagged scFv and a transmembrane intein capture handle fused to a cytoplasmic signaling domain. Figure 4C The results show that dual-targeting CD19 / CD20-specific intein CAR-T cells can kill BM185-CD19 target cells. Figure 4D The results show that dual-targeting CD19 / CD20 specific intein CAR-T cells can kill BM185-CD20 target cells. Figure 4E Another exemplary dual-vector dual-CAR intein sorting system is depicted, wherein Figure 3A The depicted cytoplasmic signaling domain shuttle construct utilizes the zeta chain-associated protein kinase 70 (Zap70) signaling domain and a tracer protein (e.g., EGFP). Figure 4F Describes the use Figure 4E Results of MACS sorting enrichment of transduced cells generated with the exemplary constructs. Figure 4G The results show that the dual-targeting CD19 / CD20-specific Zap70 intein CAR-T cells can kill C1498 target cells expressing CD19 or CD20.

[0015] 5A to 5DDepicts an exemplary three-vector intein-based ER retention sorting system and post-translational CAR assembly using secreted intein-tagged scFv. Figure 5A As shown, in this exemplary method, vector 1 encodes a dual antigen-binding scFv, each fused to an affinity tag and an orthogonal intein N-terminus; vector 2 encodes a dual intein transmembrane adaptor composed of an orthogonal intein C-terminus fused to the extracellular and transmembrane domains, the orthogonal intein N-terminus, and an ER retention motif; and vector 3 encodes a cytoplasmic signaling domain shuttle composed of an orthogonal intein C-terminus fused to a costimulatory domain and a T-cell receptor signaling domain. The intein-tagged scFv molecule undergoes trans-splicing with the dual intein transmembrane adaptor and is expelled from the ER after trans-splicing of the adaptor with the orthogonal intein of the cytoplasmic shuttle construct, thereby cleaving the ER retention motif and regenerating the full-length CAR. This enables selective MACS sorting of cells incorporating the three vectors based on the affinity tags after completing two orthogonal trans-splicing reactions. Each vector can carry additional transgenes to enhance the functionality of the engineered cells. Figure 5B Depicted are the results of MACS enrichment of triple-vector transduced cells. Figure 5C The results show that dual-targeting CD19 / CD20-specific intein CAR-T cells can kill BM185-CD19 target cells. Figure 5D The results showed that dual-targeting CD19 / CD20-specific intein CAR-T cells could kill BM185-CD19 target cells.

[0016] 6A to 6D Depicted are an exemplary three-vector hybrid leucine zipper-intein ER-based retention sorting system and post-translational CAR assembly. Figure 6A As shown, in this exemplary method, vector 1 encodes a dual antigen-binding scFv, each fused to an affinity tag and a heterodimeric leucine zipper; vector 2 encodes a leucine zipper-intein transmembrane adapter composed of a heterodimeric leucine zipper fused to a short hinge domain, a transmembrane domain, an intein N-terminus, and an ER retention motif; and vector 3 encodes a cytoplasmic signaling domain shuttle composed of an intein C-terminus fused to a costimulatory domain and a T cell receptor signaling domain. The leucine zipper-tagged scFv molecules bind to the capture zipper-intein transmembrane adapter in the ER and can be expelled from the ER after trans-splicing of the intein of the adapter and the cytoplasmic shuttle construct, thereby cleaving the ER retention motif and reproducing the full-length CAR. This enables selective MACS sorting of cells incorporating the three vectors based on affinity tags after completion of the zipper heterodimerization and intein trans-splicing reactions. Each vector can carry additional transgenes to enhance the functionality of the engineered cells. Figure 6B Show use Figure 6AResults of MACS sorting enrichment of transduced cells generated with the exemplary constructs. Figure 6C and Figure 6D Show the use of Figure 6A The efficacy of dual-targeting CD19 / CD20-specific Zap70 intein CAR-T cells generated by the method shown is shown against BM185-CD19 and BM185-CD20 target cells.

[0017] Figure 7 An exemplary three-vector intein-based ER retention sorting system and post-translational CAR assembly using intracellular intein adapters are depicted. In this exemplary method, vector 1 encodes an intein-tagged antigen-binding construct composed of an scFv fused to an affinity tag, a spacer, a transmembrane domain, an orthogonal intein N-terminus, and an ER retention motif; vector 2 encodes a dual intein cytoplasmic adapter composed of an orthogonal intein C-terminus fused to a signaling domain, an orthogonal intein N-terminus, and an ER retention motif; and vector 3 encodes a cytoplasmic signaling domain shuttle composed of an orthogonal intein C-terminus fused to a costimulatory domain and / or a T cell receptor signaling domain. The orthogonal intein-tagged molecule undergoes trans-splicing, as shown, with consecutive cleavage of two ER retention motifs to reconstitute the full-length CAR molecule. Removal of the ER retention motif enables selective trafficking of the CAR molecule to the cell surface, and after completing two orthogonal trans-splicing reactions, cells incorporating the three vectors are selectively sorted by affinity tag-based MACS. Each vector can carry an additional transgene to enhance the functionality of the engineered cells.

[0018] Figures 8A to 8D Depicted is an exemplary four-carrier intein-based sorting system for ER retention and post-translational CAR assembly using a combination of transmembrane and intracellular intein adaptors. Figure 8AAs shown, in this exemplary method, vector 1 encodes a dual antigen-binding scFv fused to an affinity tag and an orthogonal intein N-terminus; vector 2 encodes a dual intein transmembrane adapter consisting of an orthogonal intein C-terminus fused to the extracellular and transmembrane domains, the orthogonal intein N-terminus, and an ER retention motif; vector 3 encodes a dual intein cytoplasmic adapter consisting of an orthogonal intein C-terminus fused to the signaling domain, the orthogonal intein N-terminus, and an ER retention motif; and vector 4 encodes a cytoplasmic signaling domain shuttle consisting of an orthogonal intein C-terminus fused to a costimulatory domain and / or a T cell receptor signaling domain. The orthogonal intein-tagged molecule undergoes trans-splicing, as shown in the figure, with consecutive cleavage of two ER retention motifs to reconstitute the full-length CAR molecule. Sequential removal of the ER retention motif enables selective trafficking of the CAR or sorting handle to the cell surface, and after completion of three orthogonal trans-splicing reactions, cells incorporating the four vectors are selectively sorted by affinity tag-based MACS. Each vector can carry an additional transgene to enhance the functionality of the engineered cells. Figure 8B Shown are exemplary experimental results using a four-vector intein sorting system with a FLAG tag, which enables MACS sorting enrichment of transduced cells. Figure 8C Shown are exemplary experimental results using a four-vector intein sorting system with a human CD34 tag, which enables MACS sorting enrichment of transduced cells. Figure 8D Shows the use of all 4 vectors (or omitting Figure 8A Characterization of transduced cells prepared with the vectors in 3).

[0019] Figure 9A Figure 9D depicts an exemplary dual-vector intein sorting system that employs a method based on drug-regulatable CAR expression. Figure 9AIt is shown that in this exemplary method, vector 1 encodes an intein-tagged antigen-binding construct, which is composed of an scFv fused to an affinity tag, a spacer, a transmembrane domain, a low-affinity intein N-terminus, a drug dimerization domain (e.g., FKBP12), and an ER retention motif; and vector 2 encodes a drug dimerization domain (e.g., FRB*) fused to a low-affinity intein C-terminus, a costimulatory domain, and a T cell receptor signaling domain (e.g., CD3ζ). Due to the enhanced polypeptide association mediated by the drug dimerization domain, the trans-splicing incidence of the two low-affinity intein-tagged polypeptides is highest in the presence of a dimerizing agent drug. Trans-splicing results in an intracellular splicing junction and reproduces a fully functional CAR. Due to ER retention, the surface expression of the affinity-tagged antigen-binding domain polypeptide is significantly reduced until the two inteins undergo trans-splicing, which removes the ER retention motif, resulting in cell surface trafficking. The system enables drug-dependent post-translational CAR assembly and selective MACS sorting of cells that have incorporated both vectors and completed trans-splicing reactions based on affinity tags. Each vector can carry additional transgenes to enhance the functionality of the engineered cells. Figure 9B Show use Figure 9A Shown are the results of an exemplary flow cytometry experiment performed using the drug-tunable dual-vector intein sorting system, characterizing hCD34 expression in the absence or presence of A / C heterodimer (drug). Figure 9C Shown in the presence of dimerizing drugs (e.g., AP21967), CAR-T cells (effector cells, E, using Figure 9A Selective killing of BM185-CD19 (target cells, T) by the method shown.

[0020] Figures 10A to 10L Depicted is an exemplary dual-vector intein sorting system using an approach based on drug-regulatable CAR expression. Figure 10AAs shown, in this exemplary method, vector 1 encodes a fusion polypeptide containing two different antigen-binding scFv transmembrane domains with a cytoplasmic low-affinity N-terminal intein domain. Each scFv fusion polypeptide contains a different affinity tag, spacer, transmembrane domain, low-affinity intein N-terminus, and drug-dimerizable domain (e.g., FKBP12), but only one fusion polypeptide contains an ER retention motif because the ER retention motif works most effectively at the end of the vector construct (C-terminus of the fusion polypeptide and 2A sequence). Vector 2 encodes a drug-regulated cytoplasmic signaling domain shuttle consisting of a drug-dimerizable domain (e.g., FRB*) fused to the C-terminus of the low-affinity intein, a co-stimulatory domain, and a T cell receptor signaling domain (e.g., CD3ζ). Due to the enhanced polypeptide association mediated by the drug-dimerizable domain, the trans-splicing rate of the two low-affinity intein-tagged polypeptides is highest in the presence of the dimerizing agent drug. Trans-splicing results in intracellular splicing junctions and regenerates fully functional CARs targeting their respective cognate antigens. Due to ER retention, surface expression of affinity-tagged antigen-binding domain polypeptides with ER retention motifs is significantly reduced until the two inteins undergo trans-splicing, which removes the ER retention motifs and leads to cell surface trafficking. This system enables drug-dependent post-translational dual-CAR assembly and affinity-tag-based selective MACS sorting of cells that have incorporated both vectors and completed the trans-splicing reaction. Each vector can carry additional transgenes to enhance the functionality of the engineered cells. Figure 10B Show use Figure 10A Results of exemplary flow cytometry experiments performed with a drug-tunable dual-vector intein sorting system are shown, demonstrating enhanced surface expression of hCD34-tagged CAR in the presence of A / C heterodimers or rapamycin, due to cleavage of the ER retention motif of the cytoplasmic signaling shuttle intein upon proximity to the antigen-binding handle molecule by either drug. Figure 10C Show use Figure 10A The efficacy of CAR-T cells (effector cells, E) generated by the shown method in killing CD19-expressing BM185 cells (target cells, T). Figure 10D Show use Figure 10A The demonstrated efficacy of the CAR-T cells generated by this method in killing CD20-expressing BM185 cells. Figures 10E to 10L Shown are live cell imaging of BM185-CD19 and BM185-CD20 tumor cell lines co-cultured with intein CAR T cells dually targeting CD19 / CD20 in the presence or absence of activating small molecules.

[0021] Figure 11AFigures 11 through 11D depict an exemplary dual-vector intein sorting system using a method based on drug-regulatable trans-presentation of cell surface cytokine expression. Figure 11A It is shown that in this exemplary method, vector 1 encodes a cytokine that is fused to an affinity tag, a spacer, a transmembrane domain, a low-affinity intein N-terminus, a drug-dimerizable domain (e.g., FKBP12), and an endoplasmic reticulum (ER) retention motif; vector 2 encodes a drug-dimerizable domain (e.g., FRB*) that is fused to a low-affinity intein C-terminus and a co-stimulatory domain or a non-signaling intracellular domain. Due to the enhanced polypeptide association mediated by the drug-dimerizable domain, the trans-splicing incidence of the two low-affinity intein-tagged polypeptides is highest in the presence of a dimerizing agent drug. Trans-splicing results in the excision of the ER retention motif. Due to ER retention, the surface expression of the affinity-tagged cytokine trans-presented polypeptide is significantly reduced until the two inteins undergo trans-splicing, which removes the retention motif and leads to cell surface trafficking. This system enables drug-dependent hierarchical expression of cell-surface trans-presented cytokines and selective MACS sorting of cells that have incorporated both vectors and completed trans-splicing reactions based on affinity tags. Each vector can carry additional transgenes to enhance the functionality of the engineered cells. Figure 11B Results are shown for constitutive and drug-regulated expression of IL-7 on the cell surface. Figure 11C Shown are the results of T cell expansion for constitutive and drug-regulated expression of IL-7.

[0022] Figure 12A Figure 12D depicts an exemplary dual-vector degenerate intein trans-splicing that enables dual CAR formation using a drug-stabilized cytoplasmic signaling domain shuttle expression-based approach. Figure 12AAs shown, in this exemplary method, vector 1 encodes a fusion polypeptide containing two different antigen-binding scFv transmembrane domains with a degenerate cytoplasmic N-terminal intein domain. Each scFv fusion polypeptide contains a different affinity tag, spacer, transmembrane domain, and cytoplasmic degenerate intein N-terminus, but only one fusion polypeptide contains an ER retention motif because the ER retention motif works most effectively at the end of the vector construct (C-terminus of the fusion polypeptide and 2A sequence). Vector 2 encodes a cytoplasmic signaling domain shuttle consisting of an intein C-terminus, a costimulatory domain, a T cell receptor signaling domain (e.g., CD3ζ), and a drug-stable degron domain (e.g., an E. coli DHFR destabilization domain). Due to ER retention, the surface expression of the affinity-tagged antigen-binding domain polypeptide with an ER retention motif is significantly reduced until trans-splicing occurs with the cytoplasmic signaling domain shuttle, which removes the retention motif and leads to cell surface trafficking. Maximal expression of the cytoplasmic signaling domain shuttle occurs in the presence of a stable drug, which promotes trans-splicing reactions. This system enables drug-dependent post-translational dual-CAR assembly, combined with affinity tag-based selective MACS sorting of cells that have incorporated both vectors and completed trans-splicing reactions. Each vector can carry additional transgenes to enhance the functionality of the engineered cells. Figure 12B Depicts the presence and absence of trimethoprim using Figure 12A Shown are the results of an exemplary experiment using the dual-vector intein modular CAR approach. Figure 12C Show use Figure 12A The efficacy of CAR-T cells generated by the shown method in killing CD19-expressing BM185 cells in the absence and presence of trimethoprim.

[0023] Figures 13A to 13G Depicted is an exemplary dual-vector degenerate intein trans-splicing that enables dual CAR formation using a drug-destabilized cytoplasmic signaling domain shuttle-based expression approach. Figure 13AAs shown, in this exemplary method, vector 1 encodes a fusion polypeptide containing two different antigen-binding scFv transmembrane domains with a degenerate cytoplasmic N-terminal intein domain. Each scFv fusion polypeptide contains a different affinity tag, spacer, transmembrane domain, and cytoplasmic degenerate intein N-terminus, but only one fusion polypeptide contains an ER retention motif because the ER retention motif works most effectively at the end of the vector construct (C-terminus of the fusion polypeptide and 2A sequence). Vector 2 encodes a cytoplasmic signaling domain shuttle consisting of an intein C-terminus, a costimulatory domain, a T cell receptor signaling domain (e.g., CD3ζ), and a drug-activated degradation determinant domain (e.g., SMASh tag). Due to ER retention, the surface expression of the affinity-tagged antigen-binding domain polypeptide with an ER retention motif is significantly reduced until trans-splicing occurs with the cytoplasmic signaling domain shuttle, which removes the retention motif and leads to cell surface trafficking. In the presence of an inhibitor drug, expression of the cytoplasmic signaling domain shuttle is reduced, which promotes proteasomal degradation, thereby inhibiting trans-splicing and reducing CAR formation. This system enables drug-dependent post-translational dual-CAR assembly, combined with affinity tag-based selective MACS sorting of cells that have incorporated both vectors and completed trans-splicing reactions. Each vector can carry additional transgenes to enhance the functionality of the engineered cells. Figure 13B Depicted are the characterization of transfected cells pre-cultured with the hCD34 affinity tag in the presence or absence of asunaprevir. Figure 13C Depicted are the characterization of transfected cells pre-incubated with the FLAG affinity tag in the presence or absence of asunaprevir. Figure 13D Shown is the efficacy of CAR-T cells pre-cultured in the presence or absence of asunaprevir in killing CD19-expressing BM185 cells. Figure 13E Depicted are the characterization of dual-CAR transfected cells pre-cultured with hCD34 or FLAG affinity tags in the presence or absence of asunaprevir. Figure 13F Shown is the efficacy of dual CAR-T cells pre-cultured in the presence or absence of asunaprevir in killing CD19-expressing BM185 cells. Figure 13G The efficacy of dual CAR-T cells pre-cultured in the absence or presence of asunaprevir in killing CD20-expressing BM185 cells was shown.

[0024] 14A to 14C Depicted is an exemplary dual-vector dual-CAR intein sorting system comprising a drug-tunable Zap70 domain shuttle. Figure 14AAs shown, in this exemplary method, vector 1 encodes a fusion polypeptide containing two different antigen-binding scFv transmembrane domains (e.g., anti-CD19, anti-CD20), the fusion polypeptide having a degenerate cytoplasmic N-terminal intein (I N ) domain. Each scFv fusion polypeptide contains a different affinity tag, spacer, transmembrane domain, and cytoplasmic degenerate intein N-terminus, but only one fusion polypeptide contains an ER retention motif because the ER retention motif works most effectively at the end of the vector construct (C-terminal to the fusion polypeptide and 2A sequence). Vector 2 encodes a drug-tunable Zap70 domain shuttle composed of nonstructural proteinase 3 (NS3) and flanking 4A / 4B and 5A / B HCV protease cleavage recognition sequences and the intein C-terminus (I C )constitute. Figure 14B A process is shown in which, in the absence of an HCV protease inhibitor (e.g., grazoprevir), the NS3 protease cleaves the intein from the NS3-Zap70 fusion protein, thereby preventing the generation of a signaling-competent Zap70-based intein CAR. Figure 14C Shown is the efficacy of T cells transfected with the dual CD19 / CD20 CAR Zap70 construct in killing BM185 cells expressing CD19 or CD20 in the presence of grazoprevir (GZP).

[0025] 15A to 15D An exemplary intein-assisted NS3-based drug-regulated CAR is depicted. Figure 15A Shown is an exemplary method in which drug-modulated dual Zap70 intein CAR T cells exhibited killing of both BM185-CD19 and BM185-CD20 tumor cell targets in the presence of grazoprevir (GZP). Figure 15B We show the efficacy of drug-regulated CD19-targeting NS3 CD28z intein CAR T cells, which harbor two flanking HCV protease cleavage sites (5A / 5B and 4A / 4B) or used alone, in selective, drug-inducible killing of BM185-CD19 tumor cell targets in the presence of grazoprevir (GZP), without target leakage killing in the non-drug-induced state. Figures 15C to 15D Live cell imaging microscopy results are shown, showing that in the presence of grazoprevir, drug-regulated dual-targeting CD19 / CD20 NS3 CD28z intein-CAR T cells selectively killed BM185-CD19 and BM185-CD20 without "leaky" killing of non-induced targets.

[0026] 16A to 16CDepicted are exemplary intein-assisted NS3-based drug-regulated CARs generated by a nested intein approach, in which nested inteins are used to sequentially excise the drug-regulated cleavage cassette. Figure 16A As shown, in this exemplary method, the low-affinity AES split intein is spliced ​​only after the high-affinity Cfa intein is brought into proximity via initial splicing and peptide fusion between the antigen-binding handle and the cytoplasmic signaling shuttle. This second splicing step generates a trans-spliced ​​mature CAR molecule without the inserted NS3 sequence, which could affect CAR signaling. Figure 16B Shown by non-nested ( Figure 15A ) and nested methods to kill target cells expressing high-density CD19 antigens. Figure 16C Shown by non-nested ( Figure 15A ) and nested approaches to kill target cells expressing low-density CD19 antigens.

[0027] 17A to 17H Depicted is an exemplary dual-vector degenerate intein trans-splicing to enable multiple post-translational CAR and chimeric costimulatory receptor (CCR) formation. Figure 17A It is explained that in this exemplary method, vector 1 encodes two different antigen-binding scFv transmembrane domain fusion polypeptides having a degenerate cytoplasmic N-terminal intein domain. Each scFv fusion polypeptide contains a different affinity tag, spacer, transmembrane domain, and cytoplasmic degenerate intein N-terminus, but only one fusion polypeptide contains an ER retention motif because the ER retention motif works most effectively at the end of the vector construct (C-terminus of the fusion polypeptide and 2A sequence). Vector 2 encodes two cytoplasmic signaling domain shuttles, each of which is composed of a degenerate intein C-terminus and a different costimulatory domain and / or T cell receptor signaling domain (e.g., CD3ζ). The combination of two degenerate intein-tagged antigen-binding polypeptides and trans-splicing of two cytoplasmic signaling domain shuttles can generate two CAR molecules and two CCR molecules, for a total of four mature trans-splicing polypeptides. Due to ER retention, surface expression of affinity-tagged antigen-binding domain polypeptides with ER retention motifs is significantly reduced until the two inteins undergo trans-splicing and the retention motif is removed, leading to cell surface trafficking. This system enables post-translational dual-CAR and dual-CCR assembly and, combined with affinity tag-based MACS selective sorting, sorts cells that have integrated both vectors and completed the trans-splicing reaction. Further modification of the cytoplasmic signaling domain shuttle vector to include orthogonal drug destabilization domains or selective degradation determinant domains can achieve selective drug-dependent regulation of each signaling domain. Each vector can carry additional transgenes to enhance the functionality of the engineered cells. Figure 17BShown are exemplary flow cytometric analyses of CD19 CAR- and CCR-transduced cells before and after MACS sorting. Figure 17C Shown are exemplary flow cytometric analyses of only CD19 CAR-transduced cells before and after MACS sorting. Figure 17D Results show the expansion of cells expressing CD19 CAR / CCR and cells expressing CD19 CAR alone after exposure to CD19 target cells. Figure 17E Show Figure 17F Results of flow cytometric analysis of an exemplary dual CAR (CD19 / CD20) + dual CCR (dual handle + CD28z + 4-1BB) are shown. Figure 17G Shown are the efficacy of dual CAR T cells in eliminating BM185-CD19 or BM185-CD20 cell targets at the indicated E:T ratios. Figure 17H Shown are the results of expansion studies of dual CAR T cells and dual CCR T cells after exposure to BM185-CD19 or BM185-CD20 target cells at the indicated E:T ratios.

[0028] 18A to 18C Depicted is an exemplary dual-vector dual-CAR T intein sorting system comprising a degenerate Zap70 domain shuttle and a 4-1BB domain shuttle sequence that can be combined into four possible combinations: CD19-Zap70-CAR, CD20-Zap70-CAR, CD19-CCR, and CD20-CCR. This configuration represents a data compression algorithm in which the repeated use of degenerately encoded DNA elements enables the post-translational assembly of a greater number of receptors than could be encoded with the same amount of DNA. Figure 18A As shown, in this exemplary method, vector 1 encodes a fusion polypeptide containing two different antigen-binding scFv transmembrane domains (e.g., anti-CD19, anti-CD20), the fusion polypeptide having a degenerate cytoplasmic N-terminal intein (I N ) domain. Each scFv fusion polypeptide contains a different affinity tag, spacer, transmembrane domain, and cytoplasmic degenerate intein N-terminus, but only one fusion polypeptide contains an ER retention motif because the ER retention motif works most effectively at the end of the vector construct. Vector 2 encodes each of the scFvs attached at its N-terminus to the C-terminus of the intein (I C )'s Zap70 domain shuttle and 4-1BB domain sequences. Figure 18B and Figure 18CWe demonstrated enhanced killing and T cell proliferation of Zap70+BB intein-CAR / CCR T cells targeting CD19 / CD20 against BM185-CD19 and BM185-CD20 targets compared to intein-CARs that only utilize the Zap70 cytoplasmic shuttle (forming CAR but not CCR). 5. Specific implementation methods

[0029] The present application relates to multiple intein-based methods and compositions for generating and sorting modular polypeptides (e.g., CARs and CCRs). For example, but not limitation, the present application relates to multiple intein pairs, wherein each intein in a pair of inteins is fused to an extein (e.g., a modular polypeptide sequence), and wherein the paired intein catalyzes trans polypeptide splicing ("trans-splicing"), thereby fusing the corresponding extein to promote the production of the desired mature modular polypeptide. In certain embodiments, orthogonal intein pairs can be used to achieve multiple trans-splicing. Since some intein pairs (e.g., split intein pairs) only undergo trans-splicing with their paired sequences, the multiplexing of such pairs enables different exteins (e.g., different antibody binding domains) to undergo trans-splicing with paired exteins (e.g., specific transmembrane domains), while unrelated split intein pairs simultaneously enable different extein pairs (e.g., intracellular signaling domains) to undergo trans-splicing. As described herein, exemplary potential trans-splicing exteins include: antigen binding domains (which may include affinity tags); surface-presented membrane-bound cytokines; spacers and transmembrane domains (which may include affinity tags); extracellular domains of positive and negative co-stimulatory molecules; intracellular signaling domains; and intracellular non-signaling domains.

[0030] In certain embodiments, the subject matter of the present disclosure includes methods and compositions related to cell sorting systems. For example, but not limited to, according to the cell sorting system of the present disclosure, modular polypeptides containing extracellular and transmembrane domains (e.g., CAR and CCR) of affinity tags may be included, which utilize orthogonal inteins and endoplasmic reticulum (ER) retention motifs. In certain embodiments, the ER retention motif retains the construct containing the transmembrane domain in a non-spliced ​​state within the cell. In certain embodiments, the ER retention motif can be excised during trans-splicing with a homologous orthogonal intein pair, thereby enabling mature spliced ​​intein-tagged transmembrane polypeptides to be transported to the cell surface. In certain embodiments, the ER retention motif can be attached to a plurality of different intracellular polypeptides, and by utilizing orthogonal inteins, multiple rounds of trans-splicing and ER retention motif cutting can be engineered to retain the trans-splicing transmembrane polypeptides in the ER or to be transported to the cell membrane. In certain embodiments, cells are multiplexed genetically modified using multiple vectors, where each vector encodes a single subunit of a modular intein-tagged polypeptide that, when combined, reconstitute a mature affinity-tagged transmembrane polypeptide expressed on the cell surface.

[0031] In certain embodiments, magnetic bead-based sorting (MACS) targeting specific affinity tags can selectively enrich for cells that have correctly trans-spliced ​​mature affinity-tagged polypeptide products, thereby selectively isolating cells that have incorporated at least one copy of each vector encoding an intein-tagged polypeptide subunit (molecular coincidence detector). In certain embodiments, this method can selectively sort cells that have incorporated at least four unique vectors. In certain embodiments, additional transgenes can be incorporated into each vector to enhance the ability to transfer genetic information into the cell. In certain embodiments, trans-splicing of degenerate modular peptides can generate a greater number of mature trans-spliced ​​polypeptides from a given set of available precursor polypeptides.

[0032] In certain embodiments, the subject matter of the present disclosure includes methods and compositions related to the system of polypeptides (e.g., CAR and CCR) containing intracellular and transmembrane domains for drug-regulated generation and sorting modularity. In certain embodiments, an unstable degradation determinant is attached to an intracellular intein-tagged polypeptide so that the intracellular intein undergoes selective drug-regulated degradation, thereby performing drug-regulated trans-splicing with the intein-tagged transmembrane polypeptide. In certain embodiments, the intein-tagged transmembrane polypeptide contains an ER retention motif, and when the intracellular intein-tagged polypeptide containing a degradation determinant is degraded, the polypeptide remains in the cell. In certain embodiments, when there is a small molecule, the intein-tagged transmembrane polypeptide and the intracellular intein-tagged polypeptide containing a degradation determinant undergo trans-splicing, which promotes the increase in the concentration of the intracellular intein-tagged polypeptide, the ER retention motif cutting, and the cell surface transport ("drug-up" system) of the mature trans-splicing polypeptide. In certain embodiments, in the absence of a small molecule, an intein-tagged transmembrane polypeptide undergoes trans-splicing with an intein-tagged intracellular polypeptide containing a degron, which promotes elevated intracellular intein-tagged polypeptide concentrations, cleavage of the ER retention motif, and cell surface trafficking of the mature trans-spliced ​​polypeptide (a "drug-off" system). In certain embodiments, multiplexing of orthogonal drug destabilization motifs can enable alternative splicing of multiple different intein-tagged intracellular domain polypeptides by inducing selective orthogonal degradation or stabilization. In certain embodiments, MACS sorting can selectively enrich for cells that have correctly spliced ​​mature, surface-expressed, affinity-tagged trans-splicing products, thereby selectively isolating cells that have incorporated at least one copy of each vector encoding an intein-tagged polypeptide subunit.

[0033] In certain embodiments, the subject matter of the present disclosure includes methods and compositions related to a system for generating affinity-tagged modular polypeptides, which utilize low-affinity orthogonal inteins and require small molecules to regulate the dimerization of additional heterodimerization polypeptide substituents. In certain embodiments, small molecule-induced intein-tagged polypeptide subunit dimerization promotes trans-splicing to form mature modular polypeptides. In certain embodiments, trans-splicing results in cleavage of the ER retention motif, thereby causing mature trans-splicing polypeptides to migrate from the ER to the cell surface. In certain embodiments, modular polypeptides (e.g., CAR and CCR) do not contain ER retention motifs and are not dependent on trans-splicing for transport to the cell surface.

[0034] In certain embodiments, the subject matter of the present disclosure includes methods and compositions related to a system comprising a combination of embodiments described herein, which uses the orthogonal principle of intein trans-splicing to implement the reorganization of a specific molecule. For example, but not limited to, a specific antigen binding domain can be spliced ​​with an intracellular domain intein (e.g., those containing CD3z), while other antigen binding domains are spliced ​​with costimulatory domain intein (e.g., 4-1BB and CD28). In certain embodiments, a subgroup of orthogonal intein and polypeptide subunits is arranged into a sorting system, and other orthogonal intein (e.g.) is used to achieve splicing of modular polypeptide (e.g., CAR or CCR) subunits induced by small molecules or other "drugs".

[0035] 5.1. Definitions

[0036] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The following references provide general definitions of many terms used in the present invention for those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition. 1994); The Cambridge Dictionary of Science and Technology (Walker edited, 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds), Springer Verlag (1991); and Hale and Marham, The Harper Collins Dictionary of Biology (1991). Unless otherwise indicated, the following terms as used herein have the following meanings assigned to them.

[0037] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more standard deviations, according to the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within a certain order of magnitude, preferably within 5-fold, more preferably within 2-fold, of a value.

[0038] As used herein, the term "intein" refers to any of a variety of autocatalytic polypeptide sequences that can be self-excised from a larger polypeptide sequence via polypeptide splicing. As used herein, "polypeptide splicing" refers to an autocatalytic process in which an intein is self-excised from a larger polypeptide and causes the intein flanking sequences ("exteins") to be connected via new peptide bonds. Inteins exist as bifunctional sequences, wherein the endonuclease domain is present within a splicing domain that allows cis polypeptide splicing, and as a "split intein" present in a separate polypeptide sequence, which can non-covalently self-associate to catalyze trans polypeptide splicing. Exemplary inteins are described in Pinto et al., Nature Comm., 11: 1529 (2020) and Stevens et al., J. Am. Chem. Soc., 138: 2162-2165 (2016), both of which are incorporated by reference in their entirety for all purposes.

[0039] As used herein, an "orthogonal system" is a network of components (e.g., polypeptides, RNA, DNA, and small molecules) that interact with each other to achieve a specific function without hindering or being hindered by the natural functions of the host cell. Thus, "orthogonal split intein tags" will interact with each other to promote trans-polypeptide splicing of the extein to which they are fused, without hindering or being hindered by the natural functions of the host cell. The orthogonality of inteins stems from the specificity of the split N-terminal and C-terminal polypeptide sequences, which exhibit high binding affinity and high specificity. Thus, unrelated inteins can be co-expressed in the same cell without containing high background levels of non-specific trans-splicing of unrelated inteins. This enables the co-expression of multiple intein-tagged peptide constructs in cells, using orthogonal inteins to specify the desired regions of different polypeptides that will splice with each other in a defined configuration. This orthogonal intein strategy allows the construction of complex, mature, trans-spliced ​​proteins in cells from a set of proteins containing functional domains. Non-limiting examples of these domains include: antigen binding domains, spacers, transmembrane domains, signaling domains, and cytokines.The orthogonality of inteins enables the assembly of intein-tagged subunits in engineered predefined configurations.

[0040] As used herein, "degeneracy" in intein-based intracellular polypeptide assembly refers to the reuse of a particular protein domain in multiple constructs generated within a cell. For example, but not limited to any limitation, an intracellular signaling domain tagged with a particular intein (e.g., Cfa C-terminus) can be trans-spliced ​​with multiple antigen binding domains / spacers / transmembrane domains / Cfa-N-terminal intein constructs. Similarly, multiple Cfa C-terminal intein-tagged signaling domains can also be trans-spliced ​​in parallel with these antigen binding domain constructs to obtain multiple mature and modular trans-spliced ​​CAR constructs.

[0041] As used herein, the term "molecular coincidence detector" refers to the beneficial characteristic of a system that enables selective enrichment of selected cells from a mixed cell population, wherein the selected cells express all proteins encoded by a specified set of vectors. The sorting system aspect of the intein system utilizes a combination of intein-conjugated molecules that are also tagged with affinity tags or endoplasmic reticulum (ER) retention motifs. In the unspliced ​​state, cells expressing the individual components of the intein sorting system (i.e., transduced with fewer vectors than required to complete a set of co-transduced vectors) will express minimal affinity tags on the cell surface. However, in cells co-transduced with the full set of intein sorting system vectors, trans-splicing removes all ER retention motifs from the trans-spliced ​​polypeptides, and the affinity tags are attached for display on the cell surface. Thus, sorting methods (such as, but not limited to, MACS sorting using anti-affinity tag plasmids) selectively purify only cells that were co-transduced with the desired set of vectors for surface expression. Thus, in this example, the system enables MACS beads to use affinity tags as "molecular coincidence detectors" to identify cells that have incorporated all vectors from a given vector set. Thus, the multi-vector sorting implementation of the intein sorting system enables the introduction of large amounts of DNA into cells, followed by the selective purification of cells that have incorporated all delivered vectors.

[0042] As used herein, the term "modular polypeptide" refers to any polypeptide consisting of subunits that are combined to reconstruct a mature polypeptide. Non-limiting examples of such modular polypeptides include polypeptides comprising an extracellular domain, an extracellular endodomain, and a transmembrane domain, and polypeptides comprising two or more of such domains (e.g., CAR and CCR).

[0043] As used herein, "linker" refers to a functional group (e.g., a chemical substance or a polypeptide) that covalently attaches two or more polypeptides or nucleic acids to link them to each other. In certain embodiments, a linker comprises one or more amino acids for coupling two polypeptides together (e.g., for coupling V H and V Ldomain, or for coupling two dimerization domains). Linkers can typically be rich in glycine for flexibility, and serine or threonine for solubility.

[0044] As used herein, the term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which, when associated with appropriate control elements, is capable of replication and can transfer gene sequences into cells. Thus, the term encompasses cloning and expression vectors, as well as viral vectors and plasmid vectors.

[0045] As used herein, the term "expression vector" refers to a recombinant nucleic acid sequence, e.g., a recombinant DNA molecule, containing a desired coding sequence operably linked to an appropriate nucleic acid sequence required for expression of the coding sequence in a particular host organism. The nucleic acid sequences necessary for expression in prokaryotes typically include a promoter, an operator (optional), and a ribosome binding site, and typically also contain other sequences. The nucleic acid sequences necessary for expression in eukaryotic cells may include, but are not limited to, promoters, enhancers, termination, and polyadenylation signals.

[0046] In certain embodiments, nucleic acid molecules useful in the subject matter disclosed herein include nucleic acid molecules encoding antibodies or antigen-binding fragments thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides that are "substantially homologous" or "substantially identical" to an endogenous sequence are typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule.

[0047] As used herein, the term "disease" refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasia or pathogen infection of a cell, tissue, or organ.

[0048] An "effective amount" (or "therapeutically effective amount") is an amount sufficient to achieve a beneficial or desired clinical outcome after treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount sufficient to alleviate, improve, stabilize, reverse or slow the progression of a disease (e.g., neoplasia) or otherwise reduce the pathological consequences of a disease (e.g., neoplasia). The dosage comprising an effective amount is typically determined by a physician on a case-by-case basis, and such determinations are within the skill of one of ordinary skill in the art. Several factors are generally considered when determining an appropriate dosage to achieve an effective amount. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the cells (e.g., engineered immune cells) being administered.

[0049] As used herein, the term "neoplasia" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration into or invasion of other tissues or organs. The growth of neoplasia is usually uncontrolled and progressive, and occurs under conditions that do not cause normal cell reproduction or cause normal cell reproduction to stop. Neoplasia can affect a variety of cell types, tissues or organs, including but not limited to organs or their tissues or cell types selected from the group consisting of the following: skin, bladder, colon, bone, brain, chest, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestines, kidneys, liver, lungs, lymph nodes, nervous tissue, ovaries, pleura, pancreas, prostate, skeletal muscle, spinal cord, spleen, stomach, testicles, thymus, thyroid, trachea, urogenital tract, ureters, urethra, uterus and vagina. Neoplasia includes cancers such as melanoma, sarcoma, carcinoma or plasmacytoma (malignant tumor of plasma cells).

[0050] As used herein, the term "immune response cell" refers to a cell that plays a role in an immune response and includes progenitors of such cells as well as progeny of such cells.

[0051] As used herein, the term "isolated cell" refers to a cell that is separated from the molecules and / or cellular components that naturally accompany the cell.

[0052] As used herein, the terms "isolated," "purified," or "biologically pure" refer to materials that are free to varying degrees from normally associated components found in their natural state. "Isolated" refers to a degree of separation from the original source or surrounding environment. "Purified" refers to a degree of separation greater than isolated. A "purified" or "biologically pure" polypeptide is sufficiently free of other materials such that any impurities do not substantially affect the biological properties of the polypeptide or cause other adverse consequences. That is, a nucleic acid or polypeptide of the subject matter disclosed herein is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that a nucleic acid or polypeptide produces essentially a single band in an electrophoretic gel. For polypeptides that can be modified (e.g., phosphorylated or glycosylated), different modifications can produce different isolated polypeptides, which can be purified separately.

[0053] As used herein, the term "secreted" refers to the release of a polypeptide outside the cell via the secretory pathway through the endoplasmic reticulum, the Golgi apparatus, and as a vesicle that transiently fuses at the plasma membrane of the cell.

[0054] As used herein, the term "treating" or "treatment" refers to a clinical intervention that attempts to alter the course of disease in the individual or cell being treated, and can be performed for prevention or during clinical pathology. The efficacy of treatment includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. By preventing the progression of a disease or condition, treatment can prevent the affected or diagnosed subject or a subject suspected of having a condition from worsening due to the condition, and treatment can also prevent the occurrence of the condition or symptoms of the condition in a subject at risk for or suspected of having the condition.

[0055] As used herein, the term "subject" refers to any animal (eg, mammal), including but not limited to humans, non-human primates, rodents, etc., that is to be the recipient of a particular treatment.

[0056] As used herein, the term "antibody" means not only complete antibody molecules, but also antibody molecule fragments that retain immunogen binding ability. Such fragments are also well known in the art and are typically used in vitro and in vivo. Therefore, as used herein, the term "antibody" means not only complete immunoglobulin molecules, but also well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments lacking the Fc fragment of a complete antibody are cleared from the circulation faster and may have less non-specific tissue binding of a complete antibody (Wahl et al., J.Nucl.Med.24:316-325 (1983)). The antibodies of the present invention include all-natural antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single-chain V region fragments (scFv), fusion polypeptides and unconventional antibodies. In certain embodiments, an antibody is a glycopolypeptide comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as V H ) and heavy chain constant (C H ) region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as V L ) and light chain constant C L The light chain constant region contains one domain, C L . V H and V L The V domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V LIt is composed of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0057] As used herein, the term "single-chain variable fragment" or "scFv" is a fragment of a heavy chain (V H ) and light chain (V L ) are covalently linked to form V H : VL heterodimer fusion polypeptide. Heavy chain (V H ) and light chain (V L ) directly or through a peptide-encoded linker (e.g., about 10, 15, 20, 25 amino acids) that connects V H The N-terminus of V L C-terminus, or V H The C-terminus of V L N-terminus.

[0058] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule including an extracellular antigen binding domain and a transmembrane domain, which is fused to an intracellular signaling domain capable of activating or stimulating immune response cells. In certain embodiments, the extracellular antigen binding domain of CAR comprises scFv. scFv can be derived from fusing the variable heavy chain and light chain regions of an antibody. Alternatively or in addition, scFv can be derived from Fab' (rather than from an antibody, for example, obtained from a Fab library). In certain embodiments, scFv is fused to a transmembrane domain and then fused to an intracellular signaling domain. In certain embodiments, CAR is selected to have a high binding affinity or avidity for an antigen.

[0059] In certain non-limiting embodiments, the intracellular signaling domain of CAR may comprise a CD3 ζ polypeptide that can activate or stimulate cells (e.g., lymphocytes, such as T cells). CD3 ζ comprises three immunoreceptor tyrosine activation motifs (ITAMs) and transmits activation signals to cells (e.g., lymphocytes, such as T cells) after antigen is bound. The intracellular signaling domain of the CD3 ζ chain is the main transmitter of the signal of endogenous TCR.

[0060] In certain non-limiting embodiments, CAR may further include a spacer / hinge region connecting the extracellular antigen binding domain to the transmembrane domain. The spacer may be sufficiently flexible to allow the antigen binding domain to be oriented in different directions, thereby promoting antigen recognition. The spacer may be a hinge region from IgG1 or a CH2CH3 region of an immunoglobulin and a fragment of CD3, a fragment of a CD28 polypeptide, a fragment of a CD8 polypeptide, a variant thereof, or a synthetic spacer sequence.

[0061] As used herein, " co-stimulatory molecules " refer to cell surface molecules other than antigen receptors or their ligands, which are necessary for lymphocytes to respond to antigens. At least one co-stimulatory signal region may include CD28 polypeptides (e.g., CD28 intracellular domain or a fragment thereof), 4-1BB polypeptides (e.g., 4-1BB intracellular domain or a fragment thereof), OX40 polypeptides (e.g., OX40 intracellular domain or a fragment thereof), ICOS polypeptides (e.g., ICOS intracellular domain or a fragment thereof), DAP-10 polypeptides (e.g., DAP10 intracellular domain or a fragment thereof) or a combination thereof. Co-stimulatory molecules can be combined with co-stimulatory ligands. As used herein, the term " co-stimulatory ligands " refers to polypeptides expressed on the cell surface, which produce co-stimulatory responses after binding to their receptors, that is, causing activation of the intracellular response of the stimulation provided by the signal transduction domain (e.g., CD3 ζ signal transduction domain). Non-limiting examples of co-stimulatory ligands include tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members or a combination thereof. Costimulatory ligands are selected from the group consisting of tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members and combinations thereof. Non-limiting examples of TNF family members include 4-1BBL, OX40L, CD70, GITRL, CD40L and CD30L. Non-limiting examples of Ig superfamily members include CD80, CD86 and ICOSLG. For example, 4-1BBL can be combined with 4-1BB to provide an intracellular signal that binds to the CAR signal to induce CAR + Effector cell function of T cells. US7,446,190 discloses a CAR comprising an intracellular signaling domain comprising a costimulatory signaling region comprising a 4-1BB, ICOS or DAP-10 costimulatory signaling domain, which is incorporated herein by reference in its entirety.

[0062] As used herein, the term "multimerization" refers to the formation of multimers (including dimers). Multimerization includes dimerization.

[0063] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties of a polypeptide comprising an amino acid sequence disclosed herein (e.g., an extracellular antigen-binding domain of a polypeptide). Conservative modifications may include amino acid substitutions, additions, and deletions. Modifications may be introduced into the human scFv of the polypeptide disclosed herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids may be classified into groups based on their physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid in the same group. For example, amino acids may be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamine, histidine (basic polarity), lysine (basic polarity), serine, threonine and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine. Therefore, one or more amino acid residues in the CDR region can be replaced by other amino acid residues from the same group, and the retained function of the altered antibody (i.e., the function described in (c) to (l) above) can be tested using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues are changed in a specific sequence or CDR region.

[0064] In addition to full-length polypeptides, the subject matter disclosed in the present application also provides a fragment of any one of the polypeptides disclosed herein or peptide domains. As used herein, the term "fragment" means at least 5, 10, 13 or 15 amino acids. In certain embodiments, the fragment includes at least 20 continuous amino acids, at least 30 continuous amino acids or at least 50 continuous amino acids. In certain embodiments, the fragment includes at least 60 to 80, 100, 200, 300 or more continuous amino acids. Fragments can be produced by methods known to those skilled in the art or can be produced by normal polypeptide processing (for example, amino acids that are not needed for biological activity are removed from nascent polypeptides or amino acids are removed by alternative mRNA splicing or alternative polypeptide processing events).

[0065] As used herein, the homology percentage between two amino acid sequences is equivalent to the identity percentage between the two sequences. The identity percentage between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # total number of identical positions / positions × 100), which takes into account the number of spaces introduced for optimal alignment of the two sequences and the length of each space. The comparison of sequences and the determination of the identity percentage between the two sequences can be achieved using a mathematical algorithm.

[0066] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossom 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0067] Additionally or alternatively, the amino acid sequences of the presently disclosed subject matter can be further used as a "query sequence" to search public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST polypeptide searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the specific sequences disclosed herein (e.g., the heavy and light chain variable region sequences of scFv m903, m904, m905, m906, and m900). To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST programs and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0068] 5.2. Expression and sorting systems utilizing intein trans-splicing reactions

[0069] In a first aspect, the present disclosure relates to an expression and sorting system utilizing an intein trans-splicing reaction associated with the production of modular polypeptides. FIG1 depicts a split-intein-mediated trans-splicing reaction. In this exemplary reaction, a first polypeptide (extein) comprises a split-intein fused to the N-terminus of an intein, and a second polypeptide (extein) comprises a complementary split-intein fused to the C-terminus of the intein. Upon association of the split-intein, trans-splicing of the first and second polypeptides is catalyzed, resulting in the covalent attachment of the first and second polypeptides (extein) via a peptide bond and the release of a non-covalently associated intein. Figure 1D Shown is a comparison between the coding required for transduction of exogenous genes by conventional gene expression and intein-based gene expression (using CD19 and CD20 CAR as exemplary polypeptides). These constructs may include additional genes encoding domains and / or motifs (such as, but not limited to, spacers, linkers, hinges, transmembrane, ER retention, and kinases). The degeneracy of intein-based trans-splicing reactions enables the expression of large constructs with higher efficiency than when using conventional methods and enables the use of the same amount of DNA to encode a greater number of receptors, thereby providing significant advantages.

[0070] Orthogonality and degeneracy can be combined to achieve trans-splicing and combination of a large number of receptors with common binding and signaling domains (degeneracy), or to specify the splicing of a specific binding domain with a specific signaling domain (orthogonality). Drug-modulating domains can be combined with different signaling domains to achieve drug regulation of degenerate components (e.g., Zap70 and 4-1BB).

[0071] In certain embodiments, and not by way of limitation, the split inteins with complementary N- and C-termini include the Cfa intein, the gp41-1 intein, the gp41-8 intein, the Aes intein, and the Nrdj-1 intein. Table 1 lists exemplary intein amino acid sequences. Amino acid motifs involved in splicing (e.g., CLS, CFN, CLD, HNS, SVV, SVYLN, CLV) are underlined.

[0072] Table 1. Exemplary intein sequences

[0073]

[0074] In certain embodiments, the present disclosure relates to a dual-carrier intein sorting system. For example, Figure 2AAn exemplary dual-vector intein sorting system is depicted. In this exemplary method, vector 1 encodes a secreted affinity-tagged molecule fused to the N-terminal domain of an intein. Vector 2 encodes a C-terminal intein capture domain fused to a spacer, a transmembrane domain, a linker, and a non-signaling cytoplasmic domain. Trans-splicing of the two intein-tagged polypeptides results in cell-selective surface display of the affinity tag on dual-vector transduced cells, resulting from selective intracellular splicing. In certain embodiments, this method enables selective MACS sorting based on the affinity tag for cells that have incorporated both vectors and completed the trans-splicing reaction. In certain embodiments, each vector can carry additional transgenes to enhance engineered cell functionality.

[0075] In certain embodiments, the present disclosure relates to sorting and post-translational CAR assembly based on endoplasmic reticulum retention of dual-carrier inteins. For example, Figure 3A An exemplary method for sorting and post-translational CAR assembly based on dual-vector intein endoplasmic reticulum retention is described. In this exemplary method, vector 1 encodes an intein-tagged antigen-binding construct consisting of an scFv fused to an affinity tag, a spacer, a transmembrane domain, an intein N-terminus, and an endoplasmic reticulum (ER) retention motif; and vector 2 encodes a cytoplasmic signaling domain shuttle consisting of an intein C-terminus fused to a costimulatory domain and a T cell receptor signaling domain (e.g., CD3ζ). Trans-splicing of the two intein-tagged polypeptides yields an intracellular splicing junction and recapitulates a fully functional chimeric antigen receptor (CAR). Due to ER retention, the surface expression of the affinity-tagged antigen-binding domain is significantly reduced until the two inteins undergo trans-splicing, which removes the ER retention motif, resulting in cell surface transport. In certain embodiments, the method enables selective MACS sorting based on affinity tags for cells that have incorporated both vectors and completed trans-splicing reactions. In certain embodiments, each vector can carry additional transgenes to enhance the functionality of the engineered cells.

[0076] In certain embodiments, any ER retention motif can be employed in the context of the compositions, systems, and methods of the present disclosure. In certain embodiments, for example, where the ER retention motif occupies the terminus of the polypeptide chain, an E3 19K KKXX-type motif or an RXR-type ER retention motif ( Figure 3E In certain embodiments, for example, when an ER retention motif occupies a position within a polypeptide chain, an RXR-type ER retention motif may be employed. For example, but not limited to, Figures 3E to 3HThis demonstrates the ability of the RXR-type ER retention motif to function within the interior of a polypeptide chain (in this case, the 2A peptide residue remains attached to the RXR retention motif). In contrast, the E319K KKXX-type motif does not function in this context because the retention motif requires the end of the polypeptide chain to be occupied. Figures 3F to 3I As shown, cells transduced with vectors 1 and 2 encoding either RXR or KKXX motifs (co-transduced with vector 3 encoding the gp41-1 C intein) removed the ER retention motif and promoted robust surface trafficking of the fully spliced ​​sorting handle molecule. However, the RXR-type ER retention motif version only achieved high expression of the construct in triple-transduced cells, enabling selective MACS sorting of triple-transduced cells. This approach enables one-step MACS purification of cells incorporating two or more vectors with an enhanced total vector payload to generate effector cells with enhanced functionality. Furthermore, the use of RXR-type ER retention motifs enables the placement of ER-retained constructs at the 5' or 3' position of polycistronic vectors flanked by 2A peptides encoding multiple proteins.

[0077] In certain embodiments, the present disclosure relates to a dual-carrier dual-secretory intein-tagged scFv sorting system and post-translational CAR assembly. For example, Figure 4A An exemplary method for dual-vector dual-secreted intein-tagged scFv sorting and post-translational CAR assembly is described. In this exemplary method, vector 1 encodes a dual-affinity-tagged intein scFv in which each scFv is fused to an affinity tag and the N-terminus of a degenerate intein; and vector 2 encodes a transmembrane signaling domain shuttle consisting of an intein C-terminus fused to a spacer domain, a transmembrane domain, a co-stimulatory domain, and a T-cell receptor signaling domain. After capture and splicing of the intein domains, both antigen-binding domains are displayed on the cell surface, reproducing the full-length CAR molecule. Due to selective intracellular splicing, trans-splicing of the two intein-tagged polypeptides results in cell-selective surface display of the affinity tags in cells transduced with the dual vectors. In certain embodiments, this enables selective MACS sorting based on the affinity tags for cells that have incorporated both vectors and completed the trans-splicing reaction. In certain embodiments, each vector may carry additional transgenes to enhance the functionality of the engineered cells.

[0078] In certain embodiments, the present disclosure relates to intein CAR expression based on zeta chain-associated protein kinase 70 (Zap70). For example, Figure 4EAn exemplary method for dual vector degenerate intein trans-splicing to achieve expression of both Zap70-based CD19 scFv and Zap70-based CD20 scFv is depicted. In this exemplary method, vector 1 encodes two different antigen-binding scFv domain fusion polypeptides with a degenerate cytoplasmic N-terminal intein domain. Each scFv fusion polypeptide contains a different affinity tag and may also include an ER retention motif at the C-terminus of the fusion polypeptide at the end of the vector construct (see Figure 3A ). Vector 2 encodes a Zap70 domain shuttle, which consists of a degenerate intein C-terminus and a protein tag (e.g., an EGFP tag). The combination of two degenerate intein-tagged antigen-binding polypeptides and trans-splicing of the Zap70 domain shuttle can generate two CAR molecules, for a total of two mature trans-splicing polypeptides. The presence of the ER retention motif minimizes the surface expression of the affinity-tagged antigen-binding domain polypeptide until the two inteins undergo trans-splicing, thereby removing the retention motif and resulting in cell surface trafficking. This system enables post-translational dual CAR assembly, combined with affinity tag-based selective MACS sorting of cells that have incorporated the two vectors and completed the trans-splicing reaction. Each vector can carry additional transgenes to enhance the functionality of the engineered cells.

[0079] In certain embodiments, the compositions, systems and methods of the present disclosure may be facilitated by degenerate splicing of inteins. For example, but not by way of limitation, degenerate splicing of inteins sharing a common cytoplasmic signaling domain shuttle enables the reuse of DNA encoding large structural elements (e.g., Zap70 interdomain and kinase). In certain embodiments, such use may be used as a data compression algorithm because it enables a greater number of receptors to be generated per unit amount of DNA delivered by the vector. For example, but not by way of limitation, degenerate inteins that tag domains shared by multiple CAR constructs (e.g., Zap70 signaling domain and 4-1BB co-stimulatory domain) may be multiplexed with multiple molecules containing antigen binding domains to generate a greater number of trans-spliced ​​CAR and CCR molecules than would be encoded with the same amount of DNA. The amount of DNA "data" saved increases with the size of the encoded domain and the reuse of the domain in a greater number of molecules. In certain embodiments, a vector encoding a domain having a size of B i The common binding domain and size of D j The amount of DNA required to encode a series of conventional molecules (e.g., CAR and CCR molecules) for the signaling domain of C It can be described by Equation 1;

[0080] Q C =d∑B i +b∑D j Equation 1

[0081] Where d is the size D used j The number of different signaling domains is Q, and b is the number of different binding domains of size Bi used. I It can be described by Equation 2;

[0082] Q I =∑B i +∑D j +bI N +dI C Equation 2

[0083] Among them I N is the size of the DNA required to encode the N-terminal split intein, and I C is the size of DNA required to encode the C-terminal split intein. The amount of DNA saved by intein-based compact coding (Δ = Q c –Q I ) can be determined by Equation 3

[0084] Δ=(d-1)∑B i +(b-1)∑D j -bI N -bI C Equation 3

[0085] Where d is the number of encoded signaling domains, b is the number of encoded binding handle molecules, and B i is the size of each binding domain, D j For the size of each signaling domain, I N is the size of the N-terminal split intein, and I C is the size of the C-terminal intein. This is illustrated by an example of a Zap70 signaling domain and a 4-1BB co-stimulatory domain conventionally encoding two CAR and two CCR molecules regulated by an NS3 drug, the sizes of which are as follows; B1 = 1 kB, B2 = 1 kB, D1 = 2.2 kB, D2 = 0.1 kB. According to Equation 1, this requires:

[0086] 2*(B1+B2)+2*(D1+D2)=2*(1+1)+2*(2.2+0.1)=8.6kB Equation 4

[0087] Rearrangement by CAR and CCR molecules:

[0088] (B1+D1) CAR1 +(B2+D1) CAR2 +(B1+D2) CCR1+(B2+D2) CCR1 =3.2+3.2+1.1+1.1=8.6kB Equation 5

[0089] In contrast, for N = 0.3kB and I C = 0.1 intein coding, the following DNA would be required for total coding:

[0090] (B1+B2)+(D1+D2)+2(I N )+2(I C )=2+2.3+0.6+0.2=5.1kB Equation 5 This results in a DNA saving of 8.6kB-5.1kB=3.5kB. Confirmed by Equation 3, the data saving is:

[0091] 1*2+1*2.3–0.6–0.2=3.5kB Equation 6

[0092] This saving in number increases the number of antigen binding domains B and signaling domains D, or increases the size of each domain. For example, but not by way of limitation, three signaling domains of 2 kB, 0.3 kB, and 0.1 kB in size and three antigen binding domains of 1 kB in size (where 1 N = 0.3kB and I C =0.1 kB), will encode nine receptors and will result in the following DNA savings in intein encoding compared to conventional encoding:

[0093] 2*(1+1+1)+2*(2+0.3+0.1)–3*0.3–3*0.1=9.6kB Equation 7.

[0094] In certain embodiments, selective sequestration of the encoded protein (e.g., FLAG-tagged CAR) by the ER retention motif, followed by trans-splicing with the appropriate cytoplasmic signaling domain shuttle and surface trafficking after removal of the ER retention motif, enables selective MACS sorting of dual-transduced cells. In certain embodiments, such methods can be used as molecular coincidence detectors.

[0095] In certain embodiments, the present disclosure relates to a sorting system based on ER retention of a three-carrier intein and post-translational CAR assembly using secreted intein-tagged scFv. For example, Figure 5ADepicted is an exemplary method for sorting based on triple-vector intein ER retention and post-translational CAR assembly using secreted intein-tagged scFvs. In this exemplary method, vector 1 encodes a dual antigen-binding scFv, each fused to an affinity tag and an orthogonal intein N-terminus; vector 2 encodes a dual intein transmembrane adaptor composed of an orthogonal intein C-terminus fused to the extracellular and transmembrane domains, the orthogonal intein N-terminus, and an ER retention motif; and vector 3 encodes a cytoplasmic signaling domain shuttle composed of an orthogonal intein C-terminus fused to a costimulatory domain and a T-cell receptor signaling domain. The intein-tagged scFv molecule undergoes trans-splicing with the dual intein transmembrane linker and can be exported from the ER following trans-splicing of the adaptor with the orthogonal intein of the cytoplasmic shuttle construct, thereby cleaving the ER retention motif and reproducing the full-length CAR. In certain embodiments, this enables selective MACS sorting of cells incorporating three vectors based on affinity tags after completing two orthogonal trans-splicing reactions. In certain embodiments, each vector can carry additional transgenes to enhance the functionality of the engineered cells.

[0096] In certain embodiments, the compositions, systems, and methods of the present disclosure may be facilitated using an orthogonal diintein, trimolecular trans-splicing approach. For example, and not by way of limitation, such approaches (e.g., 5A to 5D ) enables selective MACS sorting of T cells incorporating three unique vectors, which can further encode additional transgenes to enhance effector cell functionality.

[0097] In certain embodiments, the present disclosure relates to a sorting system based on a three-carrier hybrid leucine zipper-intein ER retention, and post-translational CAR assembly. For example, Figure 6AAn exemplary method for sorting and post-translational CAR assembly based on a three-vector hybrid leucine zipper-intein ER retention is described. In this exemplary method, vector 1 encodes a dual antigen-binding scFv, each fused to an affinity tag and a heterodimeric leucine zipper; vector 2 encodes a leucine zipper-intein transmembrane adaptor composed of a heterodimeric leucine zipper fused to a short hinge domain, a transmembrane domain, an intein N-terminus, and an ER retention motif; and vector 3 encodes a cytoplasmic signaling domain shuttle composed of an intein C-terminus fused to a costimulatory domain and a T cell receptor signaling domain. The leucine zipper-tagged scFv molecule binds to the capture zipper-intein transmembrane adaptor in the ER and can be expelled from the ER after trans-splicing of the adaptor's intein and the cytoplasmic shuttle construct, thereby cleaving the ER retention motif and reproducing the full-length CAR. In certain embodiments, this enables selective MACS sorting of cells incorporating the three vectors based on affinity tags after completion of the zipper heterodimerization and intein trans-splicing reactions. In certain embodiments, each vector can carry additional transgenes to enhance the functionality of the engineered cells.

[0098] In certain embodiments, the compositions, systems, and methods of the present disclosure can be facilitated using orthogonal inteins and leucine zippers to achieve selective MACS sorting. For example, and not by way of limitation, such methods (e.g., 6A to 6D ) can facilitate selective MACS sorting of T cells incorporating three unique vectors, which can further encode additional transgenes to enhance effector cell functionality.

[0099] In certain embodiments, the present disclosure relates to a sorting system based on three-carrier intein ER retention and post-translational CAR assembly using intracellular intein adapters. For example, Figure 7An exemplary method for sorting based on the ER retention of a three-vector intein and post-translational CAR assembly using an intracellular intein adapter is depicted. In this exemplary method, vector 1 encodes an intein-tagged antigen-binding construct consisting of an scFv fused to an affinity tag, a spacer, a transmembrane domain, an orthogonal intein N-terminus, and an ER retention motif; vector 2 encodes a dual intein cytoplasmic adapter consisting of an orthogonal intein C-terminus fused to an optional signaling domain, an orthogonal intein N-terminus, and an ER retention motif; and vector 3 encodes a cytoplasmic signaling domain shuttle consisting of an orthogonal intein C-terminus fused to a costimulatory domain and / or a T cell receptor signaling domain. The orthogonal intein-tagged molecule undergoes trans-splicing, as shown in the figure, with consecutive cleavage of two ER retention motifs to reconstitute the full-length CAR molecule. In certain embodiments, the removal of the ER retention motif enables the CAR molecule to be selectively transported to the cell surface, and after completing two orthogonal trans-splicing reactions, the cells incorporated with the three vectors are selectively sorted by MACS based on affinity tags. In certain embodiments, each vector can carry additional transgenes to enhance the functionality of the engineered cells.

[0100] In certain embodiments, the present disclosure relates to a sorting system based on ER retention of a tetracarrier intein, and post-translational CAR assembly using a combination of transmembrane and intracellular endoplasmic reticulum adaptors. For example, Figure 8A Depicted is an exemplary method for sorting based on ER retention of four vector inteins and post-translational CAR assembly using a combination of transmembrane and intracellular intein adapters. In this exemplary method, vector 1 encodes a dual-antigen binding scFv fused to an affinity tag and the N-terminus of an orthogonal intein; vector 2 encodes a dual-intein transmembrane adapter consisting of the C-terminus of the orthogonal intein fused to the extracellular and transmembrane domains, the N-terminus of the orthogonal intein, and an ER retention motif; vector 3 encodes a dual-intein cytoplasmic adapter consisting of the C-terminus of the orthogonal intein fused to an optional signaling domain, the N-terminus of the orthogonal intein, and an ER retention motif; and vector 4 encodes a cytoplasmic signaling domain shuttle consisting of the C-terminus of the orthogonal intein fused to a costimulatory domain and / or a T cell receptor signaling domain. The orthogonal intein-tagged molecule undergoes trans-splicing, as shown, with sequential cleavage of the two ER retention motifs to reconstitute the full-length CAR molecule. In certain embodiments, the sequential removal of the ER retention motif enables the CAR molecule to be selectively transported to the cell surface, and after completing three orthogonal trans-splicing reactions, the cells incorporating the four vectors are selectively sorted by MACS based on affinity tags. In certain embodiments, each vector can carry additional transgenes to enhance the functionality of the engineered cells.

[0101] In certain embodiments, the compositions, systems, and methods of the present disclosure can be facilitated using an orthogonal three-intein splicing methodology that specifically generates trans-spliced ​​CARs or sorting handle molecules in cells incorporating four unique vectors. For example, but not by way of limitation, this recursive strategy of nested orthogonal inteins tagged with ER retention motifs can be expanded to greater than four vectors by adding additional orthogonal inteins. Furthermore, in certain embodiments, this strategy can increase the number of transgenes delivered to engineered cells due to the increased number of vectors incorporated.

[0102] In certain embodiments, the present disclosure relates to a dual-vector intein sorting system using drug-regulated CAR expression. For example, Figure 9A Describe an exemplary method for sorting dual-vector intein peptides for drug-regulated CAR expression. In this exemplary method, vector 1 encodes an intein-tagged antigen-binding construct consisting of an scFv fused to an affinity tag, a spacer, a transmembrane domain, a low-affinity intein N-terminus, a drug dimerization domain (e.g., FKBP12), and an ER retention motif; and vector 2 encodes a drug dimerization domain (e.g., FRB*) fused to a low-affinity intein C-terminus, a costimulatory domain, and a T-cell receptor signaling domain (e.g., CD3ζ). Due to the enhanced polypeptide association mediated by the drug dimerization domain, the trans-splicing incidence of two low-affinity intein-tagged polypeptides is the highest in the presence of a dimerizer drug. Trans-splicing results in intracellular splicing junctions and reproduces a fully functional CAR. Due to ER retention, the surface expression of the affinity-tagged antigen-binding domain polypeptide is significantly reduced until the two inteins undergo trans-splicing, which removes the ER retention motif and leads to cell surface trafficking. In certain embodiments, the system is capable of achieving drug-dependent post-translational CAR assembly and performing selective MACS sorting based on affinity tags for cells that have incorporated two vectors and completed the trans-splicing reaction. In certain embodiments, each vector can carry additional transgenes to enhance the functionality of the engineered cells.

[0103] In certain embodiments, the compositions, systems, and methods of the present disclosure can be used to improve the methodology of the previous FKBP / FRB-based drug-regulated CAR system by eliminating the FKBP / FRB protein after trans-splicing and generating mature CAR molecules. In certain embodiments, such methods will simplify CAR design. In addition, in certain embodiments, the signaling domain can be introduced into a membrane proximal position that is conducive to signaling.

[0104] In certain embodiments, the present disclosure relates to a dual-vector intein sorting system using drug-regulated dual CAR expression. For example, Figure 10ADescribed is an exemplary method for sorting dual vector inteins for dual CAR expression regulated by drugs. In this exemplary method, vector 1 encodes a fusion polypeptide of two different antigen-binding scFv transmembrane domains, the fusion polypeptide having a cytoplasmic low-affinity N-terminal intein domain. Each scFv fusion polypeptide contains a different affinity tag, spacer, transmembrane domain, low-affinity intein N-terminus, and a drug dimerization domain (e.g., FKBP12), but only one fusion polypeptide contains an ER retention motif because the ER retention motif works most effectively at the end of the vector construct (C-terminus of the fusion polypeptide and 2A sequence). Vector 2 encodes a drug-regulated cytoplasmic signaling domain shuttle, which is composed of a drug dimerization domain (e.g., FRB*) fused to a low-affinity intein C-terminus, a costimulatory domain, and a T cell receptor signaling domain (e.g., CD3ζ). Due to the enhanced polypeptide association mediated by the drug dimerization domain, the trans-splicing rate of the two low-affinity intein-tagged polypeptides is the highest in the presence of the dimerizing agent drug. Trans-splicing results in an intracellular splicing site and recreates a fully functional CAR. Due to ER retention, the surface expression of the affinity-tagged antigen-binding domain polypeptide with an ER retention motif is significantly reduced until the two intein peptides undergo trans-splicing, which removes the ER retention motif and leads to cell surface trafficking. In certain embodiments, the system is capable of achieving drug-dependent post-translational dual CAR assembly and performing selective MACS sorting based on affinity tags for cells that have incorporated two vectors and completed the trans-splicing reaction. In certain embodiments, each vector can carry additional transgenes to enhance the functionality of the engineered cells.

[0105] In certain embodiments, the present disclosure relates to a dual-vector intein sorting system using drug-regulated trans-presented cell surface cytokine expression. For example, Figure 11ADescribe an exemplary method for sorting dual-vector intein peptides for cell surface cytokine expression using drug-regulated trans-presentation. In this exemplary method, vector 1 encodes a cytokine that is fused to an affinity tag, a spacer, a transmembrane domain, a low-affinity intein N-terminus, a drug-dimerizable domain (e.g., FKBP12), and an endoplasmic reticulum (ER) retention motif; and vector 2 encodes a drug-dimerizable domain (e.g., FRB*) that is fused to a low-affinity intein C-terminus and a co-stimulatory domain or a non-signaling intracellular domain. Due to the enhanced polypeptide association mediated by the drug-dimerizable domain, in the presence of a dimerizer drug, the trans-splicing incidence of two low-affinity intein-tagged polypeptides is the highest. Trans-splicing results in the excision of the ER retention motif. Due to ER retention, surface expression of affinity-tagged cytokine trans-presenting polypeptides is significantly reduced until the two inteins undergo trans-splicing, which removes the retention motif and leads to cell surface trafficking. In certain embodiments, this system enables drug-dependent hierarchical expression of cell-surface trans-presenting cytokines, and selective MACS sorting based on affinity tags for cells that have incorporated both vectors and completed the trans-splicing reaction. In certain embodiments, each vector can carry additional transgenes to enhance the functionality of the engineered cells.

[0106] In certain embodiments, the compositions, systems, and methods of the present disclosure may be facilitated by methodologies that utilize ER retention motifs and drug-regulated trans-splicing to control surface expression of tethered cytokines. For example, but not by way of limitation, such methods may be used to promote cis- and trans-stimulation of T cells and / or other effector cells to enhance immune function.

[0107] In certain embodiments, the present disclosure relates to dual vector degenerate intein trans-splicing to achieve dual CAR formation using drug-stable cytoplasmic signaling domain shuttle expression. For example, Figure 12ADescribe an exemplary method for dual-carrier degenerate intein trans-splicing to express dual CAR using a drug-stable cytoplasmic signaling domain shuttle. In this exemplary method, vector 1 encodes a fusion polypeptide containing two different antigen-binding scFv transmembrane domains, the fusion polypeptide having a degenerate cytoplasmic N-terminal intein domain. Each scFv fusion polypeptide contains a different affinity tag, spacer, transmembrane domain, and cytoplasmic degenerate intein N-terminus, but only one fusion polypeptide contains an ER retention motif because the ER retention motif works most effectively at the end of the vector construct (C-terminus of the fusion polypeptide and 2A sequence). Vector 2 encodes a cytoplasmic signaling domain shuttle, which is composed of an intein C-terminus, a costimulatory domain, a T cell receptor signaling domain (e.g., CD3ζ), and a drug-stable degradation determinant domain (e.g., Escherichia coli (E. coli) DHFR destabilization domain). Due to ER retention, the surface expression of the affinity-tagged antigen-binding domain polypeptide with an ER retention motif is significantly reduced until trans-splicing occurs with the cytoplasmic signaling domain shuttle, which removes the retention motif and leads to cell surface trafficking. Maximum expression of the cytoplasmic signaling domain shuttle occurs in the presence of a stable drug, which promotes the trans-splicing reaction. In certain embodiments, the system enables drug-dependent post-translational dual CAR and assembly, combined with affinity tag-based selective MACS sorting of cells that have incorporated two vectors and completed the trans-splicing reaction. In certain embodiments, each vector can carry additional transgenes to enhance the functionality of the engineered cells.

[0108] In certain embodiments, the compositions, systems, and methods of the present disclosure can be facilitated by utilizing a method for regulating intein expression within a cell by linking a drug-regulated degron (destabilizing protein) to a cytoplasmic signaling shuttle intein. For example, but not by way of limitation, such methods can thereby control trans-splicing activity and CAR signaling.

[0109] In certain embodiments, the present disclosure relates to dual vector degenerate intein trans-splicing to achieve dual CAR formation using drug-destabilized cytoplasmic signaling domain shuttle expression. For example, Figure 13ADescribe an exemplary method for dual-carrier degenerate intein trans-splicing to express a cytoplasmic signaling domain shuttle to achieve dual CAR formation using drug destabilization. In this exemplary method, vector 1 encodes a fusion polypeptide containing two different antigen-binding scFv transmembrane domains, the fusion polypeptide having a degenerate cytoplasmic N-terminal intein domain. Each scFv fusion polypeptide contains a different affinity tag, spacer, transmembrane domain, and cytoplasmic degenerate intein N-terminus, but only one fusion polypeptide contains an ER retention motif because the ER retention motif works most effectively at the end of the vector construct (C-terminus of the fusion polypeptide and 2A sequence). Vector 2 encodes a cytoplasmic signaling domain shuttle, which is composed of an intein C-terminus, a costimulatory domain, a T cell receptor signaling domain (e.g., CD3ζ), and a drug-activated degradation determinant domain (e.g., SMASh tag). Due to ER retention, the surface expression of the affinity-tagged antigen-binding domain polypeptide with an ER retention motif is significantly reduced until trans-splicing occurs with the cytoplasmic signaling domain shuttle, which removes the retention motif and leads to cell surface trafficking. In the presence of an inhibitor drug, the expression of the cytoplasmic signaling domain shuttle is reduced, and the inhibitor drug promotes proteasomal degradation, thereby inhibiting the trans-splicing reaction and reducing CAR formation. In certain embodiments, the system is capable of achieving drug-dependent post-translational dual CAR assembly, combined with selective MACS sorting based on affinity tags for cells that have incorporated two vectors and completed the trans-splicing reaction. In certain embodiments, each vector can carry additional transgenes to enhance the functionality of the engineered cells.

[0110] In certain embodiments, the compositions, systems, and methods of the present disclosure may be administered by, for example, 13A to 13D The methodologies exemplified in are facilitated by the ability to generate T cells capable of simultaneously targeting two antigens using coordinated drug modulation. In certain embodiments, such methods enable single-step MACS purification of highly purified dual-transduced T cells incorporating two vectors.

[0111] In certain embodiments, the present disclosure relates to dual vector degenerate intein trans-splicing to achieve dual CAR formation using drug-destabilized cytoplasmic signaling domain shuttle expression. For example, Figure 14A An exemplary method for dual vector degenerate intein trans-splicing to achieve expression of Zap70-based CD19 scFv and Zap70-based CD20 scFv is depicted. In this exemplary method, vector 1 encodes two different antigen-binding scFv domain fusion polypeptides with a degenerate cytoplasmic N-terminal intein domain (I NEach scFv fusion polypeptide contains a different affinity tag and may also contain an ER retention motif at the C-terminus of the fusion polypeptide at the end of the vector construct (e.g., see Figure 3A Vector 2 encodes a drug-adjustable Zap70 domain shuttle consisting of a non-structural 3 (NS3) protease recognition sequence and an intein C-terminus (I C ) composition. Due to ER retention, the surface expression of the affinity-tagged antigen-binding domain polypeptide with an ER retention motif is significantly reduced until trans-splicing occurs with the cytoplasmic signaling domain shuttle, which removes the retention motif and leads to cell surface trafficking. In the presence of an inhibitor drug, the expression of the cytoplasmic signaling domain shuttle is reduced, and the inhibitor drug promotes proteasomal degradation, thereby inhibiting the trans-splicing reaction and reducing CAR formation. In certain embodiments, the system is capable of achieving drug-dependent post-translational dual CAR assembly, combined with affinity tag-based selective MACS sorting of cells that have incorporated two vectors and completed the trans-splicing reaction. In certain embodiments, each vector may carry additional transgenes to enhance the functionality of the engineered cells.

[0112] In certain embodiments, the present disclosure relates to an intein-assisted NS3-based drug-regulated CAR. For example, Figure 15A A method for generating drug-regulated NS3-based CARs and comprising a dual vector selection cell sorting method is described.

[0113] In certain embodiments, the present disclosure relates to an intein-assisted NS3-based drug-regulated CAR generated using a nested approach. For example, Figure 16A A method for generating dual antigen-targeted drug-regulated NS3-based CARs and comprising a dual vector selection cell sorting method is described.

[0114] In certain embodiments, the compositions, systems, and methods of the present disclosure may be administered by, for example, 16A to 16C The methodologies exemplified in are facilitated by enabling the generation of drug-regulated active signaling CARs by removing drug-regulated polypeptides (e.g., NS3 polypeptides) that may impair CAR signaling. In certain embodiments, such methods are accomplished by placing the signaling domain at a membrane distal location, such as 16A to 16C Removal of the drug-modulating peptide also prolonged CAR activity in the absence of HCV protease inhibitors.

[0115] In certain embodiments, the present disclosure relates to dual vector degenerate intein trans-splicing to achieve multiple post-translational CAR and chimeric costimulatory receptor (CCR) formation. For example, Figure 17ADescribe an exemplary method for dual-vector degenerate intein trans-splicing to achieve multiple post-translational CAR and CCR formation. In this exemplary method, vector 1 encodes a fusion polypeptide of an antigen-binding scFv transmembrane domain having a degenerate cytoplasmic N-terminal intein domain. The scFv fusion polypeptide contains different affinity tags, spacers, transmembrane domains, and cytoplasmic degenerate intein N-termini, but only one fusion polypeptide contains an ER retention motif because the ER retention motif works most effectively at the end of the vector construct (C-terminus of the fusion polypeptide and 2A sequence). Vector 2 encodes two cytoplasmic signaling domain shuttles, which are composed of an intein C-terminus, a costimulatory domain (e.g., CD28, 4-1BB), a T cell receptor signaling domain (e.g., CD3ζ), and at least one drug-activated degradation determinant domain (e.g., SMASh tag). Due to ER retention, the surface expression of the affinity-tagged antigen-binding domain polypeptide with an ER retention motif is significantly reduced until trans-splicing occurs with the cytoplasmic signaling domain shuttle, which removes the retention motif and leads to cell surface trafficking. In the presence of an inhibitor drug, the expression of the cytoplasmic signaling domain shuttle is reduced, and the inhibitor drug promotes proteasomal degradation, thereby inhibiting the trans-splicing reaction and reducing CAR formation. In certain embodiments, the system is capable of achieving drug-dependent post-translational dual CAR assembly, combined with selective MACS sorting based on affinity tags for cells that have incorporated two vectors and completed the trans-splicing reaction. In certain embodiments, each vector can carry additional transgenes to enhance the functionality of the engineered cells.

[0116] In certain embodiments, the present disclosure relates to dual vector degenerate intein trans-splicing to achieve multiple post-translational CAR and chimeric costimulatory receptor (CCR) formation. For example, Figure 17FDescribed is an exemplary method for dual-vector degenerate intein trans-splicing to achieve multiple post-translational CAR and CCR formation. In this exemplary method, vector 1 encodes two different antigen-binding scFv (CD19 / CD20) transmembrane domain fusion polypeptides having a degenerate cytoplasmic N-terminal intein domain. Each scFv fusion polypeptide contains a different affinity tag, spacer, transmembrane domain, and cytoplasmic degenerate intein N-terminus, but only one fusion polypeptide contains an ER retention motif because the ER retention motif works most effectively at the end of the vector construct (C-terminus of the fusion polypeptide and 2A sequence). Vector 2 encodes two cytoplasmic signaling domain shuttles, each consisting of a degenerate intein C-terminus and a unique costimulatory domain (e.g., CD28z, 4-1BB). Trans-splicing of the combination of two degenerate intein-tagged antigen-binding polypeptides with two cytoplasmic signaling domain shuttles can generate two CAR molecules and two CCR molecules, for a total of four mature trans-splicing polypeptides. Due to ER retention, surface expression of affinity-tagged antigen-binding domain polypeptides with ER retention motifs is significantly reduced until the two inteins undergo trans-splicing and the retention motif is removed, leading to cell surface trafficking. This system enables post-translational dual-CAR and dual-CCR assembly and, combined with affinity tag-based MACS selective sorting, sorts cells that have integrated both vectors and completed the trans-splicing reaction. Further modification of the cytoplasmic signaling domain shuttle vector to include orthogonal drug destabilization domains or selective degradation determinant domains can achieve selective drug-dependent regulation of each signaling domain. Each vector can carry additional transgenes to enhance the functionality of the engineered cells.

[0117] In certain embodiments, the compositions, systems, and methods of the present disclosure may be facilitated by a methodology that can generate four possible CAR and / or CCR combinations through trans-splicing combinations. For example, but not limited to, 17A to 17D Examples of such methodologies for generating CD19-CAR, CD20-CAR, CD19-CCR, and CD20-CCR are provided. In certain embodiments, such methodologies represent a data compression algorithm in which degenerately encoded DNA elements are reused to enable post-translational assembly of a greater number of receptors than those encoded with the same amount of DNA. Furthermore, in certain embodiments, additive CAR+CCR co-stimulation promotes enhanced T cell proliferation, and in certain embodiments, target killing is enhanced at low T cell E:T ratios, consistent with more active cell products.

[0118] In certain embodiments, the present disclosure relates to dual vector degenerate intein trans-splicing to achieve multiple post-translational CARs and chimeric costimulatory receptors (CCRs) with Zap70 domains. For example, Figure 18AAn exemplary method for dual vector degenerate intein trans-splicing to achieve expression of Zap70-based CD19 scFv and Zap70-based CD20 scFv is described. In this exemplary method, vector 1 encodes a fusion polypeptide containing two different antigen-binding scFv transmembrane domains (e.g., anti-CD19, anti-CD20) with a degenerate cytoplasmic N-terminal intein (I N ) domain. Each scFv fusion polypeptide contains a different affinity tag, spacer, transmembrane domain, and cytoplasmic degenerate intein N-terminus, but only one fusion polypeptide contains an ER retention motif because the ER retention motif works most effectively at the end of the vector construct. Vector 2 encodes each of the scFvs attached at its N-terminus to the C-terminus of the intein (I C ) Zap70 domain shuttle and 4-1BB domain sequence. Due to ER retention, the surface expression of the affinity-tagged antigen-binding domain polypeptide with the ER retention motif is significantly reduced until trans-splicing occurs with the cytoplasmic signaling domain shuttle, which removes the retention motif and leads to cell surface trafficking. In certain embodiments, each vector may carry additional transgenes to enhance the functionality of engineered cells. In certain embodiments, this configuration enables the post-translational assembly of a greater number of receptors compared to conventional methods using the same number of DNA nucleotides.

[0119] Table 2 shows exemplary sequences comprising various elements of a degenerate intein-based trans-splicing system. Table 3 lists exemplary construct sequences for an intein-based trans-splicing system.

[0120]

[0121]

[0122]

[0123] Table 3. Exemplary construct sequences for intein-based trans-splicing systems

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137] 5.3. Exemplary Modular Polypeptides

[0138] As described above, the subject matter disclosed in the present application provides a cell comprising a modular polypeptide, a system for generating such modular polypeptides, and a system for sorting cells comprising such modular polypeptides. Although certain aspects of the drawings and specific embodiments refer to exemplary embodiments using specific modular polypeptides (e.g., CAR and CCR), it should be understood that the systems outlined herein are not limited to those modular polypeptides specifically illustrated. On the contrary, the various systems described herein can be applied to the modular polypeptides incorporating various modular domains discussed herein (e.g., antigen binding domains, transmembrane domains, joints, spacers, affinity tags, ER retention motifs, intracellular signaling domains, non-signaling domains, costimulatory domains, drug regulatory domains, cytokine sequences, and chemokine sequences).

[0139] In certain embodiments, modular polypeptides are antigen recognition receptors. In certain embodiments, the antigen recognition receptors of the present disclosure are bound to specific target antigens. In certain embodiments, the target antigen may be a tumor antigen or a pathogen antigen. In certain embodiments, the antigen recognition receptor is a chimeric receptor. In certain embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In certain embodiments, the antigen recognition receptor is a chimeric co-stimulatory receptor (CCR). In certain embodiments, the antigen recognition receptor is a T cell receptor (TCR). In certain embodiments, the antigen recognition receptor is a TCR-like fusion molecule. As described in Section 5.2 above, modular polypeptides (e.g., CAR, CCR, TCR and TCR-like molecules and other modular polypeptides) can be encoded by a variety of sequences, which are incorporated into inteins at one or more positions to promote the formation of desired mature module polypeptides.

[0140] 5.3.1 Chimeric Antigen Receptor (CAR)

[0141] In certain embodiments, one or more of the modular polypeptides used in the systems described herein is a CAR. CARs are engineered receptors that transfer or confer specificity of interest to immune effector cells. CARs can be used to transfer the specificity of monoclonal antibodies to T cells; transfer of their coding sequences is facilitated by retroviral vectors.

[0142] There are three generations of CARs. A "first generation" CAR is typically composed of an extracellular antigen binding domain (e.g., scFv) that binds to a target antigen and an intracellular signaling domain. In certain embodiments, the CAR further comprises a transmembrane domain. A "first generation" CAR can provide de novo antigen recognition and induce CD4 T cell activation through its CD3 ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. + and CD8 + " second generation " CAR includes costimulatory molecules (for example, CD28, 4-1BB, ICOS, OX40, CD27, CD40, NKG2D, DAP-10, CD2, CD150, CD226) to CAR for providing the intracellular signaling domain of costimulatory signals to cells (for example, T cells or NK cells). " second generation " CAR includes providing costimulation (for example, CD28 or 4-1BB) and activation (CD3 ζ) those. " third generation " CAR includes providing multiple costimulation (for example, CD28 and 4-1BB) and activation (CD3 ζ) those.

[0143] In certain embodiments, the antigen recognition receptor is a CAR comprising an extracellular antigen binding domain and an intracellular signaling domain that is bound to an antigen. In certain embodiments, CAR further comprises a transmembrane domain. In certain embodiments, CAR further comprises a hinge / spacer. Non-limiting examples of antigens recognized by the antigen binding domains of CAR include: CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, antigens (e.g., cell surface antigens) of cytomegalovirus (CMV) infected cells, ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CC DC155, CCR1, CD10, CD117, CD123, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD30, CD300LF, CD32, CD321, CD33, C D34, CD36, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST 3. CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, COLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, E GP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-a, FOLR2, FRMD5, GABRB2, GAS2, GD2, GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5,ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B, kappa-light chain, L1CAM, LAX1, LEPR, Lewis Y(CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MYADML 2. NGFR, NKCS1, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, PRAME, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), peptidase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine peptide kinase Erb-B2, RHBDL3, RNF173, RNF183, ROR1, RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22 A5, SLC25A36, SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPA G17, STC1, STON2, SUN3, survivin, SUSD2, SYNC, TACSTD2, TAS1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycopolypeptide 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4,Wilms tumor polypeptide (WT-1), WNT4, WT1 and ZDHHC11.

[0144] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) is expressed at approximately 5×10 -7 M or smaller, approximately 1×10 -7 M or smaller, about 5×10 -8 M or smaller, approximately 1×10 -8 M or smaller, about 5×10 -9 M or smaller, or about 1×10 -9 M or smaller or about 1×10 -10 The dissociation constant (K D ) binds to the first antigen.

[0145] The combination of the extracellular antigen binding domain (for example, in scFv) can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (for example, growth inhibition) or Western blot assay. Each of these assays is typically determined by using a labeling agent (for example, antibody or scFv) specific for the target complex to detect the presence of a specific target polypeptide-antibody complex. For example, scFv can be radiolabeled and used for radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, the document is incorporated herein by reference). Radioisotopes can be detected by means of a gamma counter or scintillation counter or by autoradiography. In certain embodiments, the extracellular antigen binding domains of CAR are labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent polypeptide (GFP), blue fluorescent polypeptides (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent polypeptides (e.g., ECFP, Azure, and CyPet), and yellow fluorescent polypeptides (e.g., YFP, Citrine, Venus, and YPet).

[0146] The extracellular antigen-binding domain may comprise or may be scFv, Fab (which is optionally cross-linked) or F(ab) 2. In certain embodiments, any one of the aforementioned molecules may be contained in a fusion polypeptide having a heterologous sequence for forming the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises or is scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a mouse scFv. In certain embodiments, the extracellular antigen-binding domain is a VHH antibody (nanoantibody). Table 4 shows exemplary sequences of the extracellular antigen-binding domain.

[0147] Table 4. Exemplary extracellular antigen binding domain sequences

[0148]

[0149] In certain embodiments, antigen recognition receptor is a CAR comprising a transmembrane domain. Different transmembrane domains produce different receptor stabilities. After antigen recognition, receptors aggregate, and signals are delivered to cells. According to the subject matter disclosed herein, the transmembrane domain of antigen recognition receptor can include the following natural or modified transmembrane domains: CD8 polypeptides, CD28 polypeptides, CD3 ζ polypeptides, CD40 polypeptides, 4-1BB polypeptides, OX40 polypeptides, CD84 polypeptides, CD166 polypeptides, CD8a polypeptides, CD8b polypeptides, ICOS polypeptides, ICAM-1 polypeptides, CTLA-4 polypeptides, CD27 polypeptides, CD40 polypeptides, NKG2D polypeptides, synthetic polypeptides (not based on polypeptides associated with immune response) or combinations thereof. Table 5 shows exemplary transmembrane domains and signaling domains.

[0150]

[0151]

[0152] In certain embodiments, the signal transduction domain of CAR is a kinase. Non-limiting examples of such kinases include: Src kinase family (e.g., Blk, Fgr, Fyn, Hck, Lck, Lyn, Src, Yes, Yrk); Syk kinase family (e.g., Zap70, Syk, BTK, TEK, ITK); and receptor tyrosine (RTK) superfamily, including class I RTK (EGF receptor family or ErbB family), class II (insulin receptor family), class III (PDGF receptor family), class IV (VEGF receptor family), class V (FGF receptor family), class VI (CCK receptor family), class VII (NGF receptor family) , VIII (HGF receptor family), IX (Eph receptor family), X (AXL receptor family), XI (TIE receptor family), XII (RYK receptor family), XIII (DDR receptor family), XIV (RET receptor family), XV (ROS receptor family), XVI (LTK receptor family), XVII (ROR receptor family), XVIII (MuSK receptor family), XIX (LMR receptor), XX (undetermined), XVIII (MuSK receptor family), XIX (LMR receptor), XX and their orthologs. In certain embodiments, the signaling domain of the CAR comprises a kinase domain from one or more of PDGFR, KIT, Abl, Arg, EGFR, Raf, VEGFR, PDGFR, Flt3, Abl, Arg or ErbB2 or their orthologs.

[0153] In certain embodiments, the transmembrane domain of CAR includes a CD28 polypeptide (e.g., a transmembrane domain of CD28 or a portion thereof). In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain of human CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide includes or consists of the following: an amino acid sequence having a length of at least about 20, or at least about 25, or at least about 30 and / or at most about 220 amino acids as a continuous portion of an amino acid sequence with NCBI reference number NP_006130 (SEQID NO: 101). In certain embodiments, the CD28 polypeptide includes or consists of the following: an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 153 to 179, or 200 to 220 of SEQ ID NO: 103. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 101 ( FIG. 5 ).

[0154] In certain embodiments, antigen recognition receptor is the CAR that further comprises the hinge / spacer for connecting extracellular antigen binding domain to membrane spaning domain.Hinge / spacer can be flexible enough, to allow antigen binding domain to be oriented in different directions, so as to promote antigen recognition.In certain embodiments, the hinge / spacer of CAR can include the natural or modified hinge region of following items:CD8 polypeptides, CD28 polypeptides, CD3 zeta polypeptides, CD40 polypeptides, 4-1BB polypeptides, OX40 polypeptides, CD84 polypeptides, CD166 polypeptides, CD8a polypeptides, CD8b polypeptides, ICOS polypeptides, ICAM-1 polypeptides, CTLA-4 polypeptides, CD27 polypeptides, CD40 polypeptides, NKG2D polypeptides, synthetic polypeptides (not based on polypeptide associated with immune response) or its combination.Hinge / spacer can be the hinge region from IgG1 or the CH2CH3 district of immunoglobulin and CD3 part, a part of CD28 polypeptides (for example, a part of SEQ ID NO:101), a part of CD8 polypeptides or synthetic spacer sequence.

[0155] In certain embodiments, the antigen recognition receptor is a CAR further comprising a hinge / spacer region of a natural or modified hinge region comprising a CD28 polypeptide. In certain embodiments, the hinge / spacer region of the first antigen recognition receptor (e.g., CAR) comprises a CD28 polypeptide comprising or consisting of the following: amino acids 114 to 152 of SEQ ID NO: 101.

[0156] In certain embodiments, hinge / spacer is positioned between extracellular antigen-binding domain and membrane-spanning domain.In certain embodiments, hinge / spacer includes following items:CD8 polypeptide, CD28 polypeptide, CD3 ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 polypeptide, NKG2D polypeptide, synthetic polypeptide (not based on the polypeptide associated with immune response) or its combination.In certain embodiments, membrane-spanning domain includes following items:CD8 polypeptide, CD28 polypeptide, CD3 ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 polypeptide, NKG2D polypeptide, synthetic polypeptide (not based on the polypeptide associated with immune response) or its combination.

[0157] In certain embodiments, membrane-spanning domain and hinge / spacer are derived from identical molecule.In certain embodiments, membrane-spanning domain and hinge / spacer are derived from different molecules.In certain embodiments, hinge / spacer comprises CD28 polypeptide and membrane-spanning domain comprises CD28 polypeptide.In certain embodiments, hinge / spacer comprises CD28 polypeptide and membrane-spanning domain comprises CD28 polypeptide.In certain embodiments, hinge / spacer comprises CD84 polypeptide and membrane-spanning domain comprises CD84 polypeptide.In certain embodiments, hinge / spacer comprises CD166 polypeptide and membrane-spanning domain comprises CD166 polypeptide.In certain embodiments, hinge / spacer comprises CD8a polypeptide and membrane-spanning domain comprises CD8a polypeptide.In certain embodiments, hinge / spacer comprises CD8b polypeptide and membrane-spanning domain comprises CD8b polypeptide.In certain embodiments, hinge / spacer comprises CD28 polypeptide and membrane-spanning domain comprises ICOS polypeptide.

[0158] In certain embodiments, the antigen recognition receptor is a CAR comprising an intracellular signaling domain. In certain embodiments, the intracellular signaling domain of CAR comprises a CD3 ζ polypeptide. CD3 ζ can activate or stimulate cells (e.g., cells of the lymphoid lineage, e.g., T cells). Wild-type ("natural") CD3 ζ includes three functional immunoreceptor tyrosine-based activation motifs (ITAMs), three functional base-rich stretches (BRS) regions (BRS1, BRS2, ​​and BRS3). After the antigen is bound, CD3 ζ transmits an activation signal to the cell (e.g., a cell of the lymphoid lineage, e.g., T cell). The intracellular signaling domain of the CD3 ζ chain is the main transmitter of the signal of the endogenous TCR.

[0159] In certain embodiments, the intracellular signaling domain of the CAR comprises native CD3 zeta. In certain embodiments, the native CD3 zeta comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence of NCBI reference number NP_932170 (SEQ ID NO: 109) or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the CD3 zeta polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 109 of at least about 20, or at least about 30, or at least about 40, or at least about 50, and at most about 164 amino acids in length. In certain embodiments, the native CD3 zeta comprises or consists of an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 164, 100 to 150, or 150 to 164 of SEQ ID NO: 109. In certain embodiments, the intracellular signaling domain of the CAR contains a native CD3 zeta comprising or consisting of an amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 109 (Table 5).

[0160] In certain embodiments, native CD3ζ comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence shown in SEQ ID NO: 110 (Table 5).

[0161] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide. In certain embodiments, the modified CD3 zeta polypeptide comprises one, two, or three ITAMs. In certain embodiments, the modified CD3 zeta polypeptide comprises a native ITAM1. In certain embodiments, the native ITAM1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 116 (Table 5).

[0162] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 34 is shown in SEQ ID NO: 117 (Table 5).

[0163] In certain embodiments, the modified CD3 zeta polypeptide contains an ITAM1 variant comprising one or more loss-of-function mutations. In certain embodiments, the ITAM1 variant includes or consists of two loss-of-function mutations. In certain embodiments, each of one or more (e.g., two) loss-of-function mutations includes a mutation of a tyrosine residue in ITAM1. In certain embodiments, the ITAM1 variant consists of two loss-of-function mutations. In certain embodiments, the ITAM1 variant includes or consists of the amino acid sequence shown in SEQ ID NO: 118 (Table 5).

[0164] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 118 is shown in SEQ ID NO: 119 (Table 5).

[0165] In certain embodiments, the modified CD3 zeta polypeptide comprises native ITAM 2. In certain embodiments, native ITAM 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 120 (Table 5).

[0166] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 120 is shown in SEQ ID NO: 121 (Table 5).

[0167] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM2 variant. In certain embodiments, the ITAM2 variant comprises or consists of one or more loss-of-function mutations. In certain embodiments, the ITAM2 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM2. In certain embodiments, the ITAM1 variant consists of two loss-of-function mutations. In certain embodiments, the ITAM2 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 122 (Table 5).

[0168] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 122 is shown in SEQ ID NO: 123 (Table 3).

[0169] In certain embodiments, the modified CD3 zeta polypeptide comprises a native ITAM 3. In certain embodiments, the native ITAM 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 124 (Table 5).

[0170] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 124 is shown in SEQ ID NO: 125 (Table 5).

[0171] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM3 variant. In certain embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM3. In certain embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In certain embodiments, the ITAM3 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 126 (Table 3).

[0172] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 126 is shown in SEQ ID NO: 127 (Table 5).

[0173] Various modified CD3ζ polypeptides and CARs comprising modified CD3ζ polypeptides are disclosed in International Patent Application Publication No. WO2019 / 133969, which is hereby incorporated by reference in its entirety.

[0174] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising a native ITAM1, an ITAM2 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations, and an ITAM3 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising: a native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO: 116; an ITAM2 variant consisting of the amino acid sequence set forth in SEQ ID NO: 122; and an ITAM3 variant consisting of the amino acid sequence set forth in SEQ ID NO: 126. In certain embodiments, the CAR is designated "1XX". In certain embodiments, the modified CD3 zeta polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 111 (Table 5).

[0175] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising or consisting of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical to SEQ ID NO: 111 or a fragment thereof, and / or may optionally comprise at most one, at most two, or at most three conservative amino acid substitutions.

[0176] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 111 is shown in SEQ ID NO: 112 (Table 3).

[0177] In certain embodiments, the intracellular signaling domain of CAR further comprises at least one costimulatory signaling region. In certain embodiments, the at least one costimulatory region comprises a costimulatory molecule or a portion thereof. In certain embodiments, the at least one costimulatory region comprises at least one intracellular domain of at least one costimulatory molecule or a portion thereof. Non-limiting examples of costimulatory molecules include: CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

[0178] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide, e.g., an intracellular domain of CD28 or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising an intracellular domain of human CD28 or a portion thereof.

[0179] In certain embodiments, the CD28 polypeptide contained in the costimulatory signaling region of the first antigen recognition receptor comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical or homologous to the amino acid sequence shown in SEQ ID NO: 101 or a fragment thereof, and / or may optionally comprise at most one, at most two, or at most three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide contained in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is a continuous portion of SEQ ID NO: 101 and is at least about 20, or at least about 30, or at least about 40, or at least about 50, and at most about 220 amino acids in length. Alternatively or additionally, in certain embodiments, the CD28 polypeptide contained in the costimulatory signaling region of the CAR comprises or consists of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 180 to 220, or 200 to 220 of SEQ ID NO: 101. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide comprising or consisting of amino acids 180 to 220 of SEQ ID NO: 101.

[0180] An exemplary nucleic acid sequence encoding the amino acid sequence of amino acids 180 to 220 of SEQ ID NO: 101 is shown in SEQ ID NO: 102 (Table 5).

[0181] In certain embodiments, the intracellular signaling domain of the first antigen recognition receptor comprises a costimulatory signaling region comprising an intracellular domain of mouse CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide contained in the costimulatory signaling region comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical or homologous to an amino acid sequence having NCBI Reference Number: NP_031668.3 (or SEQ ID NO: 103) or a fragment thereof, and / or may optionally comprise at most one, at most two, or at most three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide contained in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence of at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length as a contiguous portion of SEQ ID NO: 103. In certain embodiments, the CD28 polypeptide contained in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence of amino acids 1 to 218, 1 to 50, 50 to 100, 100 to 150, 150 to 218, 178 to 218, or 200 to 218 of SEQ ID NO: 103. In certain embodiments, the costimulatory signaling region of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 178 to 218 of SEQ ID NO: 103 (Table 5).

[0182] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a 4-1BB polypeptide, e.g., an intracellular domain of 4-1BB or a portion thereof. In certain embodiments, the costimulatory signaling region comprises an intracellular domain of human 4-1BB or a portion thereof. In certain embodiments, the 4-1BB contained in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical or homologous to a sequence or fragment thereof having NCBI reference number: NP_001552 (SEQ ID NO: 104), and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the 4-1BB contained in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence of at least about 20, or at least about 30, or at least about 40, or at least about 50, and / or at most about 50, at most about 60, at most about 70, at most about 80, at most about 90, at most about 100, at most about 200, or at most about 255 amino acids in length as a contiguous portion of SEQ ID NO: 104. In certain embodiments, the costimulatory signaling region of the CAR comprises a 4-1BB polypeptide comprising or consisting of an amino acid sequence of amino acids 1 to 255, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 255 of SEQ ID NO: 104. In certain embodiments, the costimulatory signaling region of the CAR comprises a 4-1BB polypeptide comprising or consisting of an amino acid sequence of amino acids 214 to 255 of SEQ ID NO: 104 (Table 5).

[0183] In certain embodiments, the intracellular signaling domain of CAR comprises two costimulatory signaling regions, wherein the first costimulatory signaling region comprises the intracellular domain of a first costimulatory molecule or a portion thereof, and the second costimulatory signaling region comprises the intracellular domain of a second costimulatory molecule or a portion thereof. The first costimulatory molecule and the second costimulatory molecule are independently selected from the group consisting of: CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226 and NKG2D. In certain embodiments, the intracellular signaling domain of CAR comprises two costimulatory signaling regions, wherein the first costimulatory signaling region comprises the intracellular domain of CD28 or a portion thereof, and the second costimulatory signaling region comprises the intracellular domain of 4-1BB or a portion thereof.

[0184] In addition, the extracellular antigen binding domain of CAR may include a leader peptide or signal peptide that guides the nascent polypeptide into the endoplasmic reticulum. CAR is glycosylated and anchored in the cell membrane, so a signal peptide or leader peptide may be essential. The signal sequence or leader sequence may be a peptide sequence (e.g., about 5, about 10, about 15, about 20, about 25 or about 30 amino acids) present in the N-terminus of the newly synthesized polypeptide, which guides the protein into the secretory pathway. In certain embodiments, the signal peptide is covalently bonded to the 5' end (N-terminus) of the extracellular antigen binding domain of CAR. Exemplary leader sequences include, but are not limited to, a human IL-2 signal sequence (e.g., a human IL-2 signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 128), a mouse IL-2 signal sequence (e.g., a mouse IL-2 signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 129); a human κ leader sequence (e.g., a human κ leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 130), a mouse κ leader sequence (e.g., a mouse κ leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 131); a human CD8 leader sequence (e.g., a human CD8 leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 132); a truncated human CD8 signal peptide (e.g., a truncated human CD8 signal peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 133); a human albumin signal sequence (e.g., a human CD8 leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 134). NO:134 or a human albumin signal sequence consisting of the amino acid sequence shown in SEQ ID NO:134); and a human prolactin signal sequence (e.g., a human prolactin signal sequence comprising the amino acid sequence shown in SEQ ID NO:135 or a human prolactin signal sequence consisting of the amino acid sequence shown in SEQ ID NO:136). These exemplary signal sequences are shown in Table 6.

[0185] Table 6. Exemplary signal sequences

[0186] name sequence SEQ ID NO. Signal sequence 1 MYRMQLLSCIALSLALVTNS 128 Signal sequence 2 MYSMQLASCVTLTLVLLVNS 129 Signal sequence 3 METPAQLLFLLLLWLPDTTG 130 Signal sequence 4 METDTLLLWVLLLWVPGSTG 131 Signal sequence 5 MALPVTALLLPLALLLHAARP 132 Signal sequence 6 MALPVTALLLPLALLLHA 133 Signal sequence 7 MKWVTFISLLFSSAYS 134 Signal sequence 8 MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS 135

[0187] In certain embodiments, the signal peptide comprises a CD8 polypeptide, for example, the CAR comprises a truncated CD8 signal peptide. In certain embodiments, the signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 132.

[0188] 5.3.2. Chimeric costimulatory receptor (CCR)

[0189] In certain embodiments, one or more of the modular polypeptides used in the systems described herein is a CCR. The term "chimeric costimulatory receptor" or "CCR" refers to a chimeric receptor that binds to an antigen and provides a costimulatory signal but does not provide a T cell activation signal to the cell containing the CCR. Various CCRs are described in US20020018783, the contents of which are incorporated by reference in their entirety. CCRs mimic costimulatory signals, but unlike CARs, do not provide T cell activation signals. In certain embodiments, CCRs lack a CD3ζ polypeptide.

[0190] In the absence of natural costimulatory ligands on antigen presenting cells, CCR provides costimulatory signals (for example, CD28-like signals or 4-1BB-like signals). Combination antigen recognition (that is, CCR is used in combination with CAR) can enhance the T cell reactivity for the T cells expressing dual antigens, thereby improving selective tumor targeting. Kloss et al. describe a strategy, which integrates combination antigen recognition, split signal conduction and key T cell activation and costimulation balanced intensity to generate T cells, and the cells eliminate the target cells expressing antigen combination, while retaining the cells expressing each antigen individually (Kloss et al., Nature Biotechnology (2013); 31 (1): 71-75, the contents of which are incorporated by reference in their entirety). Utilizing this method, T cell activation requires recognition mediated by the CAR of a kind of antigen, but costimulation is independently mediated by CCR having specificity for the second antigen. In order to achieve tumor selectivity, the efficiency of T cell activation is reduced to such a level by the combination antigen recognition method, wherein it is invalid in the case of the rescue provided by CCR recognition while not being provided for the second antigen.

[0191] In certain embodiments, CCR includes an extracellular antigen binding domain that is combined with a second antigen, and an intracellular domain that can deliver a costimulatory signal to the cell but does not deliver an activation signal to the cell alone. In certain embodiments, CCR further includes a transmembrane domain. In certain embodiments, the intracellular domain of CCR includes an intracellular domain or a portion thereof of at least a costimulatory molecule. In certain embodiments, costimulatory molecules are selected from the group consisting of the following items: CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

[0192] In certain embodiments, the signaling domain of a CCR is a kinase. Non-limiting examples of such kinases include: the Src kinase family (e.g., Blk, Fgr, Fyn, Hck, Lck, Lyn, Src, Yes, Yrk); the Syk kinase family (e.g., Zap70, Syk, BTK, TEK, ITK); and the receptor tyrosine kinase (RTK) superfamily, which includes class I RTK (EGF receptor family or ErbB family), class II (insulin receptor family), class III (PDGF receptor family), class IV (VEGF receptor family), class V (FGF receptor family), class VI (CCK receptor family), class VII (NGF receptor family). , VIII (HGF receptor family), IX (Eph receptor family), X (AXL receptor family), XI (TIE receptor family), XII (RYK receptor family), XIII (DDR receptor family), XIV (RET receptor family), XV (ROS receptor family), XVI (LTK receptor family), XVII (ROR receptor family), XVIII (MuSK receptor family), XIX (LMR receptor), XX (undetermined), XVIII (MuSK receptor family), XIX (LMR receptor), XX and their orthologs. In certain embodiments, the signaling domain of the CAR comprises a kinase domain from one or more of PDGFR, KIT, Abl, Arg, EGFR, Raf, VEGFR, PDGFR, Flt3, Abl, Arg or ErbB2 or their orthologs.

[0193] In certain embodiments, CCR includes the intracellular domain of CD28 or a portion thereof. In certain embodiments, CCR includes the intracellular domain of 4-1BB or a portion thereof. In certain embodiments, CCR includes the intracellular domain of CD28 or a portion thereof and the intracellular domain of 4-1BB or a portion thereof.

[0194] In certain embodiments, the second antigen is selected so that the expression of the first antigen (e.g., an antigen targeted by CAR) and the second antigen are both constrained to the target cell (e.g., cancer tissue, or cancer cell, or LSC, or AML HSPC). Similar to CAR, the extracellular antigen binding domain can be a scFv, Fab, F (ab) 2 or a fusion polypeptide with a heterologous sequence for forming an extracellular antigen binding domain.

[0195] In certain embodiments, cells comprising a first antigen recognition receptor (e.g., CAR) and a second antigen recognition receptor (e.g., CAR, TCR, or TCR-like fusion molecule) are positive for both the first and second antigens and show a higher cytolytic activity than cells positive for only the first antigen. In certain embodiments, cells comprising a first antigen recognition receptor (e.g., CAR) and a second antigen recognition receptor (e.g., CAR, TCR, or TCR-like fusion molecule) do not substantially show cytolytic activity or show negligible cytolytic activity to cells positive for only the first antigen.

[0196] In certain embodiments, the first antigen recognition receptor (e.g., CAR, TCR, or TCR-like fusion molecule) binds to the antigen with low affinity (e.g., about 1×10 -8 M or larger, about 5×10 -8 M or larger, approximately 1×10 -7 M or larger, about 5×10 -7 M or larger, or about 1×10 -6 M or larger or about 1×10 -8 M is about 1×10 -6 The dissociation constant (K D )) binds to the first antigen. In certain embodiments, the first antigen recognition receptor (e.g., CAR, TCR, or TCR-like fusion molecule) binds to the first antigen with low binding affinity. In certain embodiments, the first antigen recognition receptor (e.g., CAR, TCR, or TCR-like fusion molecule) binds to the first antigen at a low accessible epitope. In certain embodiments, the first antigen recognition receptor (e.g., CAR, TCR, or TCR-like fusion molecule) binds to the first antigen with a lower binding affinity than the second antigen recognition receptor (e.g., CCR) binds to the second antigen with a lower binding affinity. In certain embodiments, the CCR binds to the first antigen with a low binding affinity of about 1×10 -9 M is about 1×10 -7 M (e.g., about 1×10 -7 M or smaller, approximately 1×10 -8 M or smaller, or about 1×10 -9 M or less) binding affinity K D Binds to a second antigen.

[0197] 5.3.3. T cell receptor (TCR)

[0198] In certain embodiments, one or more modular polypeptides used in the systems described herein are TCRs. TCR is a disulfide-linked heterodimeric polypeptide composed of two variable chains expressed as a part of a complex with an invariant CD3 chain molecule. TCR is found on the surface of T cells and is responsible for identifying antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, TCR includes α and β chains (encoded by TRA and TRB, respectively). In certain embodiments, TCR includes γ and δ chains (encoded by TRG and TRD, respectively).

[0199] Each chain of a TCR is composed of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is located close to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. The variable domains of the two chains each have three complementarity determining regions (CDRs).

[0200] In certain embodiments, the TCR can form a receptor complex with three dimeric signaling modules: CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or ζ / η. When the TCR complex engages its antigen and MHC (peptide / MHC), the T cell expressing the TCR complex is activated.

[0201] In certain embodiments, the antigen recognition receptor is an endogenous TCR. In certain embodiments, the antigen recognition receptor is a naturally occurring TCR.

[0202] In certain embodiments, the antigen recognition receptor is an exogenous TCR. In certain embodiments, the antigen recognition receptor is a recombinant TCR. In certain embodiments, the antigen recognition receptor is a non-naturally occurring TCR. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more amino acid residues. In certain embodiments, the non-naturally occurring TCR is modified from a naturally occurring TCR by at least one amino acid residue. In certain embodiments, the non-naturally occurring TCR is modified from a naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more amino acid residues.

[0203] 5.3.4. TCR-like fusion molecules

[0204] In certain embodiments, one or more of the modular polypeptides used in the systems described herein are TCR-like fusion molecules. The limiting examples of TCR-like fusion molecules include TCR-based chimeric antigen receptors (also referred to as "HIT-CAR" for example, independent of HLA, disclosed in International Patent Application No. PCT / US19 / 017525, and the international patent application is incorporated by reference in its entirety) and T cell receptor fusion constructs (TRuC) (for example, Baeuerle et al., " Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response, " Nature Communications Vol. 10, Article No.: 2087 (2019) disclosed in those, the document is incorporated by reference in its entirety).

[0205] In certain embodiments, the TCR-like fusion molecule is a recombinant T cell receptor (TCR). In certain embodiments, the recombinant TCR comprises at least one antigen binding chain. In certain embodiments, the antigen binding domain of the recombinant TCR comprises a ligand for a cell surface receptor, a receptor for a cell surface ligand, an antibody or its fragment antigen binding portion or the antigen binding portion of the TCR. In certain embodiments, the recombinant TCR comprises two antigen binding chains, i.e., a first antigen binding chain and a second antigen binding chain. In certain embodiments, the first antigen binding chain and the second antigen binding chain each comprise a constant domain. In certain embodiments, the recombinant TCR binds to an antigen (e.g., a first antigen or a second antigen) in an HLA-independent manner. Therefore, in certain embodiments, the recombinant TCR is an HLA-independent (or non-HLA-binding) TCR (referred to as "HIT-CAR" or "HIT").

[0206] In certain embodiments, the first antigen-binding chain comprises a heavy chain variable region (V H In certain embodiments, the second antigen-binding chain comprises a light chain variable region (V L In certain embodiments, the first antigen-binding chain comprises a V H The antigen-binding fragment of the present invention comprises an antigen-binding chain of the antibody V L antigen-binding fragment.

[0207] In certain embodiments, the constant domain comprises a TCR constant region selected from the group consisting of a native or modified TRAC polypeptide, a native or modified TRBC polypeptide, a native or modified TRDC polypeptide, a native or modified TRGC polypeptide, and any variants or functional fragments thereof. In certain embodiments, the constant domain comprises a native or modified TRAC polypeptide. In certain embodiments, the constant domain comprises a native or modified TRBC polypeptide. In certain embodiments, the first antigen-binding chain comprises a TRAC polypeptide, and the second antigen-binding chain comprises a TRBC polypeptide. In certain embodiments, the first antigen-binding chain comprises a TRBC polypeptide, and the second antigen-binding chain comprises a TRAC polypeptide.

[0208] In certain embodiments, the first antigen binding chain comprises the V H and a TRAC polypeptide, and the second antigen-binding chain comprises the V L and TRBC polypeptide.

[0209] In certain embodiments, the first antigen binding chain comprises the V H and a TRBC polypeptide, and the second antigen binding chain comprises the V L and TRAC peptides.

[0210] In certain embodiments, at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous. In certain embodiments, the TRAC polypeptide is endogenous. In certain embodiments, the TRBC polypeptide is endogenous. In certain embodiments, both the TRAC polypeptide and the TRBC polypeptide are endogenous.

[0211] In certain embodiments, the antigen binding chain is capable of associating with a CD3 zeta polypeptide. In certain embodiments, the antigen binding chain, upon binding to an antigen, is capable of activating the CD3 zeta polypeptide associated with the antigen binding chain. In certain embodiments, activation of the CD3 zeta polypeptide is capable of activating immune response cells. In certain embodiments, the TCR-like fusion molecule is capable of integrating with the CD3 complex and providing HLA-independent antigen recognition. In certain embodiments, the TCR-like fusion molecule replaces the endogenous TCR in the CD3 / TCR complex.

[0212] In certain embodiments, the constant domain comprises a TCR constant region, e.g., a T cell receptor alpha constant region (TRAC), a T cell receptor beta constant region (TRBC, e.g., TRBC1 or TRBC2), a T cell receptor gamma constant region (TRGC, e.g., TRGC1 or TRGC2), a T cell receptor delta constant region (TRDC), or any variant or functional fragment thereof.

[0213] In certain embodiments, the TRAC polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to an amino acid sequence encoded by a transcript expressed by the gene of NCBI Genbank ID: 28755, NG_001332.3, range 925603 to 930229 (SEQ ID NO: 136), or a fragment thereof, and / or may optionally comprise at most one, at most two, or at most three conservative amino acid substitutions. In certain embodiments, the TRAC polypeptide comprises or consists of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 136 (Table 7).

[0214] In certain embodiments, the TRBC polypeptide is a TRBC2 polypeptide. In certain embodiments, the TRBC2 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 135, or a fragment thereof, and / or may optionally comprise up to one, two, or three conservative amino acid substitutions. In certain embodiments, the TRBC2 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 137 (Table 7).

[0215] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 137 is shown in SEQ ID NO: 138 (Table 7).

[0216] In certain embodiments, the TRBC polypeptide is a TRBC1 polypeptide. In certain embodiments, the TRBC1 polypeptide contained in the costimulatory signaling region of the first antigen recognition receptor comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identical or homologous to the amino acid sequence shown in SEQ ID NO: 139, or a fragment thereof, and / or may optionally comprise up to one, two, or three conservative amino acid substitutions. In certain embodiments, the TRBC1 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 139 (Table 7).

[0217] In certain embodiments, the TRBC1 polypeptide contained in the costimulatory signaling region of the first antigen recognition receptor comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identical or homologous to the amino acid sequence shown in SEQ ID NO: 140, or a fragment thereof, and / or may optionally comprise at most one, at most two, or at most three conservative amino acid substitutions. In certain embodiments, the TRBC1 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 1408.

[0218] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 140 is shown in SEQ ID NO: 141 (Table 7).

[0219] In certain embodiments, the TRBC polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to an amino acid sequence encoded by a transcript expressed by the gene with NCBI GenBank ID: 28639, NG_001333.2 range 645749 to 647196 (TRBC1, SEQ ID NO: 142), NCBI GenBank ID: 28638, NG_001333.2 range 655095 to 656583 (TRBC2, SEQ ID NO: 143), or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRBC polypeptide comprises or consists of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 142. In certain embodiments, the TRBC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 143 (Table 7).

[0220] In certain embodiments, the TCR-like fusion molecule comprises a hinge / spacer connecting the first antigen-binding chain to the constant domain. In certain embodiments, the TCR-like fusion molecule comprises a hinge / spacer connecting the second antigen-binding chain to the constant domain. The hinge / spacer can be sufficiently flexible to allow the antigen-binding chain to be oriented in different directions, thereby promoting antigen recognition. In certain embodiments, the hinge / spacer can be a hinge region from IgG1, a CH2CH3 region of an immunoglobulin and a portion of CD3, a portion of a TCR α polypeptide, a portion of a TCR β polypeptide, a portion of a CD28 polypeptide, a portion of a CD8 polypeptide, or a synthetic spacer sequence. In certain embodiments, the hinge / spacer comprises a portion of a TCR α polypeptide. In certain embodiments, the hinge / spacer comprises a portion of a variable region (TRAV), a portion of a diversity region (TRAD), a portion of a junction region (TRAJ), a portion of a constant region (TRAC), or a combination thereof. In certain embodiments, the hinge / spacer comprises a portion of the TRAJ region of the TCR α polypeptide and a portion of the TRAC region. In certain embodiments, the hinge / spacer comprises or consists of the amino acid sequence set forth in SEQ ID NO: 144. In certain embodiments, the hinge / spacer comprises or consists of amino acids 1 to 3 of the sequence set forth in SEQ ID NO: 144. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 144 is set forth in SEQ ID NO: 145 (Table 7).

[0221] In certain embodiments, the hinge / spacer comprises a portion of a TCR beta polypeptide. In certain embodiments, the hinge / spacer comprises a portion of a variable region (TRBV), a portion of a diversity region (TRBD), a portion of a junction region (TRBJ), a portion of a constant region (TRBC), or a combination thereof. In certain embodiments, the hinge / spacer comprises a portion of a TRBJ region and a portion of a TRAC region (C) of a TCR beta polypeptide. In certain embodiments, the hinge / spacer comprises or consists of the amino acid sequence shown in SEQ ID NO: 146. In certain embodiments, the hinge / spacer comprises or consists of amino acids 1 to 2 in the sequence shown in SEQ ID NO: 146. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 146 is shown in SEQ ID NO: 147. Table 7 shows exemplary T cell receptors and hinge / spacer sequences.

[0222]

[0223]

[0224]

[0225]

[0226] In certain embodiments, the antigen binding chain does not comprise an intracellular domain. In certain embodiments, the antigen binding chain is capable of associating with a CD3 zeta polypeptide. In certain embodiments, the antigen binding chain associates with the CD3 zeta polypeptide via a constant domain. In certain embodiments, the CD3 zeta polypeptide is endogenous. In certain embodiments, the CD3 zeta polypeptide is exogenous. In certain embodiments, binding of the antigen binding chain to the target antigen is capable of activating the CD3 zeta polypeptide associated with the antigen binding chain. In certain embodiments, the exogenous CD3 zeta polypeptide is fused or integrated with a costimulatory molecule disclosed herein.

[0227] In certain embodiments, the TCR-like fusion molecule comprises an antigen binding chain comprising an intracellular domain. In certain embodiments, the intracellular domain comprises a CD3 zeta polypeptide. In certain embodiments, binding of the antigen binding chain to the antigen can activate the CD3 zeta polypeptide of the antigen binding chain.

[0228] In certain embodiments, the CD3 zeta polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to the amino acid sequence shown in SEQ ID NO: 109 or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD3 zeta polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 109 of at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 164 amino acids in length. In certain embodiments, the CD3 zeta polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 164, 100 to 150, or 150 to 164 of SEQ ID NO: 109. In certain embodiments, the CD3 zeta polypeptide comprises or consists of amino acids 52 to 164 of SEQ ID NO:109.

[0229] In certain embodiments, the CD3 zeta polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to one or more of SEQ ID NOs: 109 to 111 or 113, or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD3 zeta polypeptide comprises or consists of the amino acid sequence set forth in one or more of SEQ ID NOs: 109 to 111 or 113.

[0230] In certain embodiments, the TCR-like fusion molecule comprises an antigen binding chain comprising an intracellular domain, wherein the intracellular domain comprises a costimulatory signaling region. In certain embodiments, the intracellular domain comprises a costimulatory signaling region and a CD3 zeta polypeptide. In certain embodiments, the intracellular domain comprises a costimulatory signaling region and does not comprise a CD3 zeta polypeptide. In certain embodiments, the costimulatory signaling region comprises at least the intracellular domain of a costimulatory molecule disclosed herein.

[0231] In certain embodiments, the TCR-like fusion molecule is capable of associating with a CD3 complex (also referred to as a "T cell co-receptor"). In certain embodiments, the TCR-like fusion molecule and the CD3 complex form an antigen recognition receptor complex similar to a natural TCR / CD3 complex. In certain embodiments, the CD3 complex is endogenous. In certain embodiments, the CD3 complex is exogenous. In certain embodiments, the TCR-like fusion molecule replaces the natural and / or endogenous TCR in the CD3 / TCR complex. In certain embodiments, the CD3 complex comprises a CD3γ chain, a CD3δ chain, and two CD3ε chains. In certain embodiments, the CD3 gamma chain comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence shown in NCBI Reference No. NP_000064.1 (SEQ ID NO: 105), or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions.

[0232] In certain embodiments, the CD3 delta chain comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence shown in NCBI Reference No. NP_000723.1 (SEQ ID NO: 106), or a fragment thereof, or the amino acid sequence shown in NCBI Reference No. NP_001035741.1 (SEQ ID NO: 107), or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions.

[0233] In certain embodiments, the CD3 epsilon chain comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence shown in NCBI Reference No. NP_000724.1 (SEQ ID NO: 108), or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions.

[0234] In certain embodiments, TCR sample fusion molecules show a higher antigen sensitivity than the CAR targeting the same antigen.In certain embodiments, TCR sample fusion molecules can induce immune response when combined with low-density antigens on the surface of tumor cells.In certain embodiments, cells comprising TCR sample fusion molecules can be used to treat subjects with tumor cells (for example, from the recurrence of disease) with low expression levels of surface antigens, wherein the subject has received the treatment causing residual tumor cells.In certain embodiments, tumor cells have low density of target molecules on the surface of tumor cells.In certain embodiments, there is low density of target molecules on the cell surface with less than about 5,000 molecules / cell, less than about 4,000 molecules / cell, less than about 3,000 molecules / cell, less than about 2,000 molecules / cell, less than about 1,500 molecules / cell, less than about 1,000 molecules / cell, less than about 500 molecules / cell, less than about 200 molecules / cell or less than about 100 molecules / cell density. In certain embodiments, the target molecule with low density on the cell surface has a density of less than about 2,000 molecules / cell. In certain embodiments, the target molecule with low antigen density on the cell surface has a density of less than about 1,500 molecules / cell. In certain embodiments, the target molecule with low antigen density on the cell surface has a density of less than about 1,000 molecules / cell. In certain embodiments, the target molecule with low antigen density on the cell surface has a density of between about 4,000 molecules / cell and about 2,000 molecules / cell, between about 2,000 molecules / cell and about 1,000 molecules / cell, between about 1,500 molecules / cell and about 1,000 molecules / cell, between about 2,000 molecules / cell and about 500 molecules / cell, between about 1,000 molecules / cell and about 200 molecules / cell, or between about 1,000 molecules / cell and about 100 molecules / cell.

[0235] Various TCR-like fusion molecules are disclosed in International Patent Application Publication No. WO2019 / 133969, which is hereby incorporated by reference in its entirety.

[0236] 5.4. Antigens

[0237] In certain embodiments, the antigen bound by the modular polypeptides of the present disclosure is a tumor antigen. Any tumor antigen (antigenic peptide) can be used in the tumor-related embodiments described herein. The source of the antigen includes, but is not limited to, cancer polypeptides. The antigen can be expressed as a peptide or as a complete polypeptide or a portion thereof. The complete polypeptide or a portion thereof can be natural or mutagenized. Non-limiting examples of antigens include: CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, antigens of cytomegalovirus (CMV) infected cells (e.g., cell surface antigens), ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155, CC R1, CD10, CD117, CD123, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD30, CD300LF, CD32, CD321, CD33, CD34, C D36, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, COLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B , FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-a, FOLR2, FRMD5, GABRB2, GAS2, GD2, GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3,ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B, kappa-light chain, L1CAM, LAX1, LEPR, LewisY(CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MYAD ML2, NGFR, NKCS1, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), peptidase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine peptide kinase Erb-B2, RHBD L3, RNF173, RNF183, ROR1, RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, S LC22A5, SLC25A36, SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1 , SPAG17, STC1, STON2, SUN3, survivin, SUSD2, SYNC, TACSTD2, TAS1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycopolypeptide 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2),VLA-4, Wilms tumor polypeptide (WT-1), WNT4, WT1 and ZDHHC11.

[0238] In certain embodiments, the antigen is CD19. In certain embodiments, the antigen is expressed on acute myeloid leukemia (AML) tissue. In certain embodiments, the antigen is expressed on acute myeloid leukemia (AML) hematopoietic stem cells / progenitor cells (HSPC) and / or leukemia stem cells (LSC). In certain embodiments, AML HSPC expresses CD34. In certain embodiments, the antigen is expressed in malignant hematopoietic stem cells and / or malignant hematopoietic progenitor cells. In certain embodiments, the antigen is not expressed or is expressed at an undetectable level in non-malignant hematopoietic stem cells and / or non-malignant hematopoietic progenitor cells. In certain embodiments, the antigen is selected from the group consisting of: CD70, IL1RAP, CD19, CD33, CLEC12A, ADGRE2, CD123, and combinations thereof.

[0239] In certain embodiments, the antigen is a pathogen antigen. Non-limiting examples of viruses include: Retroviridae (e.g., human immunodeficiency virus, such as HIV-1 (also known as HDTV-III, LAVE or HTLV-III / LAV, or HIV-III); and other isolates, such as HIV-LP; Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviridae (e.g., e.g., dengue virus, encephalitis virus, yellow fever virus); Coronoviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bungaviridae (e.g., hantavirus, bungavirus, phlebovirus, and Naira virus); Arenaviridae (e.g., leptospirosis virus, flu virus); viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus); iridae) (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpes viruses); Poxviridae (variola virus, vaccinia virus, poxviruses); and Iridoviridae (e.g., African swine fever virus); as well as unclassified viruses (e.g., the causative agent of hepatitis D (thought to be a defective satellite of hepatitis B virus), the causative agents of non-A, non-B hepatitis (category 1 = internal transmission); category 2 = parenteral transmission (i.e., hepatitis C); Norwalk and related viruses, and astroviruses).

[0240] Non-limiting examples of bacteria include: Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pyloris, Borrelia burgdorferi, Legionella, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae, M. leprae), Staphylococcus aureus, Staphylococcus epidermidis, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes, pyogenes) (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Campylobacter jejuni, Enterococcus sp.), Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium spp., Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelli. Mycoplasma, Pseudomonas aeruginosa, Pseudomonas fluorescens, Corynobacteria diphtheriae, Bartonella henselae, Bartonella quintana, Coxiella burnetii, Chlamydia, Shigella, Yersinia enterocolitica, Yersinia pseudotuberculosis, Listeria monocytogenes, Mycoplasma spp.), Vibrio cholerae, Borrelia, Francisella, Brucella melitensis, Proteus mirabilis, and Proteus.

[0241] In certain embodiments, the pathogen antigen is a viral antigen present in cytomegalovirus (CMV), a viral antigen present in Epstein-Barr virus (EBV), a viral antigen present in human immunodeficiency virus (HIV), or a viral antigen present in influenza virus.

[0242] 5.5. Carrier

[0243] Genetic modification of immune response cells (e.g., T cells) can be accomplished by transducing a substantially homogeneous cell composition with a recombinant DNA construct. In certain embodiments, a retroviral vector is used to introduce the DNA construct into the cell. For example, a polynucleotide encoding any polypeptide or system disclosed herein can be cloned into a retroviral vector and expression can be driven by its endogenous promoter, a retroviral long terminal repeat sequence, or a promoter specific to the target cell type. In certain embodiments, the retroviral vector is a γ-retroviral vector. In certain embodiments, the retroviral vector is a lentiviral vector. Non-viral vectors can also be used.

[0244] For the initial genetic modification of immune response cells to include the polypeptides and / or systems disclosed herein, retroviral vectors are typically used for transduction, however any other suitable viral vector or non-viral delivery system may also be used. The polypeptides and / or systems can be constructed in a single polycistronic expression cassette, in multiple expression cassettes in a single vector, or in multiple vectors. Examples of elements for generating polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Oral IRES, Picornavirus IRES, Poliovirus IRES, and Encephalomyocarditis Virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A peptides, T2A peptides, E2A peptides, and F2A peptides, e.g., SEQ ID NOs: 23 to 26). Combinations of retroviral vectors and appropriate packaging lines are also suitable, wherein the capsid polypeptide will have the function of infecting human cells. Various cell lines that produce amphotropic viruses are known, including but not limited to PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5: 431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6: 2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85: 6460-6464). Non-amphotropic particles are also suitable, for example, particles pseudotyped with VSVG, RD114 or GALV envelopes, and any other known in the art.

[0245] Possible transduction methods also include direct co-cultivation of cells with producer cells, for example, by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culturing with viral supernatant alone or concentrated vector stock with or without appropriate growth factors and polycations, for example, by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0246] Other transduction virus vectors can be used to modify immune response cells. In certain embodiments, the selected vector shows high infection efficiency, stable incorporation into the host cell genome and persistent expression of the recombinant gene product (see, for example, Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997). Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, or herpes viruses such as Epstein-Barr virus (see also, for example, Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 23:41-42, 1986). 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well-established and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).

[0247] Non-viral methods can also be used for genetic modification of immune response cells. For example, nucleic acid molecules can be introduced into immune response cells by administering the nucleic acid in the presence of lipofectamine (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), by asialomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also have potential benefits for delivering DNA into cells. Transplanting normal genes into the affected tissues of the subject can also be accomplished by transferring normal nucleic acids in vitro to culturable cell types (e.g., autologous or heterologous primary cells or their progeny), followed by injection of cells (or their progeny) into target tissues or systemic injection. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALEN nucleases, CRISPR). Transient expression can be obtained by RNA electroporation. In certain embodiments, recombinant receptors can be introduced by transposon-based vectors. In certain embodiments, transposon-based vectors include transposons (also known as transposable elements). In certain embodiments, transposons can be recognized by transposases. In certain embodiments, transposases are "Sleeping Beauty" transposases.

[0248] The resulting cells can be grown under conditions similar to unmodified cells, allowing the modified cells to be expanded and used for a variety of purposes.

[0249] 5.6. Cells

[0250] In certain embodiments, the cell is selected from the group consisting of lymphocytes and myeloid cells. In certain embodiments, the cell is an immune response cell. In certain embodiments, the immune response cell is a lymphocyte.

[0251] In certain embodiments, the cell is a cell of the lymphoid lineage. The cell of the lymphoid lineage can produce antibodies, regulate the cellular immune system, detect foreign agents in the blood, detect foreign cells in the host, etc. Non-limiting examples of cells of the lymphoid lineage include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells can be differentiated. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., embryonic stem cell).

[0252] In certain embodiments, the cell is a T cell. T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject matter disclosed herein can be any type of T cell, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells), stem cell-like memory T cells (or stem cell-like memory T cells), and two types of effector memory T cells: for example, T cells. EM cells and T EMRA T cells, regulatory T cells (also known as suppressor T cells), tumor infiltrating lymphocytes (TILs), natural killer T cells, mucosal-associated invariant T cells, and gamma delta T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. A patient's own T cells can be genetically modified to target specific antigens by introducing antigen recognition receptors (e.g., CAR or TCR). T cells can be CD4 + T cells or CD8 + In certain embodiments, the T cells are CD4 + In certain embodiments, the T cells are CD8 + In certain embodiments, CD8 + T cells are CD4 independent. In certain embodiments, T cells are derived from induced pluripotent stem cells (iPSCs). In certain embodiments, T cells are CD8 independent of CD4. + T cells, and CD8 + T cells are derived from iPSCs.

[0253] The types of human lymphocytes of the subject matter disclosed herein include, but are not limited to, peripheral donor lymphocytes, such as those disclosed in Sadelain, M. et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CAR), Morgan, RA et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T cell receptor complex comprising α and β heterodimers), Panelli, MC et al. 2000 J Immunol 164:495-504; Panelli, MC et al. 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont, J., et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, GA et al. 2003 Blood 102:2498-2505 (disclosing the selective in vitro expansion of antigen-specific peripheral blood leukocytes using artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells).

[0254] In certain embodiments, the cells (e.g., T cells) are autologous. In certain embodiments, the cells (e.g., T cells) are non-autologous. In certain embodiments, the cells (e.g., T cells) are allogenic. In certain embodiments, the cells (e.g., T cells) are derived in vitro from engineered progenitor cells or stem cells.

[0255] In certain embodiments, the cell is a cell of the myeloid lineage. Non-limiting examples of cells of the myeloid lineage include: monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, and stem cells from which myeloid cells can differentiate.

[0256] In certain embodiments, the stem cells are pluripotent stem cells (eg, embryonic stem cells or induced pluripotent stem cells).

[0257] 5.7. Formulation and Administration

[0258] The subject matter disclosed herein provides compositions comprising the cells disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0259] The composition comprising cells disclosed herein can be conveniently provided in the form of sterile liquid preparations (e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions), which can be buffered to selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions and solid compositions. Additionally, it is slightly more convenient to use liquid compositions (especially by injection). On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact period with specific tissues. Liquid or viscous compositions can include a carrier, which can be a solvent or a dispersion medium containing, for example, water, saline, phosphate buffered saline (PBS), a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and a mixture thereof that is suitable.

[0260] The composition comprising cells disclosed herein can be provided to a subject systemically or directly for inducing and / or enhancing an immune response to an antigen and / or treating and / or preventing a vegetation. In certain embodiments, cells disclosed herein or compositions comprising them are directly injected into an organ of interest (e.g., an organ affected by a vegetation). Alternatively, cells disclosed herein or compositions comprising them are indirectly provided to an organ of interest, e.g., by being administered into a circulatory system (e.g., a tumor vasculature). Amplifiers and differentiation agents can be provided before, during, or after administering cells or compositions to increase the in vitro or in vivo production of cells.

[0261] The number of cells to be administered can vary depending on the subject being treated. In certain embodiments, approximately 10 4 and about 10 10 Between about 10 4 and about 10 7 Between about 10 5 and about 10 7 Between about 10 5 and about 10 9 Between, or about 10 6 and about 10 8 In certain embodiments, about 10 5 and about 10 7 More potent cells can be administered in even smaller quantities. Typically, at least about 1×10 5 cells, eventually reaching about l×l0 10 In certain embodiments, at least about 1×10 5 pcs, about 5×10 5 pcs, about 1×10 6 pcs, about 5×10 6 pcs, about 1×107 pcs, about 5×10 7 pcs, about 1×10 8 or about 5×10 8 In certain embodiments, about 1×10 5 In certain embodiments, about 5×10 5 In certain embodiments, about 1×10 6 The presently disclosed cells. The precise determination of what will be considered an effective dose can be based on individual factors for each subject, including their size, age, sex, weight, and the condition of the particular subject. Dosages can be readily determined by one skilled in the art from this disclosure and knowledge in the art.

[0262] Cell disclosed in the present application and composition can be used by any method known in the art, including but not limited to intravenous administration to experimenter, subcutaneous administration, intra-node administration, intratumor administration, intrathecal administration, intrapleural administration, intraosseous administration, intraperitoneal administration, pleural administration and direct administration.Cell disclosed in the present application can be used with any physiologically acceptable medium (usually intravascular), although it can also be introduced into other convenient positions (for example, thymus) of the appropriate position for regeneration and differentiation that cell can find.Cell can be introduced by injection, conduit etc.

[0263] The compositions comprising cells disclosed herein can be provided to subject systemically or directly for inducing and / or enhancing immune response to antigen and / or treating and / or preventing vegetation (for example, cancer), pathogen infection or infectious disease. In certain embodiments, cells disclosed herein, compositions or nucleic acid compositions are directly injected into an organ of interest (for example, an organ affected by vegetation). Alternatively, cells disclosed herein, compositions or nucleic acid compositions are indirectly provided to an organ of interest, for example, by being administered into the circulatory system (for example, tumor vasculature). Amplifiers and differentiation agents can be provided before, during or after administering cells, compositions or nucleic acid compositions to increase the in vitro or in vivo production of cells (for example, T cells (for example, CTL cells)).

[0264] Compositions disclosed herein can be a pharmaceutical composition comprising cells disclosed herein or its progenitor cells and a pharmaceutically acceptable carrier. Administration can be autologous or allogenic. For example, cells or progenitor cells can be obtained from a subject and applied to the same subject or different compatible subjects. Peripheral blood-derived cells or their offspring (for example, in vivo, ex vivo or in vitro derived) can be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection or parenteral administration. When administering the therapeutic composition of the subject disclosed herein (for example, a pharmaceutical composition comprising cells disclosed herein), it can be formulated into unit dose injectable forms (solutions, suspensions, emulsions).

[0265] 5.8. Treatment

[0266] The subject matter disclosed herein provides various methods of using the cells disclosed herein or compositions comprising the same. The cells disclosed herein and compositions comprising the same can be used in therapy or medicine. For example, the subject matter disclosed herein provides methods for inducing and / or increasing an immune response in a subject in need thereof. The cells disclosed herein and compositions comprising the same can be used to reduce tumor burden in a subject. The cells disclosed herein and compositions comprising the same can reduce the number of tumor cells, reduce tumor size, and / or eradicate tumors in a subject. The cells disclosed herein and compositions comprising the same can be used to treat and / or prevent tumors (or neoplasms) in a subject. The cells disclosed herein and compositions comprising the same can be used to extend the survival of a subject suffering from a tumor. In certain embodiments, the tumor is cancer. The cells, compositions, and nucleic acid compositions disclosed herein can also be used to treat and / or prevent pathogen infection or other infectious diseases in a subject (such as an immunocompromised human subject). The cells, compositions, and nucleic acid compositions disclosed herein can also be used to treat and / or prevent autoimmune diseases in a subject. In certain embodiments, each of the above methods includes administering the cells disclosed herein or a composition comprising the same (e.g., a pharmaceutical composition) to achieve a desired effect, e.g., to alleviate an existing condition or prevent recurrence. For treatment, the amount administered is an amount that effectively produces the desired effect. An effective amount can be provided in a single administration or a series of administrations. An effective amount can be provided in a bolus or by continuous perfusion.

[0267] Non-limiting examples of tumors (or neoplasms) include: blood cancers (e.g., leukemias, lymphomas, and myelomas), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcomas, and various carcinomas (including prostate cancer and small cell lung cancer). Suitable cancers further include any cancer known in the art of oncology, including, but not limited to, astrocytomas, fibrosarcomas, myxosarcoma, liposarcoma, oligodendroglioma, ependymomas, medulloblastomas, primitive neuroectodermal tumors (PNETs), chondrosarcomas, osteogenic sarcomas, pancreatic ductal adenocarcinomas, small cell and large cell lung adenocarcinomas, chordomas, angiosarcomas, endotheliosarcomas, squamous cell carcinomas, bronchoalveolar carcinomas, epithelial adenocarcinomas and their liver metastases, lymphangiosarcomas, lymphangioendotheliosarcomas, hepatomas, bile duct carcinomas, synoviomas, mesotheliomas, Ewing's tumors, rhabdomyosarcomas, colon carcinomas, basal cell carcinomas, sweat gland carcinomas, papillary carcinomas, sebaceous gland carcinomas, papillary adenocarcinomas, cystadenocarcinomas, Medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia and heavy chain disease, breast tumors such as ductal and lobular adenocarcinoma, cervical squamous and adenocarcinoma, uterine and ovarian epithelial carcinoma, prostate adenocarcinoma, bladder transitional squamous cell carcinoma, B-cell and T-cell lymphoma (nodular and diffuse) plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma and leiomyosarcoma. In certain embodiments, the neoplasm is a cancer. In certain embodiments, the neoplasm is selected from the group consisting of blood cancer (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, and laryngeal cancer. In certain embodiments, the cells, compositions, and nucleic acid compositions disclosed herein can be used to treat and / or prevent blood cancer (e.g., leukemia, lymphoma, and myeloma) or ovarian cancer that is not suitable for conventional therapeutic intervention.

[0268] In certain embodiments, the tumor and / or neoplasm is a solid tumor. Non-limiting examples of solid tumors include: renal cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, lung neuroendocrine carcinoma, small cell lung cancer, pancreatic cancer, breast cancer, astrocytoma, glioblastoma, laryngeal / pharyngeal cancer, EBV-associated nasopharyngeal carcinoma, and ovarian cancer.

[0269] In certain embodiments, the tumor and / or neoplasm is a blood cancer. Non-limiting examples of blood cancers include multiple myeloma, leukemia, and lymphoma. Non-limiting examples of leukemias include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia. Lymphoma can be Hodgkin's lymphoma or non-Hodgkin's lymphoma. In certain embodiments, lymphoma is non-Hodgkin's lymphoma, including B-cell non-Hodgkin's lymphoma and T-cell non-Hodgkin's lymphoma.

[0270] In certain embodiments, the tumor and / or neoplasm is a B-cell malignancy. Non-limiting examples of B-cell malignancies include: B-cell non-Hodgkin's lymphoma (NHL), B-cell Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter's transformation, and CNS lymphoma.

[0271] In certain embodiments, the tumor and / or neoplasm is a B cell-associated neoplasm. Non-limiting examples of B cell-associated tumors include chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), B cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic B cell lymphoma / leukemia (unclassified), splenic diffuse red pulp small B cell lymphoma, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, monoclonal gammopathy of undetermined significance (MGUS, IgM), heavy chain disease (α heavy chain disease, γ heavy chain disease, μ heavy chain disease), M GUS (IgG / A), plasma cell myeloma, solitary plasmacytoma of bone, extraskeletal plasmacytoma, monoclonal immunoglobulin deposition disease, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, follicular lymphoma, follicular neoplasm in situ, duodenal follicular lymphoma, pediatric follicular lymphoma, large B-cell lymphoma with IRF4 rearrangement, primary cutaneous follicle center cell lymphoma, mantle cell lymphoma, primary Mantle cell tumor, diffuse large B-cell lymphoma (DLBCL) (not otherwise specified (NOS)), germinal center B-cell type, activated B-cell type, T-cell / histiocyte-rich large B-cell lymphoma, primary DLBCL of the central nervous system (CNS), primary cutaneous DLBCL (leg type), Epstein-Barr virus (EBV)-positive DLBCL (NOS), EBV-positive mucocutaneous ulcers, DLBCL associated with chronic inflammation, lymphomatoid granuloma, primary longitudinal Septal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, human herpes virus 8 (HHV-8)-associated DLBCL (NOS), Burkitt lymphoma, Burkitt-like lymphoma with 11q aberration, high-grade B-cell lymphoma with rearrangement of MYC and BLC2 and / or BCL6, high-grade B-cell lymphoma (NOS), and B-cell lymphoma (unclassifiable).

[0272] In certain embodiments, the tumor and / or neoplasm is a myeloid disorder. Non-limiting examples of myeloid disorders include myelodysplastic syndrome, myeloproliferative neoplasms, chronic myelomonocytic leukemia, acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasms, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, chronic myeloid leukemia, and polycythemia vera.

[0273] In certain embodiments, the bone marrow disorder is acute myeloid leukemia (AML). In certain embodiments, the first antigen and / or the second antigen are independently selected from the group consisting of CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155, CCR1 , CD10, CD117, CD123, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD30, CD300LF, CD32, CD321, CD33, CD34, CD36, CD38, CD41 , CD44, CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2 , COL15A1, COLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-a, FOLR2, FRMD5, GABRB 2, GAS2, GD2, GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2,KDR, KIF19, KIF26B, kappa-light chain, L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MYADML2, NGFR, N KCS1, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), peptidase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine peptide kinase Erb-B2, RHBDL3, RNF173, RNF183, RO R1, RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, SLC25A41 , SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, survivin, SUSD2, SYNC, TACSTD2, TAS1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycopolypeptide 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms' tumor polypeptide (WT-1), WNT4, WT1, and ZDHHC11.

[0274] In certain embodiments, the myeloid disorder is acute myeloid leukemia (AML), and the first antigen and / or the second antigen are expressed on AML hematopoietic stem cells / progenitor cells (HSPCs) and / or leukemic stem cells (LSCs). In certain embodiments, AML HSPCs express CD34. In certain embodiments, the first antigen and / or the second antigen are not expressed or expressed at undetectable levels in non-hematopoietic stem cells and / or non-hematopoietic progenitor cells. In certain embodiments, the first antigen and / or the second antigen are independently selected from the group consisting of CD70, IL1RAP, CD33, CLEC12A, ADGRE2, CD123, and combinations thereof.

[0275] The subject disclosed in the present application provides a method for treating and / or preventing viral infection in a subject. The method may include: applying an effective amount of cell disclosed in the present application, compositions disclosed in the present application, or nucleic acid compositions disclosed in the present application to a subject suffering from viral infection. The non-limiting examples of viral infection include viral infection caused by the following items: cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis F or hepatitis G, human immunodeficiency virus (HIV), adenovirus, BK polyomavirus, coronavirus, coxsackievirus, poliovirus, herpes simplex virus type 1, herpes simplex virus type 2, human cytomegalovirus, human herpes virus type 8, varicella zoster virus, influenza virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, papillomavirus, rabies virus, and rubella virus. Other viral targets include: Paramyxoviridae (e.g., pneumovirus, measles virus, metapneumovirus, respiratory virus, or mumps virus), Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., arenaviruses such as lymphocytic choriomeningitis virus), Arteriviridae (e.g., porcine respiratory and reproductive syndrome virus or equine arteritis virus), Bunyaviridae (e.g., phlebovirus or hantavirus), Caliciviridae (e.g., Norwalk virus), Coronaviridae (e.g., coronavirus or torovirus), Filoviridae (e.g., Ebola-like virus), Flaviviridae (e.g., hepatitis virus or flavivirus), Viruses), Herpesviridae (e.g., herpes simplex virus, varicella virus, cytomegalovirus, roseophagitis virus, or lymphocytic cryptovirus), Orthomyxoviridae (e.g., influenza virus or togovirus), Parvoviridae (e.g., parvovirus), Picomaviridae (e.g., enterovirus or hepatovirus), Poxviridae (e.g., orthopoxvirus, fowlpox virus, or leporipoxvirus), Retroviridae (e.g., lentivirus or spumavirus), Reoviridae (e.g., rotavirus), Rhabdoviridae (e.g., rabies virus, granulorhabdovirus, or vesiculovirus), and Togaviridae (e.g., alphavirus or rubella virus).In certain embodiments, viral infections include human respiratory coronaviruses, influenza viruses A through C, hepatitis A through G, and herpes simplex viruses 1 through 9. In certain embodiments, the subject is immunocompromised.

[0276] The subject matter disclosed herein provides a method for treating and / or preventing a bacterial infection in a subject. The method may comprise administering an effective amount of a cell disclosed herein, a composition disclosed herein, or a nucleic acid composition disclosed herein to a subject suffering from a bacterial infection. Bacterial infections include, but are not limited to, Mycobacteria, Rickettsia, Mycoplasma, Neisseria meningitides, Neisseria gonorrheoeae, Legionella, Vibrio cholerae, Streptococci, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Corynobacteria diphtheriae, Clostridium spp., enterotoxigenic Eschericia coli, Bacillus anthracis, and Enterotoxigenic Escherichia coli. anthracis, Rickettsia, Bartonella henselae, Bartonella quintana, Coxiella burnetii, Chlamydia, Mycobacterium leprae, Salmonella, Shigella, Yersinia enterocolitica, Yersinia pseudotuberculosis; Legionella pneumophila; Mycobacterium tuberculosis; Listeria monocytogenes; Mycoplasma spp.), Pseudomonas fluorescens, Vibrio cholerae, Haemophilus influenzae, Bacillus anthracis, Treponema pallidum, Leptospira, Borrelia, Corynebacterium diphtheriae, Francisella, Brucella melitensis, Campylobacter jejuni, Enterobacter, Proteus mirabilis, Proteus, and Klebsiella pneumoniae.

[0277] The subject matter disclosed herein provides methods for treating and / or preventing autoimmune diseases in subjects. The methods may include administering an effective amount of cells, compositions, or nucleic acid compositions disclosed herein to a subject suffering from an autoimmune disease.

[0278] The subject matter disclosed herein provides methods for treating and / or preventing infectious diseases in subjects. The methods may include administering an effective amount of cells disclosed herein, compositions disclosed herein, or nucleic acid compositions disclosed herein to a subject suffering from an infectious disease.

[0279] Non-limiting examples of autoimmune and inflammatory diseases or conditions thereof include arthritis (e.g., rheumatoid arthritis (RA)), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, ulcerative colitis, psoriasis, psoriatic arthritis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease, multiple sclerosis, systemic sclerosis, asthma, organ transplant rejection, diseases or conditions associated with transplantation, Takayasu's arteritis, giant cell arteritis, Kawasaki disease, polyarteritis nodosa, Behçet's syndrome, Wegener's granulomatosis, ANCA vasculitis, Chalger-Strauss syndrome, microscopic polyangiitis, connective tissue disease vasculitis, Hennoch-Schonlein purpura, cryoglobulinemic vasculitis, cutaneous leukocytoclastic vasculitis, sarcoidosis, Cogan's syndrome, Wiskott-Aldrich syndrome, primary CNS vasculitis, thrombotic occlusion, and the like. Thrombotic vasculitis, paraneoplastic arteritis, myelodysplastic syndrome, erythema eminentum, amyloidosis, autoimmune myositis, Guillain-Barré syndrome, histiocytosis, atopic dermatitis, pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still's disease, Sjögren's syndrome, myelofibrosis, chronic inflammatory demyelinating polyneuropathy, Kimura's disease, systemic sclerosis, chronic periaortitis, chronic prostatitis, idiopathic pulmonary fibrosis chemoattractant, chronic granulomatous disease, idiopathic bleomycin-induced lung inflammation, cytarabine-induced lung inflammation, autoimmune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, autoimmune lymphopenia, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto's thyroiditis, atopic thyroiditis, Graves' disease, autoimmune polyglandular syndrome, autoimmune Addison's syndrome and / or myasthenia gravis. According to the subject matter disclosed in the present application, the various methods described above may include: administering a checkpoint immune blocker to the subject.

[0280] The subject may have advanced disease, in which case the goal of treatment may include slowing or reversing disease progression, and / or ameliorating side effects. The subject may have a history of prior treatment, in which case the goal of treatment will generally include reducing or delaying the risk of relapse.

[0281] Further modifications can be introduced into the cells disclosed herein to avoid or minimize the risk of immunological complications (referred to as "malignant T cell transformation"), for example, graft-versus-host disease (GvHD), or when healthy tissue expresses the same target antigen as the tumor cell, causing results similar to GvHD. A potential solution to this problem is to engineer suicide genes into the cells disclosed herein. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible caspase 9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptides. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can achieve T cell elimination by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). EGFRt can be covalently linked to the upstream of an antigen recognition receptor. The suicide gene can be contained in a vector comprising a nucleic acid encoding an antigen recognition receptor disclosed herein. In this way, administration of a prodrug designed to activate a suicide gene (e.g., a prodrug (e.g., AP1903 that can activate iCasp-9)) during malignant T cell transformation (e.g., GVHD) triggers apoptosis in suicide gene-activated cells expressing the antigen recognition receptor disclosed herein. Incorporation of a suicide gene into the antigen recognition receptor disclosed herein confers an increased level of safety by enabling the elimination of the majority of receptor-expressing cells within a very short period of time. Cells disclosed herein that incorporate a suicide gene can be pre-eliminated at a given time point after cell infusion or eradicated at the earliest sign of toxicity.

[0282] 5.9. Kit

[0283] The subject matter disclosed in the present application provides a kit for inducing and / or enhancing immune response and / or treating and / or preventing vegetation or pathogen infection (e.g., autoimmune disease or infectious disease) in a subject. In certain embodiments, the kit includes a composition, buffer, nucleic acid, carrier and reagent for producing cells disclosed in the present application. In certain embodiments, the kit includes a sterile container; such container can be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack or other suitable container forms known in the art. Such container can be made of plastic, glass, laminated paper, metal foil or other materials suitable for accommodating medicine.

[0284] If desired, the cell, composition, or nucleic acid composition is provided with instructions for administering the cell, composition, or nucleic acid composition to a subject having a tumor (e.g., cancer) or a pathogen infection (e.g., an infectious disease) or an immune disorder (e.g., an autoimmune disease) or a risk of developing a tumor, pathogen infection, or immune disorder. The instructions generally include information about the use of the cell, composition, or nucleic acid composition for treating and / or preventing a neoplasm or pathogen infection (e.g., an infectious disease) or an immune disorder (e.g., an autoimmune disease). In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; dosage regimens and methods of administration for treating or preventing tumors, pathogen infection (e.g., an infectious disease), or immune disorders (e.g., an autoimmune disease), or symptoms thereof; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. These instructions may be printed directly on the container (if present), or as a label applied to the container, or as a separate sheet, booklet, card, or folder provided with or in the container.

[0285] 5.10. Example Examples

[0286]

[0287] In certain non-limiting embodiments, the subject matter disclosed herein relates to systems comprising two or more nucleic acid constructs, each comprising a nucleotide sequence encoding at least one extein; at least one of a complementary N-split intein and C-split intein pair; at least one endoplasmic reticulum (ER) retention motif; and a regulator motif or regulatable domain that regulates expression of the extein. In some of such systems, the extein comprises one or more of an extracellular antigen binding domain, an extracellular cytokine, a transmembrane domain, or a signaling domain. In some of such systems, the complementary split intein pair is selected from the group consisting of a Cfa intein, a gp41-1 intein, a gp41-8 intein, an Aes123PolB1 intein (Aes intein), an NrdJ-1 intein, an IMPDH-1 intein, an SspGyrB intein, a DNA polymerase III (DnaE) intein, orthologs thereof, and variants thereof. In some of these systems, the N-split intein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, and 9. In some of these systems, the C-split intein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, and 10. In some of these systems, the N-split intein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, and 9, and the complementary C-split intein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, and 10, respectively. In some of these systems, the ER retention motif is an E319K motif (KKXX) or an RXR motif. In some of these systems, the E319K motif comprises the sequence of SEQ ID NO: 18. In some of these systems, the RXR motif comprises a sequence selected from any one of SEQ ID NOs: 19 to 22. In some of these systems, the N-terminal amino acid of the ER retention motif is linked to the C-terminal amino acid of the N-split intein. In some of these systems, the ER retention motif is an RXR motif flanked by sequences encoding exteins. In some of these systems, the regulator motif or regulatable domain comprises a drug-stabilized signaling domain, a drug-destabilized degradation determinant domain, or a drug-regulatable self-cleavage domain. In some of these systems, the extein comprises a CAR domain and a CCR domain. In some of these systems, the extein comprises at least two extracellular antigen-binding CAR domains. In some of these systems, the protein extein comprises at least two extracellular antigen-binding CCR domains. In some of these systems, the extein comprises two CAR domains and two CCR domains. In some of these systems, the extein comprises at least one signaling domain.In some systems of such systems, the extein comprises at least two signaling domains. In some systems of such systems, the signaling domain further comprises a kinase. In some systems of such systems, the signaling domain is one or more of CD3δ, CD3γ, CD3ε, CD3ζ, CD28, 4-1BB, ICOS, OX40, CD27, CD40, NKG2D, DAP-10, CD2, CD150, CD226, NKG2D, Zap70, or an ortholog thereof, or a variant thereof. In some systems of such systems, the nucleic acid construct comprises: a nucleotide sequence encoding a regulator motif comprising a non-structural 3 (NS3) protease cleavage site and a drug-regulatable HCV protease; and a signaling domain comprising a kinase. In some systems of such systems, the nucleic acid construct comprises: a nucleotide sequence encoding a regulator motif comprising a non-structural 3 (NS3) protease cleavage site and a drug-regulatable HCV protease; and a Zap 70 signaling domain. In certain of such systems, the nucleic acid construct comprises a nucleotide sequence encoding a regulator motif comprising a dimerization domain that dimerizes in the presence of a drug; and at least one signaling domain, wherein the signaling domain comprises a kinase.

[0288] In certain non-limiting embodiments, the subject matter disclosed herein relates to methods for modifying cells, comprising delivering the disclosed system to the cells. In certain of these methods, the cells are mammalian cells. In certain of these methods, the mammalian cells are immune cells. In certain of these methods, the immune cells are T cells.

[0289] In certain non-limiting embodiments, the subject matter disclosed herein relates to methods for enriching a modified cell population, wherein the enrichment comprises: modifying the cell population as disclosed herein; culturing the cell population; and enriching the modified cell population by selecting for surface expression of an extein. In certain of these methods, the cells are mammalian cells. In certain of these methods, the mammalian cells are immune cells. In certain of these methods, the immune cells are T cells.

[0290] In certain non-limiting embodiments, the subject matter disclosed herein relates to methods for treating a disease, comprising providing a modified cell population comprising a system as described herein, or cells modified according to the modification methods disclosed herein, or an enriched cell population enriched according to the methods disclosed herein to a subject in need thereof. In certain embodiments of such embodiments, the subject is a human subject. In certain embodiments of such embodiments, the disease is cancer, an autoimmune disease, an inflammatory disease, or a graft-versus-host disease. In certain embodiments of such embodiments, the cancer is leukemia, lymphoma, myeloma, ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, testicular cancer, anal cancer, skin cancer, gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, or soft tissue sarcoma. In certain embodiments of such embodiments, the leukemia is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, or B-cell prolymphocytic leukemia. In certain embodiments of such embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. In certain embodiments of such embodiments, the non-Hodgkin's lymphoma is B-cell non-Hodgkin's lymphoma or T-cell non-Hodgkin's lymphoma. In certain embodiments of such embodiments, the cancer comprises cells expressing CD19 or CD20. In certain of such embodiments, the cancer comprises cells expressing at least one antigen selected from the group consisting of: CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, an antigen of cytomegalovirus (CMV) infected cells, ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, C CDC155, CCR1, CD10, CD117, CD123, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD30, CD300LF, CD32, CD321, CD33, C D34, CD36, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3,CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, COLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-a, FOLR2, FRMD5, GABRB2, GAS2, GD 2, GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B, kappa-light chain, L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MY ADML2, NGFR, NKCS1, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, PRAME, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), peptidase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine peptide kinase Erb-B2,RHBDL3, RNF173, RNF183, ROR1, RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5 , SLC25A36, SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STO N2, SUN3, survivin, SUSD2, SYNC, TACSTD2, TAS1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycopolypeptide 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms' tumor polypeptide (WT-1), WNT4, WT1, and ZDHHC11.

[0291] 6. Examples

[0292] Materials and methods

[0293] 6.1.1. Plasmid construction

[0294] The intein and other proteins of interest (e.g., antigen binding domains, spacers, transmembrane domains, cytoplasmic signaling domains, drug regulatory domains, reporter proteins) are combined into vector sequences using standard molecular cloning techniques and arranged into a polycistronic configuration with a self-splicing 2A peptide.

[0295] 6.1.2 Retroviral transduction

[0296] The retroviral DNA plasmid encoding the intein and other construct elements was stably transfected into the retroviral packaging system. Viral supernatant was generated and concentrated with polyethylene glycol. T cells were stimulated with plate-bound antibodies or antibody-coated beads. After T cell stimulation, T cells were incubated with recombinant human fibronectin ( Takara Bio Inc.) and centrifuged onto coated culture plates. After transduction, T cells were assessed for construct expression by flow cytometry. Other methods for vector incorporation include lentiviral or adeno-associated viral (AAV) vectors or non-viral targeted DNA incorporation based on CRISPR / Cas. Transposon delivery and non-incorporated lentiviral vector template delivery combined with site-specific incorporation based on CRISPR / Cas are also compatible with the intein sorting system approach.

[0297] 6.1.3 Intein-based cell sorting

[0298] T cells co-transduced with multiple vector components in the intein sorting system were incubated with anti-affinity tag beads (e.g., anti-FLAG and hCD34) and positively selected on magnetic separation columns (Miltenyi Biotec). Post-sort cell purity and construct expression were confirmed by flow cytometry.

[0299] 6.1.4 Luciferase-based target cleavage assay

[0300] Target cells are genetically modified to express the luciferase gene (firefly red or click beetle red) and then incubated with CAR T cells at different effector:target cell (E:T) ratios for 24 hours. After the addition of D-luciferin, the surviving residual target cells are quantified by light flux.

[0301] 6.1.5 Live cell imaging

[0302] Target cells were genetically modified to express iRFP713 and Different effector / target cell ratios were incubated with CAR T cells co-expressing EGFP in a live cell imaging system. T cell and target cell counts were assessed by continuous microscopic imaging.

[0303] Data Compression

[0304] Degenerate inteins that use tags for domains shared by multiple CAR constructs (e.g., Zap70 signaling domain and 4-1BB co-stimulatory domain) can be multiplexed with multiple molecules containing antigen binding domains to generate a greater number of trans-spliced ​​CAR and CCR molecules than those encoded by the same amount of DNA. The amount of DNA "data" saved increases with the size of the encoded domain and the reuse of the domain in a greater number of molecules.

[0305] The code has a size of B i The common binding domain and size of D jThe number of DNA Q required to routinely encode a series of signaling domains for CAR and CCR molecules C It can be described by Equation 1;

[0306] Q C =d∑B i +b∑D j Equation 1

[0307] Where d is the size D used j is the number of different signaling domains, and b is the number of different binding domains of size Bi used.

[0308] The amount of DNA required to encode a series of intein-encoded CAR and CCR molecules is Q I It can be described by Equation 2;

[0309] Q I =∑B i +∑D j +dI N +dI C Equation 2

[0310] Among them I N is the size of the DNA required to encode the N-terminal split intein, and I C The size of the DNA required to encode the C-terminal split intein.

[0311] DNA savings (Δ = Q c –Q I ) can be determined by Equation 3

[0312] Δ=(d-1)∑B i +(b-1)∑D j -bI N -dI C Equation 3

[0313] Where d is the number of encoded signaling domains, b is the number of encoded binding handle molecules, and B i is the size of each binding domain, D j For the size of each signaling domain, I N is the size of the N-terminal split intein, and I C is the size of the C-terminal intein.

[0314] This is illustrated by an example of a Zap70 signaling domain and a 4-1BB co-stimulatory domain that routinely encode two CAR and two CCR molecules regulated by an NS3 drug, with the following domain sizes: B1 = 1 kB, B2 = 1 kB, D1 = 2.2 kB, D2 = 0.1 kB. According to Equation 1, this requires:

[0315] 2*(B1+B2)+2*(D1+D2)=2*(1+1)+2*(2.2+0.1)=8.6kB Equation 4

[0316] Rearrangement by CAR and CCR molecules:

[0317] (B1+D1) CAR1 +(B2+D1) CAR2 +(B1+D2) CCR1 +(B2+D2) CCR1 =3.2+3.2+1.1+1.1=8.6kB Equation 5

[0318] In contrast, for N = 0.3kB and I C = 0.1 intein coding, the following DNA would be required for total coding:

[0319] (B1+B2)+(D1+D2)+2(I N )+2(I C ) = 2 + 2.3 + 0.6 + 0.2 = 5.1 kB Equation 5

[0320] This resulted in a DNA saving of 8.6 kB - 5.1 kB = 3.5 kB.

[0321] Confirmed by Equation 3, the data savings is:

[0322] 1*2+1*2.3–0.6–0.2=3.5kB Equation 6

[0323] This saving in number increases the number of antigen binding domains B and signaling domains D, or increases the size of each domain.

[0324] Three signaling domains of 2 kB, 0.3 kB, and 0.1 kB in size and three antigen binding domains of 1 kB in size were used. N = 0.3kB and I C =0.1 kB), will encode nine receptors and will result in the following DNA savings in intein encoding compared to conventional encoding:

[0325] 2*(1+1+1)+2*(2+0.3+0.1)–3*0.3–3*0.1=9.6 kB Equation 7.

[0326] 6.3. Dual-carrier intein sorting system - secretory affinity tag and intein tag, transmembrane intein capture handle

[0327] In this example, mouse T cells were transduced with the following two vectors ( Figure 2A ): (Vector 1) encodes a secreted FLAG-tagged intein construct (which consists of a FLAG affinity tag fused to the N-terminus of the gp41-1 intein) and also encodes a BFP reporter gene; while (Vector 2) encodes a capture intein construct consisting of the C-terminus of the gp41-1 intein fused to an IgG1 hinge spacer, a CD28 transmembrane domain, and a truncated CD3ζδ non-signaling cytoplasmic domain. Vector 2 also encodes a Thy1.1 reporter gene. The two intein-tagged constructs are covalently linked during the trans-splicing of the gp41-1 intein to obtain an extracellular splicing junction. Only when the constructs are expressed by the same cell will the transmembrane intein-tagged construct capture the FLAG-tagged intein-tagged construct, thereby achieving a trans-splicing reaction. Cells transduced with the two vectors were enriched by MACS sorting using anti-FLAG magnetic beads to obtain a highly pure BFP+ / Thy1.1+ cell population ( Figure 2B ).

[0328] 6.4. Dual and Triple Carrier Intein Modular CAR and Sorting Systems—Extracellular scFv with Spacer Fusion to Transmembrane Intein Capture Handle Trans-splicing with Cytoplasmic Signaling Domain Shuttle Intein and Cytoplasmic Intein Adapter Molecules

[0329] This example involves an intein sorting system using the inward rectifier potassium (Kir6.2) RXR ER retention motif ( Figure 3A), in which mouse T cells were transduced with the following two vectors: (Vector 1) encodes the Thy1.1 reporter gene and also encodes a transmembrane intein-tagged construct composed of the following: a human CD34 affinity tag (QBEND epitope) flanked by two human CD20 affinity tags (rituximab binding mimotope) to form an RQR domain, a FLAG affinity tag, a CD8 hinge and transmembrane domain, a consensus sequence from the N-terminus of the aligned (Cfa) intein, and a Kir6.2 RXR or E3 19K KKXX endoplasmic reticulum (ER) retention motif; while (Vector 2) encodes the Cfa intein C-terminus fused to the CD28 costimulatory domain and the CD3-ζ chain, and also encodes the EGFP reporter gene. Trans-splicing of the two Cfa intein-tagged constructs results in an intracellular splice junction and forms a transmembrane-bound construct with both RQR and FLAG affinity tags on the cell surface. Due to ER retention of the transmembrane construct until the two inteins undergo trans-splicing (which removes the ER retention motif), the surface expression of these affinity tags is significantly reduced. Cells transduced with both vectors were enriched via MACS sorting using anti-FLAG magnetic beads to obtain highly pure T cell populations expressing each affinity tag on their surface.

[0330] Mouse T cells were transduced with the following two vectors: (Vector 1) encodes the Thy1.1 reporter gene and encodes an antigen-binding intein-tagged construct comprising a CD19-targeting scFv fused to an hCD34 affinity tag, a CD8 transmembrane domain, a Cfa intein N-terminus, and Kir6.2 RXR or E319K KKXX (ER retention motif); and (Vector 2) encodes a Cfa intein C-terminus fused to a CD28 costimulatory domain and a CD3-ζ chain, and also encodes an EGFP reporter gene. Trans-splicing of the two Cfa intein-tagged constructs yields an intracellular splicing junction, allowing the formation of a full-length CD19 chimeric antigen receptor (CAR). Due to the ER retention motif, the surface expression of the CD19 antigen-binding domain is significantly reduced until the two inteins undergo trans-splicing, which removes the ER retention motif. The cells transduced with the two vectors were enriched by MACS sorting using anti-hCD34 magnetic beads to obtain high-purity hCD34+CD19-CAR T cells ( Figures 3B to 3C ). CD19-CAR T cells (effector cells) generated by trans-splicing of Cfa intein or untransduced control T cells were cultured with target cells expressing CD19 (e.g., BM185 cells) at different E / T ratios for 24 hours. Figure 3D Demonstrated the efficacy of CD19-CAR T cells in killing BM185-CD19 target cells.

[0331] Figure 3EAn exemplary three-vector based intein retention is shown, using either the RXR or E319K (KKXX) retention motif. Figures 3F to 3I The ability of RXR-type ER retention motifs to function within a polypeptide chain is demonstrated (in this case, the 2A peptide residue remains attached to the RXR retention motif). In contrast, E3 19K KKXX-type motifs do not work in this context because the retention motif needs to occupy the end of the polypeptide chain. In this example, T cells were transduced with a CD20 mimetic epitope, hCD34 tag, and FLAG tag containing a sorting handle molecule with a cytoplasmic Cfa N intein and an E319K retention motif (vector 1). Cells were co-transduced with a second vector encoding a Cfa C intein fused to a gp41-1 N intein (cytoplasmic adapter intein), with a fused Kir6.2 RXR or E319K KKXX ER retention motif, followed by a 2A peptide and an EGFP reporter gene (vector 2). In this context, the KKXX retention motif is ineffective in retaining the spliced ​​sorting arbor protein in the ER, whereas the RXR motif tolerates the addition of C-terminal amino acids, retaining the protein within the cell. For cells transduced with vectors 1 and 2 encoding either the RXR or KKXX motif, co-transduction with vector 3 encoding the gp41-1 C intein eliminated the ER retention motif and promoted robust surface trafficking of the fully spliced ​​sorting arbor molecule. However, the RXR-type ER retention motif version achieved high expression of this construct only in triple-transduced cells, enabling selective MACS sorting of triple-transduced cells. This approach enables the generation of effector cells with enhanced functionality by one-step MACS purification of cells incorporating two or more vectors with increased total vector payload. Furthermore, the use of the RXR-type ER retention motif enables the placement of ER-retained constructs at the 5' or 3' position of polycistronic vectors containing multiple protein-encoding 2A peptides flanking them.

[0332] 6.5. Dual-vector, dual-secretory intein-tagged modular scFv sorting system for post-translational assembly of CAR.

[0333] 6.5.1 Dual secretory CAR fused to cytoplasmic signaling domains

[0334] In this example, mouse T cells were transduced with the following two vectors ( Figure 4A ): (Vector 1) encodes a dual-affinity tagged intein-tagged scFv (targeting CD19 or CD20) fused to the FLAG tag and the gp41-1 endoplasmic reticulum N-terminus, and also encodes the BFP reporter gene. (Vector 2) encodes a transmembrane signaling domain shuttle (composed of the gp41-1 intein C-terminus fused to the CD8 spacer and transmembrane domain, the CD28 costimulatory domain, and the CD3-ζ chain), and also encodes the Thy1.1 reporter gene ( Figure 4A After splicing of the two gp41-1 intein-tagged constructs, both the CD19 and CD20 binding domains are displayed on the cell surface, reproducing the full-length CAR molecule ( Figure 4A Cells containing both vectors were enriched by MACS sorting using anti-FLAG magnetic beads to obtain highly pure FLAG-positive dual CAR T cells ( Figure 4B ). Figure 4C and Figure 4D The results show that CD19 / CD20-targeted dual CAR T cells kill BM185-CD19 and BM185-CD20 cells respectively. Unexpectedly, the intein-scFv molecule appears to be spliced ​​only inside the ER (probably because a reducing environment is required to keep the intein sulfhydryl groups in a reduced state, while the extracellular environment is oxidative). This allows the intein-CAR cells with affinity-tagged intein-scFv arms to be uniquely expressed only in dual-transduced T cells and enables selective MACS sorting of dual-transduced T cells.

[0335] 6.5.2 Dual secretory CAR fused to Zap70

[0336] In this example, mouse T cells were transduced with the following two vectors ( Figure 4E ): (Vector 1) encodes two different antigen-binding scFv domains (e.g., recognizing CD19 or CD20 antigens), each containing a polypeptide fused to a transmembrane domain and a degenerate cytoplasmic N-terminal intein domain and a different affinity tag. Vector 2 encodes a Zap70 domain shuttle, which consists of a degenerate intein C-terminus and an EGFP protein tag. After trans-splicing of the two degenerate intein-tagged antigen-binding polypeptides with the Zap70 domain shuttle, a CD19 CAR and a CD20 CAR are generated, each attached to the Zap70 domain, and expressed on the cell surface. Cells containing the two vectors are enriched by MACS sorting using anti-FLAG magnetic beads to obtain highly pure FLAG-positive dual CAR T cells ( Figure 4F T cells co-expressing trans-spliced ​​CD19 / Zap70 and CD20 / Zap70 CARs (effector cells) or untransduced T cells (control) were cultured with C1498 target cells expressing both CD19 and CD20 antigens for 24 hours. Figure 4GThe efficacy of effector T cells killing target cells is shown. This use of the degenerate splicing of the intein of the common cytoplasmic signaling domain shuttle enables the reuse of DNA encoding large structural elements (e.g., Zap70 interdomain and kinase) as a data compression algorithm. This enables the per unit DNA delivered by the vector to generate a larger amount of receptors and is associated with the extremely high expression of the trans-splicing CAR construct. The ER retention motif selectively isolates the CAR of the FLAG tag, and subsequently trans-splicing with the appropriate cytoplasmic signaling domain shuttle and removing the surface transport after the ER retention motif, and selective MACS sorting (molecular coincidence detector) can be performed on the double transduced cells.

[0337] 6.6. Three-vector intein CAR and sorting system - secreted affinity-tagged intein-tagged scFv, transmembrane intein adapter with ER retention motif, and cytoplasmic signaling domain shuttle

[0338] In this example, mouse T cells were transduced with the following three vectors ( Figure 5A ): (Vector 1) encodes a dual CD19 and CD20 binding scFv fused to a FLAG tag and the N-terminus of a gp41-1 intein, and also encodes a BFP reporter gene; while (Vector 2) encodes a Thy1.1 reporter gene and a dual intein transmembrane adapter consisting of a gp41-1 intein C-terminus fused to the CD8 extracellular and transmembrane domains, the Cfa intein N-terminus, and the E319K ER retention motif; and (Vector 3) encodes a cytoplasmic signaling domain shuttle consisting of a Cfa intein C-terminus fused to the CD28 costimulatory domain and the CD3-ζ chain, and also encodes an EGFP reporter gene. The gp41-N intein-tagged scFv molecule undergoes trans-splicing with the dual intein transmembrane adapter in the ER, which can be exported from the ER after trans-splicing of the adapter with the Cfa intein of the cytoplasmic shuttle construct, thereby cleaving the ER retention motif and reproducing the full-length CAR. The triple-transduced cells were selected by MACS sorting using anti-FLAG magnetic beads to obtain highly pure FLAG-positive dual-CAR T cells ( Figure 5B These dual CAR T cells targeting CD19 / CD20 showed an effect on CD19-expressing cells in a 24-hour co-culture experiment. Figure 5C ) or CD20( Figure 5D Thus, this orthogonal two-intein, three-molecule trans-splicing methodology enables selective MACS sorting of T cells incorporating three unique vectors, which can further encode additional transgenes to enhance effector cell functionality.

[0339] 6.7. Modular Leucine Zipper-ZipR-CAR-Dual Antigen Specificity-Secretionary Affinity-Tagged Leucine Zipper-Tagged scFv, Transmembrane Zipper-Intein Adaptor

[0340] In this example, mouse T cells were transduced with the following three vectors ( Figure 6A ): (Vector 1) encodes a dual CD19 and CD20 binding scFv fused to a FLAG tag and an RR12EE345L leucine zipper, and also encodes a BFP reporter gene; while (Vector 2) encodes a Thy1.1 reporter gene and a leucine zipper-intein transmembrane adapter consisting of an EE12RR345L leucine zipper fused to an IgG1 hinge, a CD28 transmembrane domain, a Cfa intein N-terminus, and an E319K ER retention motif; and (Vector 3) encodes a cytoplasmic signaling domain shuttle consisting of a Cfa intein C-terminus fused to a CD28 costimulatory domain and a CD3-ζ chain, and also encodes an EGFP reporter gene. The leucine zipper-tagged scFv molecule binds to the capture zipper-intein transmembrane adapter in the ER, which can be expelled from the ER after trans-splicing of the Cfa intein of the adapter and the cytoplasmic shuttle construct, thereby cleaving the ER retention motif and reproducing the full-length CAR. The triple-transduced cells were enriched by MACS sorting using anti-FLAG magnetic beads to obtain highly pure FLAG-positive dual-CAR T cells ( Figure 6B These T cells showed cytotoxicity against BM185 target cells expressing CD19 or CD20 antigens after 24 hours of co-culture ( Figures 6C to 6D Thus, this methodology, utilizing orthogonal inteins and leucine zippers, enables selective MACS sorting of T cells incorporating three unique vectors that can further encode additional transgenes to enhance effector cell functionality.

[0341] 6.8. Four-carrier intein sorting system

[0342] In this example, mouse T cells were transduced with the following four vectors ( Figure 8A): (vector 1) encodes a secreted FLAG affinity-tagged gp41-1 intein N-terminus with a BFP reporter gene; (vector 2) encodes a Thy1.1 reporter gene and a dual intein-tagged transmembrane intein adaptor composed of a V5 reporter gene tag, a gp41-1 intein C-terminus, a CD8 spacer, a CD28 transmembrane domain, a Cfa intein N-terminus, and an E319K ER retention motif; (vector 3) encodes an EGFP reporter gene and a dual intein-tagged cytoplasmic intein adaptor containing a Cfa intein C-terminus, a gp41-8 intein N-terminus, and an E319K retention motif; and (vector 4) encodes a truncated EGFR (EGFRt) reporter gene and a cytoplasmic chain terminator construct composed of the gp41-8 intein C-terminus fused to the CD3-ζ non-signaling delta stalk. Trans-splicing of the homologous orthogonal intein leads to the assembly of surface-expressed molecules with the ER retention motif removed. The quadruple-transduced cells were enriched by MACS sorting using anti-FLAG (or anti-CD34) magnetic beads to obtain a highly pure cell population that incorporated all four vectors ( Figure 8B ). Additional transgenes can be encoded on each vector to enhance the functionality of the engineered cells. Antigen binding domains and signaling domains can be added to transform the sorting system into a modular CAR system.

[0343] Figure 8C The example shown is similar to Figure 8B Similar, except that the secreted FLAG affinity tag on vector 1 is replaced by a truncated hCD34 molecule, which serves as the antibody binding epitope. The quadruple-transduced cells are enriched via MACS sorting using anti-hCD34 magnetic beads to obtain a highly pure cell population that incorporates all four vectors. Additional transgenes can be encoded on each vector to enhance the function of the engineered cells. Antigen binding domains and signaling domains can be added to convert the sorting system into a modular CAR system.

[0344] exist Figure 8D In the example shown, mouse T cells expressed Figure 8BThe example shown is transduced in a similar manner, except that vector 3 (cytoplasmic intein adapter) is omitted, thereby cutting off the connection between the constructs encoded by vector 2 (transmembrane intein adapter) and vector 4 (intracellular chain terminator). In the absence of vector 3, the EGFP reporter gene is not expressed. In the absence of the cytoplasmic intein adapter encoded by vector 3, the transmembrane intein adapter encoded by vector 2 is retained in the ER, inhibiting the expression of FLAG (encoded by vector 1) on the cell surface. BFP / Thy1.1-positive cells (vectors 1 and 2) can only express sufficient levels of FLAG when vector 3 is present. Therefore, this orthogonal three-intein splicing methodology specifically generates trans-splicing CAR or sorting handle molecules in cells incorporating four unique vectors. This recursive strategy of nested orthogonal inteins with ER retention motif tags can, in principle, be extended to more than 4 vectors by adding additional orthogonal inteins. Due to the increase in the number of incorporated vectors, this strategy greatly increases the number of transgenics delivered to engineered cells.

[0345] 6.9. Dual-vector Intein Sorting System - Drug-regulated CAR Expression

[0346] In this example, mouse T cells were transduced with the following two vectors ( Figure 9A ): (Vector 1) encodes a Thy1.1 reporter gene and a drug-activated intein-tagged antigen-binding construct consisting of a CD19-targeting scFv fused to an hCD34 affinity tag, a CD8 hinge and transmembrane domain, an AES CL low-affinity intein N-terminus, a FKBP12 drug-induced heterodimerization domain (DmrA), and an E319K ER retention motif; whereas (Vector 2) encodes an EGFP reporter gene and a cytoplasmic signaling shuttle consisting of a FRB drug-induced heterodimerization domain (DmrC), an AES CL low-affinity intein C-terminus, a CD28 costimulatory domain, and a CD3-ζ chain. In the absence of a dimerizing drug, cell surface hCD34 expression is low due to the presence of the ER retention motif encoded by Vector 1 ( Figure 9B Addition of 1 μM A / C heterodimerization drug (AP21967) resulted in heterodimerization of the FKBP12 (DmrA) and FRB (DmrB) domains of the two intein-tagged constructs (i.e., A / C heterodimerization), promoting drug-induced splicing of the low-affinity AES CL intein ( Figure 9B ). Thus, this system is able to regulate post-translational CAR assembly through drug-regulated splicing. Double-transduced cells were cultured with 1 μM A / C heterodimerization drug and subsequently enriched by MACS sorting using anti-hCD34 magnetic beads to obtain highly pure hCD34+CD19-CAR T cells ( Figure 9B Using CD19-expressing BM185 cells as targets, the ability of this system to modulate target cell killing through drug-dependent cell surface expression of CAR was demonstrated ( Figure 9C This methodology improves upon previous FKBP / FRB-based drug-regulated CAR systems by eliminating the FKBP / FRB protein after trans-splicing and generating mature CAR molecules. This simplifies CAR design and introduces the signaling domain into a membrane-proximal location that facilitates signaling.

[0347] 6.10. Dual-Carrier Intein Sorting System - Drug-Regulated CAR Expression, Dual CARs, Dual Antigen Specificity

[0348] This example is designed with Figure 9A Similar to the example shown in Figure 9D, but with dual CD19 and CD20 antigen specificity. Mouse T cells were transduced with the following two vectors ( Figure 10A ): (Vector 1) encodes two antigen-binding constructs consisting of: (1) (1) A CD19-targeting scFv fused to an affinity tag, CD28 hinge and transmembrane domain, AES intein N-terminus, FKBP12 heterodimerization domain, and (2) a CD20-targeting VHH (variable heavy chain single domain binding domain) fused to an hCD34 tag, CD8 hinge and transmembrane domain, AES intein N-terminus, FKBP12 heterodimerization domain, and E319K ER retention motif. (Vector 2) encodes a drug-regulated cytoplasmic signaling domain shuttle consisting of the FRB* heterodimerization domain, AES intein C-terminus, CD28 co-stimulatory domain, and CD3-ζ chain, and also encodes an EGFP reporter gene. In the absence of a dimerizing drug, hCD34 expression at the cell surface is low due to the presence of the encoded ER retention motif fused to the cytoplasmic side of the CD20 antigen binding construct ( Figure 10B Addition of 1 μM A / C heterodimerization drug (AP21967) or rapamycin (50 nM) enables heterodimerization of FRB* encoded by vector 2 with the FKBP12 domain of the CD19 and CD20 targeting construct encoded by vector 1 ( Figure 10C). This enables the low-affinity AES intein to be efficiently spliced ​​due to the enhanced proximity induced by dimerization, thereby reconstituting the full-length CD19 and CD20 CARs on the cell surface. The ER retention motif on the construct targeting CD20 enables hCD34 surface expression to reach sortable levels only after drug-induced splicing. Due to the lack of an ER retention motif, the stalk targeting CD19 can be constitutively transported to the cell surface and cells transduced with vector 1 can be identified. In the absence of heterodimerizing drugs, the stalk targeting CD19 lacks the trans-splicing signaling domain and is unable to direct T cell activation. The dual-transduced cells were cultured in 1 μM A / C heterodimers and enriched by MACS sorting using anti-hCD34 magnetic beads to obtain highly pure hCD34+CD19 CAR T cells ( Figure 10B ). 24-hour co-culture studies demonstrated that dual CAR T cells targeting CD19 / CD20 could kill BM185-CD19 ( Figure 10C ) and BM185-CD20 cells ( Figure 10D ) in heterodimerization-dependent target cell killing. Figures 10E to 10L Live cell microscopy was demonstrated, showing the kinetics of eliminating CD19+ or CD20+ target cells by dual CART cells targeting CD19 / CD20 in the presence of heterodimerized drugs. This methodology improves the previous FKBP / FRB-based drug-regulated CAR system by eliminating FKBP / FRB proteins after trans-splicing and generating mature CAR molecules. This simplifies CAR design and introduces signaling domains into membrane-proximal positions that are conducive to signal transduction. The design also promotes the generation of drug-regulated multi-antigen-targeting CAR T cells.

[0349] 6.11. Dual-carrier intein sorting system - trans-presented cytokines

[0350] This example relates to constitutive and drug-regulated presentation of IL-7 on the cell surface. For the constitutive expression system, mouse T cells were transduced with the following two vectors: (Vector 1) encoding the Thy1.1 reporter gene and the IL-7 presentation construct (which consists of an IL-7 molecule fused to the hCD34 affinity tag, the CD8 hinge region and the transmembrane domain, the Cfa intein N-terminus, and the E319K ER retention motif). (Vector 2) encoding the cytoplasmic chain terminator construct (which consists of the Cfa intein C-terminus, the CD3-ζ non-signaling δ handle), and also encoding the EGFP reporter gene. The two Cfa inteins are spliced ​​together in the cell, removing the ER retention motif and enabling IL-7 to be presented on the cell surface. For the drug-regulated expression system, mouse T cells were transduced with the following two vectors ( Figure 11A): (Vector 1) encodes the Thy1.1 reporter gene, an IL-7 presentation construct consisting of an IL-7 molecule fused to an hCD34 affinity tag, the CD8 hinge and transmembrane domains, the N-terminus of the AES intein, the FKBP12 heterodimerization domain (DmrA), and the E319K ER retention motif. (Vector 2) encodes a drug-regulated cytoplasmic chain terminator construct consisting of the FRB* heterodimerization domain (DmrC), the C-terminus of the AES intein, and the CD3-ζ non-signaling delta stalk, and also encodes the EGFP reporter gene. Addition of the A / C heterodimerization drug (AP21967) at a concentration of 1 μM enables splicing of the low-affinity AES intein due to drug-induced construct heterodimerization, resulting in removal of the ER retention motif and presentation of IL-7 on the cell surface. Both constitutive and drug-regulated IL-7 trans-presenting cells were enriched by MACS sorting using anti-hCD34 magnetic beads to obtain highly pure IL-7 trans-presenting T cells ( Figure 11B To assess the expansion of these T cells, equal numbers of sorted and activated T cells were plated in the absence of IL-2 for 72 hours. Additionally, drug-conditioned cells were maintained in the presence of A / C heterodimerization drug (1 μM) for 3 days. Cells were counted using flow cytometry. Both constitutively and drug-conditioned trans-presenting IL-7 cells exhibited robust expansion compared to untransduced T cells ( Figure 11C This approach exploits ER retention motifs and drug-regulated trans-splicing to control surface expression of tethered cytokines that promote cis- and trans-stimulation of T cells or other effector cells to enhance immune function.

[0351] 6.12. Dual-vector intein modular CAR with drug-regulated splicing - single antigen specificity - trimethoprim regulation

[0352] In this example, mouse T cells were transduced with the following two vectors ( Figure 12A ): (Vector 1) encodes a Thy1.1 reporter gene and an intein-tagged antigen-binding construct (composed of a CD19-targeting scFv fused to an hCD34 affinity tag, a CD8 hinge region and transmembrane domain, a Cfa intein N-terminus, and an E319K ER retention motif). (Vector 2) encodes a cytoplasmic signaling domain shuttle (composed of a Cfa C-terminus, a CD28 costimulatory domain, a CD3-ζ1XX chain (ITAM 2 and 3 with a loss-of-function mutation), a dihydrofolate reductase destabilizing domain (DHFR-DD), and an EGFP reporter gene. In the absence of the stabilizing drug trimethoprim, the DHFR-DD domain is unstable, which leads to degradation of the construct, thereby impairing expression of the hCD34-tagged CAR on the cell surface. Figure 12BTrimethoprim (10 μM) was included to stabilize DHFR-DD, thereby enabling more efficient trans-splicing of the Cfa split intein domain to reconstitute the full-length CD19 CAR ( Figure 12B The double-transduced cells were cultured with 10 μM trimethoprim and then enriched by MACS sorting using anti-hCD34 magnetic beads to obtain highly pure hCD34+CD19-CAR T cells ( Figure 12B ). Figure 12C They showed that BM185 target cells expressing CD19 were killed only in the presence of trimethoprim. This approach utilizes a drug-regulated degron (a destabilized protein) in combination with a cytoplasmic signaling shuttle intein to regulate intein expression within the cell, thereby controlling trans-splicing activity and CAR signaling.

[0353] 6.13. Dual-vector intein modular CAR with drug-regulated splicing - single antigen specificity - asunaprevir regulation

[0354] 6.13.1 hCD34 affinity tag

[0355] In this example, mouse T cells were transduced with the following two vectors ( Figure 13A ): (Vector 1) encodes the Thy1.1 reporter gene and an intein-tagged antigen binding construct (which consists of a CD19-targeting scFv fused to an hCD34 tag, a CD8 hinge region and transmembrane domain, a Cfa intein N-terminus, and an E319K retention motif). (Vector 2) encodes a cytoplasmic signaling domain shuttle consisting of a Cfa intein C-terminus, a CD28 co-stimulatory domain, a CD3-ζ1XX chain (ITAM 2 and 3 with loss-of-function mutations), a hepatitis C virus (HCV) NS3 protease cleavage site, and a degradation determinant-tagged asunaprevir-regulated HCV protease (small molecule assist, SMASh tag). Splicing of the two Cfa split intein domains reconstitutes the full-length CD19 CAR on the cell surface. In the absence of asunaprevir, the protease will cleave the peptide at the cleavage site, removing the degradation determinant ( Figure 13A ), thereby preventing the cytoplasmic signaling domain shuttle from being degraded. Adding 3 μM asunaprevir to the culture medium blocks protease activity, which can maintain the connection between the degradation determinant and the cytoplasmic signaling domain shuttle, leading to its degradation, thereby reducing the generation of mature trans-splicing CAR molecules ( Figure 13B ). Double-transduced T cells cultured in the absence of asunaprevir were enriched using anti-hCD34 magnetic beads to obtain highly pure hCD34+CD19CAR T cells ( Figure 13B ).

[0356] 6.13.2 FLAG affinity tag

[0357] In this example, mouse T cells were transduced with the following two vectors: (Vector 1) encoding a Thy1.1 reporter gene and an intein-tagged antigen binding construct consisting of a CD19-targeting scFv fused to a FLAG tag, a CD8 hinge and transmembrane domain, a Cfa intein N-terminus, and an E319K retention motif; (Vector 2) encoding a cytoplasmic signaling domain shuttle consisting of a Cfa intein C-terminus, a CD28 co-stimulatory domain, a CD3-ζ1XX chain (ITAM 2 and 3 with loss-of-function mutations), a hepatitis C virus (HCV) NS3 protease cleavage site, and a degradation determinant-tagged asunaprevir-regulated HCV protease (small molecule assist, SMASh tag). Splicing of the two Cfa inteins reconstitutes the full-length CD19 CAR on the cell surface. As described above, the cytoplasmic signaling domain shuttle will be degraded in the presence of 3 μM asunaprevir, but not in the absence of asunaprevir ( Figure 13C ). Double-transduced T cells cultured in the absence of asunaprevir were enriched using anti-FLAG magnetic beads to obtain highly pure FLAG+CD19 CAR T cells ( Figure 13C ). Next, I checked Figure 13C Compared with T cells pre-cultured with 3 μM asunaprevir, T cells without asunaprevir pre-culture showed higher efficacy in lysing BM185 cells expressing CD19 ( Figure 13D ).

[0358] In the second example, mouse T cells were transduced with the following two vectors: (Vector 1) encoding two intein-tagged antigen-binding constructs consisting of the following: a CD20-targeting nanobody VHH domain fused to the hCD34 tag, the CD28 hinge and transmembrane domain, and the Cfa N-terminus, followed by a CD19-targeting scFv fused to the FLAG tag, the CD8 hinge and transmembrane domain, the Cfa intein N-terminus, and the E319K retention motif; (Vector 2) encoding a cytoplasmic signaling domain shuttle consisting of the Cfa intein C-terminus, the CD28 costimulatory domain, the CD3-ζ1XX chain (with loss-of-function mutations in ITAM 2 and 3), the hepatitis C virus (HCV) NS3 protease cleavage site, and a degradation determinant-tagged asunaprevir-regulated HCV protease (small molecule assist, SMASh tag). Splicing of the two Cfa inteins reconstitutes the full-length CD20 and CD19 CARs on the cell surface. As described in the examples above, the cytoplasmic signaling domain shuttle was degraded in the presence of 3 μM asunaprevir, but not in its absence ( Figure 13E ). Thus, the retention motif on the CD19-targeting portion of vector 1 reduced FLAG expression in the presence of drug. Dual-transduced T cells were cultured for 4 days with and without 3 μM asunaprevir and then co-cultured with BM185 expressing either CD19 or CD120. Figure 13F and Figure 13G As shown, T cells displayed specific cytotoxicity against BM185 expressing both CD19 and CD20, and this cytotoxicity was abolished when T cells were pre-incubated with asunaprevir. This methodology enables the generation of T cells capable of simultaneously targeting two antigens using coordinated drug regulation and further enables the purification of highly purified dual-transduced T cells incorporating both vectors via a single-step MACS purification.

[0359] 6.13.3 NS3-based drug-regulated Zap70CAR

[0360] In a third example, mouse T cells were transduced with the following two vectors ( Figure 14A ): (Vector 1) encodes a fusion polypeptide containing two different antigen-binding scFv transmembrane domains with a degenerate cytoplasmic N-terminal intein domain. Each scFv fusion polypeptide contains a different affinity tag and may also include an ER retention motif at the end of the vector construct at the C-terminus of the fusion polypeptide; and (Vector 2) encodes a drug-regulatable ζ chain-associated protein kinase 70 (Zap70) domain shuttle consisting of a degenerate intein C-terminus, a hepatitis C virus (HCV) NS3 protease with flanking cleavage sites ( Figure 14B ) and a tracer protein tag (e.g., EGFP tag). Splicing of two Cfa inteins reconstitutes the full-length CD19 CAR on the cell surface. Figure 14C The efficacy of NS3-regulated Zap70-CAR T cells dually targeting CD19 / CD20 in killing either CD19- or CD20-expressing BM185 cells was shown only in the presence of grazoprevir (preventing NS3-based cleavage of the CAR construct). Figure 14B ). This use of degenerate splicing of inteins that share a common cytoplasmic signaling domain shuttle enables the reuse of DNA encoding large structural elements (e.g., NS3 protease fused to Zap70 interdomain and kinase) as a data compression algorithm. This enables the generation of a greater number of receptors per unit DNA delivered by the vector and is associated with extremely high expression of trans-splicing CAR constructs. The selective sequestration of FLAG-tagged CARs by the ER retention motif, followed by trans-splicing with the cytoplasmic signaling domain shuttle and removal of the ER retention motif followed by surface transport, enables selective MACS sorting (molecular coincidence detector) of dual-transduced cells.

[0361] 6.14. NS3-Based Drug-Regulated CD28ζ-Based Intein-CAR

[0362] 6.14.1 Intein-assisted NS3-based drug-regulated CAR-non-nested approach

[0363] In this example, mouse T cells were transduced with vector 1, which encodes the CD19 antigen-binding handle consisting of the CD19 scFv, the CD8 hinge and transmembrane domains, the Cfa intein, and the E319K ER retention motif, and vector 2, which encodes the NS4a cofactor fused to the HCV NS3 protease and the CD28 costimulatory domain and the CD3ζ signaling domain followed by an EGFP reporter gene. In different variants, the NS4a-NS3 fusion is flanked by a combination of 5A / 5B and 4A / 4B cleavage sites for the NS3 protease ( Figure 15A ). Figure 15B Shown is the efficacy of CAR-transfected T cells in selectively killing BM185-CD19 cell targets in the presence of grazoprevir (GZP). Figures 15C to 15D Live cell microscopy images are shown showing the efficacy of NS3-regulated Zap70-CAR T cells dual-targeting CD19 / CD20 in killing BM185 cells expressing CD19 or CD20 only in the presence of grazoprevir (preventing NS3-based cleavage of the CAR construct). The high efficiency of cleavage by the NS3 protease avoids the "leaky" killing of targets by intein CARs in the absence of protease inhibitors. These T cells were generated by transduction with vector 1, a dual intein-tagged dual antigen-binding construct consisting of a CD20-targeting nanobody VHH domain fused to an hCD34 tag, CD28 hinge and transmembrane domains, and the Cfa N-terminus, followed by a CD19-targeting scFv fused to a FLAG tag, CD8 hinge and transmembrane domains, the Cfa intein N-terminus, and an E319K retention motif; and vector 2 encoding a Cfa C 5A-5B NS4a NS3 4A-4BCD28z EGFP drug-regulated cytoplasmic signaling domain shuttle.

[0364] This use of degenerate splicing of the intein of the shared common cytoplasmic signaling domain shuttle enables the reuse of DNA encoding large structural elements (e.g., NS3 protease fused to CD28ζ) as a data compression algorithm. This enables the generation of a greater number of receptors per unit DNA delivered by the vector and is associated with the extremely high expression of the trans-splicing CAR construct. The selective isolation of the FLAG-tagged CAR by the ER retention motif, and subsequent trans-splicing with the cytoplasmic signaling domain shuttle and removal of the surface transport after the ER retention motif, enables selective MACS sorting (molecular coincidence detector) of the dual transduced cells.

[0365] 6.14.2 Intein-assisted NS3-based drug-regulated CAR-nested approach

[0366] In this example, mouse T cells were transduced with vector 1, which encodes a Thy1.1 transduction reporter gene and an antigen-binding handle targeting CD19 (with CD8 hinge and transmembrane domains, nested cytoplasmic AES CL N intein, cytoplasmic Cfa N intein, and E3 19K ER retention motif), and vector 2, which encodes a Cfa C-terminal split intein fused to an HCV NS4a-NS3 protease domain fusion (flanked by 5A / 5B and 4A / 4B cleavage sites), an AES CL C-terminal intein, and the CD28ζ signaling domain. Figure 16A Following trans-splicing of the high-affinity intermolecular Cfa intein, the low-affinity AES CL intein can be spliced ​​to remove the intermediate NS3 protease-based drug regulation system. Figure 16B and Figure 16C Shown are the non-nested ( Figure 15A ) and nested methods to kill CD19-expressing BM185 target cells at high and low densities. This methodology enables the generation of drug-regulated CARs that actively signal by removing drug-regulating peptides, which can weaken CAR signal transduction by placing the signaling domain at a distal position on the membrane. Removal of drug-regulating peptides can also prolong the activity of CARs in the absence of HCV protease inhibitors.

[0367] 6.15. Modular CARs with Dual-Carrier Intein and Chimeric Costimulatory Receptor (CCR) Orthogonal Signaling Domains

[0368] In this example, mouse T cells were transduced with the following two vectors ( Figure 17A): (Vector 1) encodes a Thy1.1 reporter gene and an intein-tagged antigen-binding construct (composed of a CD19-targeting scFv fused to a FLAG tag, a CD8 hinge and transmembrane domain, a Cfa intein N-terminus, and an E319K retention motif); (Vector 2) encodes a cytoplasmic signaling domain shuttle composed of a Cfa C intein terminus, a 4-1BB costimulatory domain fused to a BFP reporter gene, a 2A cleavage peptide followed by a second Cfa C intein terminus, a CD28 costimulatory domain, a CD3-ζ1XX chain (with loss-of-function mutant ITAMs 2 and 3) fused to an EGFP reporter gene, a hepatitis C virus (HCV) NS3 protease cleavage site, and a degron-tagged asunaprevir-regulated HCV protease (small molecule assisted shutoff (SMASh) tag). Splicing of the Cfa intein recombined full-length CD19 CAR or a chimeric costimulatory receptor (CCR) targeting CD19. CD19 CAR enables cells to kill CD19 target cells, while CD19 CCR enables T cells to expand and survive after recognizing CD19. Anti-FLAG magnetic beads are used to enrich the double-transduced cells to obtain high purity FLAG + CD34 + CD19 CAR-CCR T cells ( Figure 17B Control CD19CD281XX intein CAR T cells lacking 4-1BB co-stimulation were also transduced and enriched to high purity using anti-FLAG magnetic beads ( Figure 17C ). After sorting, 20,000 T cells were cultured in the absence of IL-2 and in the presence or absence of 60,000 BM185 target cells expressing CD19. After 3 days of culture, the cells were counted by flow cytometry. Compared with T cells containing only CD19 CAR, T cells expressing both CD19 CAR and CCR were present in greater numbers after exposure to CD19-expressing targets ( Figure 17D ). Figure 17E Showing dual CAR (CD19 / CD20) + dual CCR dual handle + CD28z + 4-1BB (as shown Figure 17F Flow cytometry analysis results. Figure 17G Shown are the efficacy of dual CAR T cells in eliminating BM185-CD19 or BM185-CD20 target cells at the indicated E:T ratios. Figure 17HThe results of the expansion study of dual CAR-T cells and dual CCR-T cells after exposure to BM185-CD19 or BM185-CD20 target cells at the indicated E:T ratios are shown. The methodology can be combined by trans-splicing to generate four possible combinations: CD19-CAR, CD20-CAR, CD19-CCR and CD20-CCR. This configuration represents a data compression algorithm in which degenerately encoded DNA elements are reused to enable post-translational assembly of a greater number of receptors than those encoded with the same amount of DNA. Additive CAR+CCR co-stimulation promoted enhanced T cell proliferation and enhanced target killing at low T cell E:T ratios, consistent with a more active cell product.

[0369] 6.16. Dual-vector Intein Modular Zap70-CAR and Chimeric Costimulatory Receptor (CCR) Expression

[0370] In this example, mouse T cells were transduced with the following two vectors ( Figure 18A ): (Vector 1) encodes a fusion polypeptide containing two different antigen-binding scFv transmembrane domains (e.g., anti-CD19 and anti-CD20) with a degenerate cytoplasmic N-terminal intein (I N ) domain. Each scFv fusion polypeptide contains a different affinity tag and can also contain an ER retention motif (not shown) at the C-terminus of the fusion polypeptide at the end of the vector construct. (Vector 2) encodes a Zap70 domain shuttle containing a degenerate intein C-terminus and a CCR (e.g., 4-1BB). Figure 18B and Figure 18C The Zap70 intein-CAR+CCR combination showed enhanced target killing and T cell proliferation, targeting CD19 + and CD20 +Target both (dual CAR / dual CCR activity). The results show that the co-expression of 4-1BB CCR improves the activity of Zap70 CAR-T cells. This methodology can be combined by trans-splicing to generate four possible combinations: CD19-Zap70-CAR, CD20-Zap70-CAR, CD19-CCR, CD20-CCR. This use of the degenerate splicing of the intein of the common cytoplasmic signaling domain shuttle enables the reuse of DNA encoding large structural elements (e.g., Zap70 interdomain and kinase) as a data compression algorithm. This enables the vector to deliver more receptors per unit DNA and is associated with the extremely high expression of the trans-splicing CAR construct. The selective isolation of the CAR of the ER retention motif to the FLAG-tagged CAR, and the subsequent trans-splicing with the cytoplasmic signaling domain shuttle and the removal of the surface transport after the ER retention motif, can selectively MACS sort the double-transduced cells (molecular coincidence detector).

Claims

1. A system comprising two nucleic acid constructs, wherein: A. Each nucleic acid construct comprises a nucleic acid sequence encoding an extein; B. The first nucleic acid construct comprises a nucleic acid sequence encoding one of a complementary pair of N-split intein and C-split intein; C. a second nucleic acid construct comprising a nucleic acid sequence encoding the other of the complementary N-split intein and C-split intein pair; and D. At least one nucleic acid construct encodes an endoplasmic reticulum (ER) retention motif.

2. The system according to claim 1, comprising: A. A first nucleic acid construct comprising a nucleic acid sequence encoding: i. first extein; and ii. the first N-split intein of the first complementary split intein pair; B. A second nucleic acid construct comprising a nucleic acid sequence encoding: i. Second extein; ii. the first C-split intein of the first complementary split intein pair; and iii. a second N-split intein, wherein the second N-split intein belongs to a second complementary split intein pair; and C. A third nucleic acid construct comprising a nucleic acid sequence encoding: i. third extein; and ii. a second C-split intein, wherein the second C-split intein belongs to the second complementary split intein pair.

3. The system of claim 1 , comprising: A. A first nucleic acid construct comprising a nucleic acid sequence encoding: a. first extein; and b. first leucine zipper motif; B. A second nucleic acid construct comprising a nucleic acid sequence encoding: a. a second leucine zipper motif; and b. N-split intein of a complementary split intein pair; and C. A third nucleic acid construct comprising a nucleic acid sequence encoding: a. second extein; and b. The C-split intein of the complementary split intein pair.

4. The system of claim 1, comprising: A. A first nucleic acid construct comprising a nucleic acid sequence encoding: a. first extein; and b. the first N-split intein of the first complementary split intein pair; B. A second nucleic acid construct comprising a nucleic acid sequence encoding: a. Second extein; b. the first C-split intein of the first complementary split intein pair; and c. a second N-split intein, wherein the second N-split intein belongs to a second complementary split intein pair; C. A third nucleic acid construct comprising a nucleic acid sequence encoding: a. Third extein; b. a second C-split intein, wherein the second C-split intein belongs to a second complementary split intein; and c. a third N-split intein, wherein the third N-split intein belongs to a third complementary split intein pair; and D. A fourth nucleic acid construct comprising a nucleic acid sequence encoding: a. Extein IV; and b. a third C-split intein, wherein the third C-split intein belongs to the third complementary split intein pair.

5. The system according to any one of claims 1 to 4, wherein the N-terminal amino acid of the N-split intein is linked to the C-terminal amino acid of the extein, or the N-terminal amino acid of the extein is linked to the C-terminal amino acid of the C-split intein.

6. The system of any one of claims 1 to 5, wherein the complementary split intein pair is selected from the group consisting of: Cfa intein, gp41-1 intein, gp41-8 intein, Aes123PolB1 intein (Aes intein), NrdJ-1 intein, IMPDH-1 intein, SspGyrB intein, DNA polymerase III (DnaE) intein, orthologs thereof, and variants thereof.

7. The system of any one of claims 1 to 6, wherein the complementary pair of N-split and C-split inteins comprises a splicing motif selected from the group consisting of: CLS, CFN, CLD, HNS, SVV, SVYLN, and CLV.

8. The system according to any one of claims 1 to 7, wherein the N-split intein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7 and 9.

9. The system according to any one of claims 1 to 8, wherein the C-split intein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10.

10. The system of any one of claims 1 to 7, wherein the N-split intein has an amino acid sequence selected from SEQ ID NOs: 1, 3, 5, 7, and 9, and the complementary C-split intein has an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, and 10, respectively.

11. The system according to any one of claims 1 to 10, wherein the ER retention motif is a KKXX motif or a RXR motif.

12. The system of claim 11, wherein the E319K motif comprises SEQ ID No 18.

13. The system of claim 11, wherein the RXR motif comprises a sequence selected from any one of SEQ ID NOs: 19 to 22.

14. The system of any one of claims 1 to 13, wherein the N-terminal amino acid of the ER retention motif is linked to the C-terminal amino acid of the N-split intein.

15. The system of any one of claims 1 to 11, 13 or 14, wherein the ER retention motif is a RXR motif flanked by extein-encoding sequences.

16. The system of any one of claims 1 to 12, wherein at least one nucleic acid construct further comprises a nucleotide sequence encoding one or more of an affinity tag, a spacer, or a linker, wherein the amino acid sequence of one or more of the affinity tag, the spacer, or the linker is disposed on the polypeptide chain between the C-terminal amino acid of the extein and the N-terminal amino acid of the N-split intein, or between the N-terminal amino acid of the extein and the C-terminal amino acid of the C-split intein.

17. The system of claim 16, wherein the affinity tag is one or more of: (i) a FLAG tag comprising the amino acid sequence DYKDDDDK; (ii) a Strep tag comprising the amino acid sequence of SEQ ID NO: 27; (iii) a V5 tag comprising the amino acid sequence of SEQ ID NO: 28; (iv) a CD34 tag comprising the amino acid sequence of SEQ ID NO: 29; (v) a CD20 mimotope tag comprising the amino acid sequence of SEQ ID NO: 30; (vi) tagBFP comprising the amino acid sequence of SEQ ID NO:

31.

18. The system of claim 16 or claim 17, wherein the spacer is a CD8 spacer comprising the amino acid sequence of SEQ ID NO: 32, a CD28 spacer comprising the amino acid sequence of SEQ ID NO: 33, or a PD-1 spacer comprising the amino acid sequence of SEQ ID NO:

34.

19. The system of any one of claims 16 to 18, wherein the linker has an amino acid sequence selected from any one of SEQ ID NOs: 11 to 17, or comprises the amino acid sequence GGGGSGGGS, GGGGSGTG, GTSRAKRGS, ALGGSGGGS, GGGGSTS, SGGGGSD, GSSGSG, GSGGTR, GSGTR, TSGSG, GSGGS, GSS, GTG, LES, GSG, GSL, or ALG.

20. The system of claim 16, wherein the linker is a cleavable linker.

21. The system of claim 20, wherein the cleavable linker comprises a sequence selected from one or more of SEQ ID NOs: 23, 24, 25, or 26.

22. The system of any one of claims 1 to 21, wherein at least one nucleic acid construct comprises a nucleotide sequence encoding a regulatable genetic element.

23. The system of claim 22, wherein the regulatable genetic element encodes a regulator motif that regulates expression of an extein.

24. The system of claim 23, wherein the regulator motif comprises a drug-stabilized signaling domain, a drug-destabilizing degron domain, or a drug-regulatable self-cleavage domain.

25. The system of claim 24, wherein the drug destabilizing degron domain is a dihydrofolate reductase destabilizing domain (DHFR-DD).

26. The system of claim 25, wherein the drug is trimethoprim or an analog thereof.

27. The system of claim 23, wherein the regulator motif comprises a nonstructural 3 (NS3) protease cleavage site and a drug-regulated protease.

28. The system of claim 27, wherein the drug-regulated protease is HCV protease regulated by asunaprevir, grazoprevir, or an analog thereof.

29. The system of claim 23, wherein the regulator motif comprises a dimerization domain that dimerizes in the presence of a drug.

30. The system of any one of claims 1 to 22 or 29, wherein the N-terminal amino acid of the ER retention motif is linked to the C-terminal amino acid of the dimerization domain.

31. The system of claim 29 or claim 30, wherein the dimerization domain is selected from FK506 binding protein 12 (FKBP12) and FKBP-rapamycin binding (FRB).

32. The system of any one of claims 29 to 31 , wherein the dimerization domain is regulated by rapamycin or an analog thereof.

33. The system of any one of claims 15 to 25, wherein the regulatable genetic element comprises a sequence selected from SEQ ID NOs: 38 to 47, or the sequence DEMEECSQH.

34. The system of any one of claims 1 to 34, wherein the extein comprises an extracellular antigen binding domain, an extracellular cytokine, a transmembrane domain, a signaling domain, or a combination thereof.

35. The system of claim 34, wherein the extracellular antigen binding domain further comprises a hinge region.

36. The system of claim 35, wherein the hinge region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144 to 147.

37. The system of any one of claims 34 to 36, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv), Fab, F(ab)2, or a single domain VHH antibody.

38. The system of any one of claims 34 to 37, wherein the extein comprises a chimeric antigen receptor (CAR), a chimeric co-stimulatory receptor (CCR), a T cell receptor (TCR), a TCR-like fusion molecule, an ortholog thereof, or a variant thereof.

39. The system of any one of claims 34 to 38, wherein the extracellular antigen binding domain binds one or more antigens selected from the group consisting of: CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, an antigen of cytomegalovirus (CMV) infected cells, ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC 155. CCR1, CD10, CD117, CD123, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD30, CD300LF, CD32, CD321, CD33, CD34, CD36, CD38 , CD41, CD44, CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNI H2, COL15A1, COLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-a, FOLR2, FRMD5, GABRB2, GA S2, GD2, GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19,KIF26B, kappa-light chain, L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MYADML2, NGFR, N KCS1, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), peptidase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine peptide kinase Erb-B2, RHBDL3, RNF173, RNF183, ROR 1. RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, survivin, S USD2, SYNC, TACSTD2, TAS1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycopolypeptide 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms tumor polypeptide (WT-1), WNT4, WT1, and ZDHHC11.

40. The system of claim 38 or 39, wherein the CAR further comprises a signal sequence.

41. The system of claim 40, wherein the signal sequence comprises the amino acid sequence of one of SEQ ID NOs: 128 to 135.

42. The system of any one of claims 38 to 41, wherein the CAR comprises an extracellular antigen binding domain of an anti-CD19 antibody.

43. The system of claim 42, wherein the extracellular antigen binding domain of the CAR is an anti-CD19 scFv.

44. The system of claim 42 or claim 43, wherein the extracellular antigen binding domain of the CAR is an anti-CD19 scFv having a heavy chain variable domain (VH) of SEQ ID NO: 94 and a light chain variable domain (VL) of SEQ ID NO:

95.

45. The system of any one of claims 42 to 44, wherein the extracellular antigen binding domain of the CAR is an anti-CD19 scFv having the sequence of SEQ ID NO:

96.

46. ​​The system of any one of claims 38 to 41, wherein the CAR comprises an extracellular antigen binding domain of an anti-CD20 antibody.

47. The system of claim 46, wherein the extracellular antigen binding domain of the CAR is an anti-CD20 scFv.

48. The system of claim 46 or 47, wherein the extracellular antigen binding domain of the CAR is an anti-CD20 scFv having a heavy chain variable domain (VH) of SEQ ID NO: 97 and a light chain variable domain (VL) of SEQ ID NO:

98.

49. The system of any one of claims 46 to 48, wherein the extracellular antigen binding domain of the CAR is an anti-CD20 scFv having the sequence of SEQ ID NO:

99.

50. The system of claim 42, wherein the extracellular antigen binding domain of the CAR is a VHH antibody.

51. The system of claim 50, wherein the VHH antibody has the sequence of SEQ ID NO:

100.

52. The system of claim 38, wherein the TCR comprises an alpha chain constant region (TRAC) and a beta chain constant region (TRBC), wherein: A. the TRAC is encoded by the nucleotide sequence of SEQ ID NO: 136; B. the TRBC is encoded by a nucleotide sequence selected from any one of SEQ ID NOs: 138, 141, 142, or 143; C. The TRBC comprises an amino acid sequence selected from any one of SEQ ID NO: 137, 139 or 140.

53. The system of claim 34, wherein the cytokine is selected from the group consisting of IL-7, IL-15, and IL-18.

54. The system of any one of claims 34 to 53, wherein the signaling domain is one or more of CD3δ, CD3γ, CD3ε, CD3ζ, CD28, 4-1BB, ICOS, OX40, CD27, CD40, NKG2D, DAP-10, CD2, CD150, CD226, NKG2D, Zap70, orthologs thereof, or variants thereof.

55. The system of any one of claims 34 to 54, wherein the signaling domain further comprises a kinase.

56. The system of claim 55, wherein the kinase is one or more of a Src kinase, a Syk kinase, or a receptor tyrosine kinase (RTK).

57. The system of claim 55 or claim 56, wherein the kinase is one or more of a Src kinase, a Syk kinase, or a receptor tyrosine kinase (RTK).

58. The system of any one of claims 55 to 57, wherein the signaling domain comprises a kinase domain from one or more of PDGFR, KIT, Abl, Arg, EGFR, Raf, VEGFR, PDGFR, Flt3, Abl, Arg, or ErbB2, or an ortholog thereof.

59. The system of any one of claims 34 to 43, wherein: a. the CD3γ signaling domain comprises the amino acid sequence of SEQ ID No. 105; b. the CD3δ signaling domain comprises an amino acid sequence selected from any one of SEQ ID NO: 106 or 107; c. the CD3ε signaling domain comprises the amino acid sequence of SEQ ID NO: 108; d. the CD3 zeta signaling domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 109 to 111 or 113; e. the CD28 signaling domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 101 to 103; f. the 4-1BB signaling domain comprises the amino acid sequence of SEQ ID NO: 104; g. The Zap70 signaling domain comprises an amino acid sequence selected from any one of SEQ ID NO: 114 or 115.

60. The system of any one of claims 34 to 58, wherein the signaling domain further comprises an immunoreceptor tyrosine-based activation motif (ITAM).

61. The system of claim 60, wherein the ITAM comprises an amino acid sequence selected from any one of SEQ ID NOs: 116, 118, 120, 122, 124, 126.

62. The system of any one of claims 1 to 61, wherein the nucleic acid construct encodes an amino acid sequence selected from any one of SEQ ID NOs: 55 to 93.

63. A system comprising two or more nucleic acid constructs, each nucleic acid construct comprising a nucleotide sequence encoding: A. At least one extein; B. at least one of a complementary N-split intein and C-split intein pair; C. at least one endoplasmic reticulum (ER) retention motif; and D. A regulator motif or regulatable domain that regulates the expression of the extein.

64. The system of claim 63, wherein the extein comprises one or more of an extracellular antigen binding domain, an extracellular cytokine, a transmembrane domain, or a signaling domain.

65. The system of claim 63 or claim 64, wherein the complementary split intein pair is selected from the group consisting of: Cfa intein, gp41-1 intein, gp41-8 intein, Aes123PolB1 intein (Aes intein), NrdJ-1 intein, IMPDH-1 intein, SspGyrB intein, DNA polymerase III (DnaE) intein, orthologs thereof, and variants thereof.

66. The system of any one of claims 63 to 65, wherein the N-split intein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, and 9.

67. The system of any one of claims 63 to 66, wherein the C-split intein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, and 10.

68. The system of any one of claims 63 to 65, wherein the N-split intein has an amino acid sequence selected from SEQ ID NOs: 1, 3, 5, 7, and 9, and the complementary C-split intein has an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, and 10, respectively.

69. The system of any one of claims 63 to 68, wherein the ER retention motif is an E319K motif (KKXX) or a RXR motif.

70. The system of claim 6, wherein the E319K motif comprises the sequence of SEQ ID NO:

18.

71. The system of claim 69, wherein the RXR motif comprises a sequence selected from any one of SEQ ID NOs: 19 to 22.

72. The system of any one of claims 63 to 71, wherein the N-terminal amino acid of the ER retention motif is linked to the C-terminal amino acid of the N-split intein.

73. The system of any one of claims 63 to 69 or 71, wherein the ER retention motif is a RXR motif flanked by extein encoding sequences.

74. The system of any one of claims 63 to 73, wherein the regulator motif or the regulatable domain comprises a drug-stabilized signaling domain, a drug-destabilizing degron domain, or a drug-regulatable self-cleavage domain.

75. The system of any one of claims 63 to 74, wherein the extein comprises a CAR domain and a CCR domain.

76. The system of any one of claims 63 to 75, wherein the extein comprises at least two extracellular antigen-binding CAR domains.

77. The system of any one of claims 63 to 76, wherein the extein comprises at least two extracellular antigen-binding CCR domains.

78. The system of any one of claims 63 to 77, wherein the extein comprises two CAR domains and two CCR domains.

79. The system of any one of claims 63 to 78, wherein the extein comprises at least one signaling domain.

80. The system of any one of claims 63 to 79, wherein the extein comprises at least two signaling domains.

81. The system of any one of claims 63 to 80, wherein the signaling domain further comprises a kinase.

82. The system of any one of claims 63 to 81, wherein the signaling domain is one or more of CD3δ, CD3γ, CD3ε, CD3ζ, CD28, 4-1BB, ICOS, OX40, CD27, CD40, NKG2D, DAP-10, CD2, CD150, CD226, NKG2D, Zap70, an ortholog thereof, or a variant thereof.

83. The system of any one of claims 63 to 82, wherein the nucleic acid construct comprises: A. a nucleotide sequence encoding a regulator motif comprising a nonstructural 3 (NS3) protease cleavage site and a drug-regulatable HCV protease; and B. A signaling domain comprising a kinase.

84. The system of claim 83, wherein the nucleic acid construct comprises: A. a nucleotide sequence encoding a regulator motif comprising a nonstructural 3 (NS3) protease cleavage site and a drug-regulatable HCV protease; and B. Zap 70 signaling domain.

85. The system of any one of claims 63 to 82, wherein the nucleic acid construct comprises: A. a nucleotide sequence encoding a regulator motif comprising a dimerization domain that dimerizes in the presence of a drug; and B. at least one signaling domain, wherein the signaling domain comprises a kinase.

86. A method of modifying a cell comprising delivering to the cell the system of any one of claims 1 to 85.

87. The method of claim 86, wherein the cell is a mammalian cell.

88. The method of claim 87, wherein the mammalian cell is an immune cell.

89. The method of claim 88, wherein the immune cell is a T cell.

90. A method for enriching a modified cell population, comprising: A. Modifying a cell population according to the method of claim 56; B. culturing the cell population; as well as C. Enrichment of the modified cell population by selecting for surface expression of exteins.

91. The method of claim 90, wherein the cell is a mammalian cell.

92. The method of claim 91, wherein the mammalian cell is an immune cell.

93. The method of claim 92, wherein the immune cell is a T cell.

94. A method for treating a disease comprising providing to a subject in need thereof a modified cell population comprising the system of any one of claims 1 to 85, or cells modified by the method of any one of claims 86 to 89, or a cell population enriched by the method of any one of claims 90 to 93.

95. The method of claim 94, wherein the subject is a human subject.

96. The method of claim 94 or claim 95, wherein the disease is cancer, an autoimmune disease, an inflammatory disease, or graft-versus-host disease.

97. The method of claim 96, wherein the cancer is leukemia, lymphoma, myeloma, ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, testicular cancer, anal cancer, skin cancer, stomach cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, or soft tissue sarcoma.

98. The method of claim 97, wherein the leukemia is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, or B-cell prolymphocytic leukemia.

99. The method of claim 97, wherein the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma.

100. The method of claim 99, wherein the non-Hodgkin's lymphoma is a B-cell non-Hodgkin's lymphoma or a T-cell non-Hodgkin's lymphoma.

101. The method of any one of claims 96 to 100, wherein the cancer comprises cells expressing CD19 or CD20.

102. The method of any one of claims 96 to 101, wherein the cancer comprises cells expressing at least one antigen selected from the group consisting of: CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, an antigen of cytomegalovirus (CMV) infected cells, ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155 , CCR1, CD10, CD117, CD123, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD30, CD300LF, CD32, CD321, CD33, CD34, CD36, CD38, CD4 1. CD44, CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2, C OL15A1, COLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-a, FOLR2, FRMD5, GABRB2, GAS2, GD 2. GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B,κ-light chain, L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MYADML2, NGFR, N KCS1, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), peptidase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine peptide kinase Erb-B2, RHBDL3, RNF173, RNF183, ROR 1. RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, survivin, S USD2, SYNC, TACSTD2, TAS1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycopolypeptide 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms tumor polypeptide (WT-1), WNT4, WT1, and ZDHHC11.

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