35 results about "Antigen receptor gene" patented technology

The invention discloses a CAR-T transgene vector based on replication defective recombinant lentivirus. The CAR-T transgene vector comprises an original nuclear replicon pUCOri sequence, a resistance gene AmpR sequence containing ampicillin, a virus replicon SV40 Ori sequence, a lentivirus packaging cis element, ZsGreen1 green fluorescent protein, an IRES ribosome binding sequence, a human EF1 alpha promoter , a chimeric antigen receptor of second-generation CAR or third-generation CAR and a regulating element, wherein the original nuclear replicon pUCOri sequence is used for plasmid replication; the resistance gene AmpR sequence is used for massively proliferating target strains; the virus replicon SV40 Ori sequence is used for enhancing replication in eukaryocyte; the lentivirus packaging cis element is used for lentivirus packaging; the ZsGreen1 green fluorescent protein is used for expressing green fluorescent for eukaryocyte; the IRES ribosome binding sequence is used for jointly transcribing and expressing protein; the human EF1 alpha promoter is used for conducting eukaryotic transcription on antigen receptor genes; the chimeric antigen receptor is used for forming the second-generation CAR or the third-generation CAR integrating recognition, transfer and start; the regulating element is used for enhancing expression efficiency of transgenes and used after eWPRE-enhanced type woodchuck hepatitis b virus is transcribed. Besides, the invention further discloses a construction method and application of the vector. By means of the CAR-T transgene vector and the construction method and application of the vector, secretion of cell factors and an in vitro killing effect of CAR-T cells can be remarkably improved, and the clinical treatment effect is remarkable.

The invention provides a preparation method of a double chimeric antigen receptor gene modified T lymphocyte targeting breast cancer stem cells. The preparation method is characterized in that integrin-associated protein (CD47) and transcriptional coactivator (TAZ) are both highly expressed in breast cancer tissues, especially in the breast cancer stem cells; by established CD47 and TAZ over-expressed breast cancer cells, higher proliferation and metastasis capacity and cancer stem cell features are shown. Extracellular domain of the two breast cancer stem cell antigens CD47 and TAZ are targeted to generate monoclonal strains in specific binding under immunity action, and single-chain antibodies in specific binding with the CD47 and TAZ by genetic recombination are obtained to construct a human CD47 and TAZ containing double chimeric antigen receptor gene recombined to a virus vector to transfect human T lymphocyte. By high expression of the double chimeric antigen receptor gene in specific binding with human CD47 and TAZ expressing breast cancer stem cells, a first signal and a costimulatory signal can be activated to trigger anti-breast-cancer cytotoxicity, and high cytotoxicity in in-vivo and in-vitro anti-cancer experiments is achieved.

The invention provides a preparation method of bispecific chimeric antigen receptor gene modified natural killer cells, comprising the features: constructing a bispecific chimeric antigen receptor gene containing specific-binding signaling lymphocyte activation molecule family member 7 and fibronectin mutants, recombining the bispecific chimeric antigen receptor gene to a viral vector, transfecting with human natural killer cells, and highly expressing the bispecific chimeric antigen receptor gene, thus specifically binding tumor cells that express the signaling lymphocyte activation molecule family member 7 and fibronectin mutants, inhibiting expression of natural killer cell inhibitory receptors, and preventing immune escape of tumor cells; meanwhile, activating a first signal and a co-stimulatory signal to trigger toxic activity of the tumor cells, and great anti-tumor killing toxicity is shown in in-vivo and in-vitro experiments; the invention also provides a kit for the preparation of autologous natural killer cells through the above method, and with the kit, it is possible to highly express the bispecific chimeric antigen receptor gene and trigger great anti-tumor effect.

The invention provides a micro-annullus DNA recombinant plasmid containing a recombinant chimeric antigen receptor gene expression box and a preparation method of the micro-annullus DNA recombinant plasmid. The micro-annullus DNA recombinant plasmid can be used for implementing site-specific recombination in host bacteria, eliminating a prokaryotic plasmid part and forming micro-annullus DNA containing the recombinant chimeric antigen receptor gene expression box. The invention also provides the micro-annullus DNA containing the recombinant chimeric antigen receptor gene expression box as well as the preparation method and application of the micro-annullus DNA; the micro-annullus DNA can be used for stably and lastingly expressing a recombinant chimeric antigen receptor gene in a host cell and is an efficient and safe gene treatment vector; moreover, the invention also provides the host cell containing the micro-annullus DNA as well as the preparation method and application of the host cell; besides, the invention also provides a recombinant chimeric antigen receptor as well as the preparation method and application thereof.

The invention relates to a method for efficiently knocking chimeric antigen receptor (CAR) genes into T cell specific genome loci through clusters of regularly interspaced short palindromic repeats (CRISPR)-Cas9, and application of the method, in particular to a method for accurately knocking the CAR genes into target genome DNAs of T cells. The method comprises the steps: (a) the target genome DNAs are shorn through gene editing substances, wherein the target genome DNAs are located behind a gene promoter region and between initiation codons and exon 3-ends where the initiation codons are located; and (b) the CAR genes are cloned to a repair template vector and then guided into the T cells, the shorn target genome DNAs are repaired by the cells in a homologous recombination mode, thus theCAR genes are embedded, and an adeno-associated virus vector is adopted as the repair template vector. The method can be applied to preparation of the various CAR-T cells.

The invention discloses a natural killer cell modified by specific chimeric antigen receptor gene and a preparation method and use thereof. The natural killer cell modified by specific chimeric antigen receptor gene comprises a NY-ESCO-1 specific chimeric antigen receptor gene and a truncated spleen tyrosine kinase Syk gene. The preparation method simultaneously infects the natural killer cell with a slow virus containing the NY-ESCO-1 specific chimeric antigen receptor gene and a slow virus containing the truncated spleen tyrosine kinase Syk gene. By the combined action of the NY-ESCO-1 specific chimeric antigen receptor gene and the truncated spleen tyrosine kinase Syk gene, the natural killer cell modified by the specific chimeric antigen receptor gene improves the targeted tumor cell specific lethality of the natural killer cell, improves the ability of activating downstream signals of the natural killer cell, avoids the escape of tumor immunity, and accordingly enhances the killing activity on the tumor cell.

The invention discloses a construction method and an application of a recombinant gene of a bispecific chimeric antigen receptor for treating HIV infection complicated with blood tumor. The chimeric antigen receptor is formed by sequentially connecting a signal peptide, an HIV gp120 antigen specific single-chain antibody and an anti-CD19 single-chain antibody and then sequentially connecting witha CD28 transmembrane region, a CD28 intracellular structural domain (ICD), a 4-1BB costimulatory structural domain and a CD3zeta intracellular signal transduction structural domain in series, or the chimeric antigen receptor comprises: first CAR composed of the signal peptide, the chimeric antigen receptor, the HIV gp120 antigen specific single-chain antibody, the CD28 transmembrane region, the CD28-ICD, the anti-CD19 single-chain antibody, the CD8 transmembrane region, the CD28-ICD, the 4-1BB costimulatory structural domain and the CD3zeta intracellular signal transduction domain; and secondCAR composed of and the signal peptide, the anti-CD19 single-chain antibody, the CD8 transmembrane region in parallel, connecting the CD28-ICD, the 4-1BB costimulatory structural domain and the CD3zeta intracellular signal transduction domain, wherein the first CAR and the second CAR are sequentially connected in parallel.

The invention belongs to the field of cell therapy, and discloses a chimeric antigen receptor gene engineering vector and immune cells, and applications thereof, wherein the chimeric antigen receptorgene engineering vector comprises a lentivirus vector skeleton, regulatory elements connected onto the lentivirus vector skeleton and a chimeric antigen receptor gene sequence. The regulatory elementsinclude a WPRE element, a cPPT element and an RRE element. The chimeric antigen receptor gene sequence comprises a costimulatory signal pathway sequence and an effector signal pathway sequence whichare independently expressed; or the chimeric antigen receptor gene sequence comprises a costimulatory signal pathway sequence and an effector signal pathway sequence which are in fused expression. Thechimeric antigen receptor gene engineering vector has the advantages of good safety, high transfection efficiency, rapid amplification of transfected immune cells, and great improvement of killing effect. In addition, the recognition on target cells by the chimeric antigen receptor immune cells is more precise than the recognition on target cells by chimeric antigen receptor immune cells with single target, and the off-target effect of single target can be prevented.

The invention discloses a NK (Natural Killer) cell with a chimeric CEA (Carcino-Embryonic Antigen) antigen receptor and a preparation method and applications thereof. The NK cell with the chimeric CEAantigen receptor, which is disclosed by the invention, is obtained by expressing the chimeric CEA antigen receptor in a NK cell. The NK cell with the chimeric CEA antigen receptor has the following characteristics that: 1, tumor cells can be efficiently and specifically killed; 2, while the CEA-CAR (Chimeric Antigen Receptor) is expressed, the regulatory mechanism of the NK cell per se is not affected, that is, the ability to inhibit the effect on homologous normal cells is not affected; and 3, after in vitro expansion of lymphocytes, the NK cell accounts for more than 90% of the harvested total cells and the CEA-CAR-NK accounts for more than 34% of the harvested total cells. The NK cell with the chimeric CEA antigen receptor, which is disclosed by the invention, plays an important role in clinical treatment of various solid tumors when serving as a biological agent.

The present invention provides a production method of a functional differentiated cell having a post-rearrangement genotype of a particular antigen receptor gene, which includes culturing a stem cell having the genotype in a medium to give the differentiated cell derived from the stem cell. As the stem cell having the genotype, a stem cell (e.g., ES cell) established by transplantation of the nucleus of a cell having the genotype is preferable. As the differentiated cell, NKT cell is preferable.

The invention relates to a multi-signal embedded antigen receptor and an expression gene thereof, an NK (Natural Killer) cell modified by the multi-signal embedded antigen receptor and application. The embedded antigen receptor comprises a first segment, a second segment and a self-pyrolysis polypeptide T2A for connecting the first segment and the second segment, wherein the first segment comprises a first signal peptide, a TCRgamma chain and a TCRdelta chain, which are connected in sequence; the second segment comprises a second signal peptide SP, a tumor antibody, a CD8 structure domain, a 41BB intracellular domain and a DAP12-ITAM structural domain, which are connected in sequence; a C end of the self-pyrolysis polypeptide T2A is connected with the TCRdelta chain; an N end of the self-pyrolysis polypeptide T2A is connected with the second signal peptide SP. An experiment result shows that the multi-signal embedded antigen receptor can be stably expressed in the NK cell and can be used for more rapidly and specifically identifying malignant tumor cells and killing the malignant tumor cells, and the toxic side effect of treatment is reduced.

The invention belongs to the field of immunotherapy of tumor cells, and provides a recombination chimeric antigen receptor (CAR) gene, a carrier comprising the CAR gene, a CAR-T cell and application thereof. The recombination CAR gene comprises nucleotide sequences encoding the following parts: an antigen binding part of a CD30 antibody, a transmembrane part, a CD137 cytoplasm functional area anda CD3zeta cytoplasm functional area, the CD137 cytoplasm functional area and the CD3zeta cytoplasm functional area are connected in arbitrary order; the invention further provides the application, andthe application applies a separated recombinant nucleic acid, a CAR fusion protein or a recombination T-lymphocyte to preparing drugs used for the treatment or adjuvant treatment of Hodgkin disease lymphoma or anaplastic large cell lymphoma or other CD3O positive tumors.

The present application discloses a universal PCR primer combination for detecting chimeric antigen receptor genes, and an application thereof. The primer combination comprises a primer pair for specifically detecting the chimeric antigen receptor genes, sequences of the primer pair are shown in SEQ ID No.1-2, chimeric antigen receptors comprise a costimulatory signal molecule ligand CD3-zeta, anda target of the primer pair is located on a coding gene of the costimulatory signal molecule ligand CD3-zeta. The primer pair provided by the present application has advantages of good specificity, high sensitivity and high accuracy, can be widely applied to qualitative and quantitative detections of one or more of the chimeric antigen receptor genes, and has important application values in the fields of T cell immunotherapy of CAR-T cell quality control, companion diagnostics and detection of CAR-T in blood during a treatment process of clinical patients using one or more of the CAR-T, etc.

The invention relates to a specific lymphocyte content analysis method and device based on TCR/BCR high-throughput sequencing, and belongs to the technical field of gene detection. The analysis methodcomprises the following steps of performing high-throughput sequencing: extracting genome DNA of a biological sample to be detected, adding a predetermined amount of artificially synthesized TCR/BCRcoding gene internal standard sequences, and carrying out high-throughput sequencing; and performing data analysis: obtaining high-throughput sequencing data and TCR/BCR gene sequences obtained by analysis, calculating the amplification multiple according to the input internal standard sequences, comparing the TCR/BCR gene sequences of the sample to be detected with antigen receptor gene sequencesA of target lymphocytes, obtaining the number of original antigen receptor gene sequences consistent with the antigen receptor gene sequences A of the target lymphocyte, the number of sequences withsequencing errors, the number of sequences subjected to secondary rearrangement and the number of mutated sequences, obtaining the total number of target lymph sequences, and calculating the number ofthe contained target lymphocytes according to the total number of the target lymph sequences and the amplification multiple, so that the content of each T/B cell can be accurately determined.

The invention discloses a construction method for a recombination gene of a bispecific chimeric antigen receptor for treating a human immunodeficiency virus (HIV) infection and application thereof. The bispecific chimeric antigen receptor comprises an anti-CD32a single-chain antibody and an anti-HIV gp120 single-chain antibody; and a coding nucleotide sequence of the anti-CD32a single-chain antibody and a coding nucleotide sequence of the anti-HIV gp120 single-chain antibody are connected in series in a coding gene of the bispecific chimeric antigen receptor. According to the bispecific chimeric antigen receptor of the invention, by taking a marker fusion protein gp120 of HIV replication and proliferation and a marker envelop protein CD32a of the latent period as target points, a dual-target point CAR-T cell is designed, and thus, HIV infected target cells can be thoroughly eliminated; and the bispecific chimeric antigen receptor has great clinical promotion value.

The invention provides a recombinant chimeric antigen receptor (CAR) gene, a vector containing the same, a CAR-T cell and a use thereof. The recombinant CAR gene comprises a nucleic acid sequence encoding an antigen-binding portion of a CD30 antibody, a transmembrane portion and a CD137 cytoplasmic functional region and a CD3zeta cytoplasmic functional region linked in any order; also provides a method of treating Hodgkin's lymphoma or anaplastic large cell lymphoma or other CD30-positive tumors using the CAR-T cells of the invention.

The invention discloses a lentiviral vector for expressing CD19 and CD20 antibody gene CARs (chimeric antigen receptors) and a preparation method of the lentiviral vector. The lentiviral vector for expressing the CD19 and CD20 antibody gene CARs is formed through cotransfection of 293 T cell by a shuttle expression vector and an auxiliary packaging plasmid, recombination harvest and concentration, wherein the shuttle expression vector contains CD19 and CD20 antibody gene CAR genes. The preparation method of the lentiviral vector expressing the CD19 and CD20 antibody gene CARs is simple, feasible, short in consumed time and high in repeatability; a produced lentivirus has high infection efficiency, can express a target gene efficiently, causes little immune response of a body and is quite wide in application range.