CN112679618B - TRBC 1-targeted humanized chimeric antigen receptor, T cell and application - Google Patents

Abstract

The invention discloses a TRBC 1-targeted humanized chimeric antigen receptor, a T cell and application, wherein the chimeric antigen receptor comprises scFv, the scFv sequence is derived from a murine antibody JOVI.1, and CDR regions of the murine antibody JOVI.1 are transplanted to human antibody frameworks VH1_1-02 and VK2_ A17. The invention provides a CAR-T product with an antibody sequence for humanization, specific recognition and killing TRBC1+ T cells, and for normal cells (including normal T cells expressing TRBC2) which do not express TRBC1, the CAR-T product hardly has the condition of non-specific killing, so that the normal cellular immune function of a patient is reserved, and the condition of low immunity or lack of immunity of the patient is avoided.

Description

TRBC 1-targeted humanized chimeric antigen receptor, T cell and application

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to a TRBC 1-targeted humanized chimeric antigen receptor, a T cell and application.

Background

CAR-T Cell therapy (Chimeric Antigen Receptor T-Cell therapy), i.e., Chimeric Antigen Receptor T-Cell immunotherapy, is to link a single-chain Antigen recognition sequence (scFv) of a specific antibody to the transmembrane region and the intracellular signal sequence of CD3 ζ and express the antibody on T cells, so that the antibody has a function of specifically recognizing a tumor Antigen and killing tumor target cells independently of MHC molecules.

In 2011, CAR-T therapy by the professor Carl June, university of pennsylvania, led to a breakthrough in the treatment of 3 patients with Chronic Lymphocytic Leukemia (CLL): second generation CAR-T cells fused to the 4-1BB (CD137) costimulatory domain achieve efficient target cell killing and long-term proliferation in patients; in 2012, the CAR-T product was used by the Carl June team to treat Acute Lymphocytic Leukemia (ALL) patients who did not respond to chemotherapy, eminence girl, Emily controlled their disease: leukemia is in complete remission, which remains cancer-free to survive to date.

In 2017, the U.S. Food and Drug Administration (FDA) approved two CAR-T products suitable for relapse of refractory B cell tumors to be marketed, indicating the acceptance and expectation of society for this emerging tumor treatment approach.

According to the statistics of the World Health Organization (WHO), the annual growth rate of the global lymphoma is 7.5 percent, the lymphoma is one of the malignant tumors with the fastest growth rate at present, about 35 million new cases exist worldwide every year, and the death number exceeds 20 ten thousand. In recent years, the incidence of lymphoma in China also tends to rise year by year, and the incidence of the disease in China currently exceeds 6.68/10 ten thousand people and is frequently generated in children and young and strong years. If leukemia and lymphoma are combined and analyzed, the incidence rate of malignant lymphoma in China is about 25/12 ten thousand per year, namely about 20 thousand people suffer from lymphoma or leukemia per year, and great threat is brought to the life health of common people.

Among patients with malignant lymphoma, the incidence rate of non-Hodgkin lymphoma accounts for more than 90%, wherein the T lymphoma patients in China account for 25% of all non-Hodgkin lymphoma, which is higher than about 15% in European and American countries. T cell lymphoma (T cell lymphoma) is a lymphatic system malignant tumor with strong invasiveness, fast progress, low response rate to radiotherapy and chemotherapy, poor prognosis and high recurrence rate, and the average 5-year survival rate is less than 25%. At present, no clear and effective standard treatment scheme exists for the disease, and a targeted treatment medicament is lacked. Therefore, the exploration of a safer and more effective treatment scheme is the direction of common efforts of clinical and scientific research institutions at home and abroad.

The CAR-T products currently undergoing clinical or preclinical experiments mostly target B cell-derived tumors such as lymphoma, leukemia, multiple myeloma and the like, and the CAR-T products targeting T cell lymphoma are rarely reported. Among them, the main difficult points include:

1. lack of suitable tumor associated antigens as targets. Since tumor T cells are derived from variant T cells, their surface antigens are substantially similar to normal T cells, and lack specific tumor antigens. At present, it has been reported that CAR-T products suitable for T cell lymphoma, or target T cell common antigens (such as CD5, CD3, CD7, etc.), can kill normal T cells of patients while killing tumor T cells, resulting in acquired immune deficiency or low level of patients; or surface antigens targeting T cell subsets (e.g. CD4, CD30, CCR4, etc.), with rather limited applicability and risk of antigen deletion, tumor escape; meanwhile, the CAR-T cells express antigens at the same time, so that the CAR-T cells can also cause 'suicide' between the CAR-T cells, reduce the functional activity of the CAR-T cells and influence the sustained action of the CAR-T cells.

2. During the preparation of CAR-T cells from autologous lymphocytes, contamination of tumor T cells needs to be strictly avoided: if tumor cells are mixed in the prepared CAR-T, the CAR has already been clinically practiced in CD19 CAR-T products, because the single-chain antibody sequence (scFv) on the CAR, which recognizes tumor antigens, binds to antigens expressed by the cells themselves, so that the antigens cannot be recognized by other CAR-T cells, thereby causing the tumor cells to escape and the tumor to recur; if the CAR-T cells are prepared from allogeneic lymphocytes, immune rejection issues need to be considered.

The T cell receptor beta chain constant region is encoded by two genes (TRBC1 and TRBC2), only one of which is expressed by mature T cells. Due to the nature of monoclonal origin of tumor cells, TRBC1 and TRBC2 become a CAR-T treatment target which can be applied to T-cell malignant lymphoma, so that the problem of tumor cell pollution is effectively avoided, and meanwhile, part of normal T-cell immune functions of a patient are retained. At present, a CAR-T product targeting TRBC1 is developed and appeared, but because the CAR-T product uses a murine antibody as an antigen recognition domain, the CAR-T product has certain immunogenicity, is easy to be recognized and eliminated by immune cells of a host, reduces the long-term curative effect of the CAR-T cell in vivo, and increases the risk of disease recurrence.

4. TRBC 1-targeted CAR is available, but has a high self-activation level, and is easy to cause premature exhaustion of T cell functions, so that the proliferation capacity of the T cell is insufficient, and the tumor killing function and the persistent disease control capacity of the T cell are influenced.

The ongoing development work on the relevant CAR-T products has been broken through in several areas, for example:

1. development of NK CAR-T cell system: because NK T cells also have the function of killing target cells, have limited proliferation capacity and short service life, can effectively avoid long-term influence on an immune system and normal T cells, and can effectively avoid the pollution of tumor T cells. But also has the characteristics of limited proliferation capacity and short service life, so that the infusion frequency and the cell quantity of the NK CAR-T cells need to be increased compared with the common CAR-T cells; and the difficulties of gene editing and cell amplification of NK T cells are higher, and higher requirements are provided for production and process optimization.

2. A gene editing technology is adopted to knock out the target antigen expressed by the CAR-T cell while preparing the CAR-T cell. However, due to the application of gene editing technology, potential off-target risks still exist, and the technical requirements are high.

3. The development of a universal allogeneic CAR-T cell system. By using a gene editing technology, antigen proteins and T Cell Receptors (TCR) expressed by T cells are knocked out simultaneously so as to prevent the returned T cells from generating Graft-versus-host reaction (GVHD) and CAR-T self-killing conditions; clinical application of the preparation of CAR-T cells using allogeneic T cells was achieved with Major Histocompatibility Complex (MHC) differentiation allowing, thereby circumventing tumor T cell contamination.

Disclosure of Invention

The invention aims to provide a CAR-T product targeting TRBC1 gene aiming at the condition that the existing market has small quantity of CAR-T products aiming at target T cell lymphoma and the effect is not ideal.

In order to achieve the aim, the invention provides a humanized chimeric antigen receptor targeting TRBC1, which comprises scFv, wherein the scFv sequence is derived from a murine antibody JOVI.1, and CDR regions of the murine antibody JOVI.1 are grafted to human antibody frameworks VH1_1-02 and VK2_ A17.

Preferably, the scFv comprises a heavy chain and a light chain; the sequences of the heavy chain and the light chain of the scFv are respectively shown as SEQ ID NO.11 and 13; or as shown in SEQ ID NO.11 and 12; or as shown in SEQ ID NO.12 and 13; or as shown in SEQ ID NO.12 and 14.

Preferably, the chimeric antigen receptor comprises: an extracellular domain structure formed by serially connecting a CD8 alpha signal peptide sequence, a tag sequence, the scFv sequence and a hinge sequence of a CD8 alpha chain in sequence, a transmembrane region sequence of a CD8 alpha chain, and an intracellular domain structure formed by serially connecting an intracellular domain sequence of a human 41BB molecule and an intracellular domain sequence of a human CD3 zeta chain.

Preferably, the nucleic acid sequence encoding the chimeric antigen receptor is as shown in any one of SEQ ID NO.3, 5, 7 and 9.

Preferably, the amino acid sequence of the chimeric antigen receptor is shown in any one of SEQ ID No.4, 6, 8 and 10.

Preferably, the chimeric antigen receptor is cloned into a vector.

The present invention also provides a T cell comprising: the humanized chimeric antigen receptor targeted to TRBC1 described above.

Preferably, the T cells are purified by magnetic beads; the magnetic beads are coated with TRBC1 specific antibodies.

Preferably, the method for preparing T cells comprises the steps of:

(1) binding TRBC1 antibody to magnetic beads;

(2) mixing T cells to be purified with magnetic beads combined with TRBC1 antibody;

(3) and standing on a magnetic frame, and taking the supernatant to obtain the T cells.

The invention also provides application of the TRBC 1-targeted humanized chimeric antigen receptor, which is characterized in that the chimeric antigen receptor is used for preparing a targeted anti-tumor medicament.

Compared with the prior art, the invention has the beneficial effects that:

(1) the CAR-T product which is developed and optimized by the invention and can specifically recognize and kill TRBC1+ T cells can be used for patients with T lymphocytoma with TRBC1 positive tumor T cells.

(2) The invention removes cells (including normal T cells and T lymphoma cells) of TRBC1+ in T cells provided by patients or donors by using a negative selection method through magnetic beads coupled with TRBC1 specific antibodies, thereby avoiding the pollution of tumor T cells in the prepared CAR-T cells.

(3) The CAR-T product of the antibody sequence humanization, specific recognition and killing TRBC1+ T cells provided by the invention hardly has the condition of non-specific killing on normal cells (including normal T cells expressing TRBC2) not expressing TRBC1, thereby retaining the normal cellular immune function of a patient and avoiding the condition of low or missing immunity of the patient.

(4) Compared with the CAR constructed by other three antibodies, the humanized antibody H104 provided by the invention has lower background activation level and higher activation level after encountering antigens, can effectively and specifically recognize and kill TRBC1+ tumor target cells, reduces the self-activation level of CAR-T cells, relieves the condition of function exhaustion of the CAR-T cells, and has more advantages in the aspects of tumor killing capacity in a tumor-bearing mouse in-vivo model, continuous anti-tumor effect and the like.

Drawings

FIG. 1 is a graph showing the sorting effect of T cell samples from human Peripheral Blood Mononuclear Cells (PBMC) sorted by TRBC1 specific antibody coupled magnetic beads and detected and evaluated by flow cytometry.

FIG. 2 is a graph showing the results of the detection of functional specificity of TRBC1 CAR.

FIG. 3 is a graph of the results of measurements of background activation levels of TCR knockout Jurkat T cell lines expressing TRBC1 CAR.

Figure 4 is a graph of the results of levels of activation indicators that identify and kill target cells following expression of different TRBC1 CARs on human primary T cells.

FIG. 5 (a) is a graph comparing the results of different CAR-T cells killing TRBC1+ tumor target cells in vitro; FIG. 5 (b) is a graph comparing the results of different CAR-T cells killing TRBC2+ tumor target cells in vitro.

FIG. 6 is a graph comparing the results of IL-2 secretion and IFN- γ secretion by different CAR-T cells.

FIG. 7 is a graph of the effect of H104 CAR-T cells on killing TRBC1+ tumor target cells on a tumor-bearing mouse animal model.

FIG. 8 is a graph showing the results of measuring the fluorescence intensity of a substrate oxidized by firefly light-emitting enzyme.

Detailed Description

The technical solution of the present invention is further described below with reference to the accompanying drawings and examples.

Mature T cells each express a specific T Cell Receptor (TCR) consisting of two chains, alpha and beta, with the constant region of the beta chain (constantregion) encoded by two genes: TRBC1 and TRBC 2. In developmentally mature T cells, only one of them is expressed to construct a complete TCR. The CAR-T product which specifically recognizes and kills TRBC1+ T cells is developed and optimized, and can be used for patients with T lymphocytoma with TRBC1 positive tumor T cells.

Example (b): humanized engineering of TRBC1 antibody sequences

The variable region of IgG-type antibodies consists of 4 Framework Regions (FRs) and 3 Complementary Determining Regions (CDRs) of each of the light and heavy chains: wherein 6 CDR regions constitute the main antigen binding surface, the diversity of sequence and structure is extremely high; the FR region functions mainly to provide a supporting framework for the structure of the CDR region, and the sequence of the FR region has certain conservation in species. Therefore, the amino acid sequence of the FR region of the original antibody can be replaced by the FR region sequence of the human antibody by a method of homology modeling and structure analysis without significantly affecting the structure of the antibody and the ability of the antibody to bind to an antigen, so as to reduce the possibility that the antibody derived from other species will cause a human anti-antibody response. This technique of humanizing antibodies is also known as CDR-grafted antibody technique.

The process of constructing a CDR-grafted humanized antibody of the present invention comprises: CDR regions of a murine antibody JOVI.1 were grafted to human antibody frameworks VH1_1-02 and VK2_ A17(vBASE Database, Retter I, Althaus HH, Munch R, Muller W: VBASE2, an endogenous V gene Database nucleic Acids Res.2005Jan 1; 33(Database issue) D671-4), which have high homology to the original antibody framework and which constitute the closest 3D structure prediction of the antibody; meanwhile, in order to ensure the integrity of the CDR structure and maintain the binding capacity of the CDR region and the antigen, besides the CDR region, a part of sites of the mouse-derived antibody close to the CDR part are reserved; through antibody 3D structural simulation, residues in the framework region, the side chains of which may affect the CDR conformation of the antibody through electrostatic attraction, hydrophobic interaction and hydrogen bonding force, are analyzed, and are retained. For example: VH1_1-02- #55, #66, #67, #68, #69, #70, #72, thereby obtaining a humanized heavy chain sequence hHC1 (the sequence is shown in SEQ ID NO. 11); further retains #80, #82 and hHC2 (shown in SEQ ID NO. 12) which are closer to the CDR portions; retains VK2_ A17#40, #56, thus obtaining a humanized light chain sequence hLC1 (the sequence is shown as SEQ ID NO. 13); further retains #42, #52 and hLC2 (the sequence is shown in SEQ ID NO. 14). The numbering of the amino acids of the antibodies follows the numerical designation KABAT (Kabat E.A., Wu, T.T., Perry, H., Gottesman, K.and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, Fifth edition.NIH Publication No.91-3242, Kabat E.A.et al,1991, Supra). The obtained humanized antibodies light and heavy chains are combined in pairs to obtain 4 humanized antibodies with different degrees, which are respectively named as H101(hHC1+ hLC1), H102(hHC1+ hLC2), H103(hHC2+ hLC1) and H104(hHC2+ hLC 2).

First, CAR expression vector construction

The antigen-specific single Chain antibody (scFv) sequence of TRBC1 CAR used in the present invention was derived from mouse HYBRIDOMA cell JOVI.1 (journal LV, Haydn MP, Colin RA, etc. Generation of Monoclonal Antibodies Against Agents a Human T cell Receptor. beta. Chain Expressed in Transgenic Rice, HYBRIDAM 1992) and the framework of the antibody sequence was optimized for humanized sequence. The extracellular domain structure of the CAR is formed by sequentially connecting a CD8 alpha signal peptide sequence, a myc tag sequence, an scFv sequence and a hinge sequence of a CD8 alpha chain in series; the transmembrane region sequence is that of the CD8 alpha chain; the intracellular domain structure consists of the intracellular domain sequence of the human 41BB molecule concatenated with the intracellular domain sequence of the human CD3 zeta chain (CD 247). The base sequences of all CARs of the invention were cloned into the pHR-hEF1 α -IRES-EGFP vector by means of Gibson ligation (NEB # E2611L) and confirmed by one-generation sequencing.

The sequence from mouse hybridoma JOVI.1 (murine antibody JOVI.1) is named as M001, the nucleic acid sequence of CAR constructed by M001 is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2. The nucleic acid sequence of the CAR constructed by the H101 is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4. The nucleic acid sequence of the CAR constructed by the H102 is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO. 6. The nucleic acid sequence of the CAR constructed by H103 is shown as SEQ ID NO.7, and the amino acid sequence is shown as SEQ ID NO. 8. The nucleic acid sequence of the CAR constructed by the H104 is shown as SEQ ID NO.9, and the amino acid sequence is shown as SEQ ID NO. 10.

The CAR nucleic acid sequence constructed as described above is composed of a signal peptide sequence, a Myc tag sequence, a scFv sequence, a CD8 hinge and transmembrane region sequence, a 41BB co-stimulatory domain, and a CD3 zeta signal domain, all of which are connected in series in this order. The scFv adopts GGSG linker to connect the light chain and the heavy chain of the antibody, and the sequence of the GGSG linker is shown in SEQ ID NO. 15. The amino acid sequence of the signal peptide is shown as SEQ ID NO.16, the amino acid sequence of the Myc tag is shown as SEQ ID NO.17, the amino acid sequences of the CD8 hinge and the transmembrane region are shown as SEQ ID NO.18, the amino acid sequence of the 41BB co-stimulation structure domain is shown as SEQ ID NO.19, and the amino acid sequence of the CD3 zeta signal structure domain is shown as SEQ ID NO. 20.

Second, human primary T cell culture and lentivirus infection

Human primary T cells were all taken from healthy informed volunteers. Primary T cells were cultured in RPMI-1640(Gibco #11875-093) medium containing 10% fetal bovine serum (Gibco #16000044), 100U/ml penicillin and 100. mu.g/ml streptomycin sulfate (Gibco #10378016), 1mM pyruvate (Gibco # 11360070). To maintain proliferation of T cells, 100U/ml of recombinant IL-2(Sigma-Aldrich # SRP3085) was added to the medium.

Preparing lentivirus: Lenti-X293T cells (TaKaRa #632180) were digested, resuspended in antibiotic-free DMEM medium (Gibco #11995- ⁵ The cells were plated on 6-well cell culture plates (Corning # CLS3516) at 37 ℃ with 5% CO ₂ And (5) standing and culturing for 24 hours in an incubator. Using a lipofection system (Mirus #2300), 500ng of lentiviral packaging plasmids pCMVdR8.92(Addgene #8455) and 50ng of pMD2.G (Addgene #12259) were formulated with 500ng of lentiviral plasmids to be packaged as a transfection complex according to the protocol of the lipofection protocol, added to a culture medium of Lenti-X293T cells at 37 ℃ with 5% CO ₂ And (5) standing and culturing in an incubator. 18 hours after transfection, the medium was replaced with fresh medium. And collecting cell supernatant 48 hours after liquid change, determining virus titer, and freezing and storing in a refrigerator at-80 ℃ for later use. If necessary, the virus can be concentrated by ultracentrifugation, PEG precipitation, etc.,

lentivirus infection of primary T cells: the T cells were purified using a human T cell magnetic bead negative selection enrichment kit (Stemcell #19051HLA) according to the protocol and subjected to TRBC1 depletion purification using rProteinA magnetic beads (Life Technologies #10001D) coated with TRBC1 specific antibodies as follows: adding 100 μ l rPreteinA magnetic beads (containing half volume of preservation solution) into 1ml PBST (PBS + 0.1% TWEEN-20), placing on a magnetic frame to adsorb the magnetic beads, removing supernatant, repeating for 2 times to balance the magnetic beads; diluting 10 μ g of TRBC1 specific antibody in 100-200 μ l PBST, adding into the balanced magnetic beads, placing in a 4 ℃ rotating wheel, and combining overnight; the following day, the beads were placed on a magnetic rack, the supernatant discarded, and the beads were washed with 1ml of PBST to remove free antibody, and after 2 repeated washes, resuspended in the appropriate amount of media. Human peripheral blood mononuclear cells are recovered in advance, purified T cells are obtained by a T cell enrichment kit, 100 mu l of rPreteinA wall molecules combined with TRBC1 specific antibodies are added into every 2x10 ^ 6T cells, PBS is added to 2.5ml after the rPreteinA wall molecules are combined for 10 minutes by a room temperature rotating wheel, the mixture is placed on a magnetic frame and stands for 5 minutes, so that TRBC1+ T cells are attracted to the tube wall due to the magnetic bead effect, and the rest cells are poured out along with supernatant.

Magnetic beads coated with anti-human CD3 and anti-human CD28 antibodies (Life Technologies #11132D) were used, and the ratio of T cells in 1: 1 for 24 hours, the prepared lentivirus was added for infection. After 18 hours, the supernatant containing the lentiviral cells was removed and replaced with fresh T cell complete medium. After 5 days of T cell stimulation with magnetic beads, the beads were removed and fresh complete T cell culture medium containing 100U/ml hIL2 was replenished every 2-3 days.

Three, flow cytometry analysis

Staining for cell surface markers: diluting the antibody in FACS buffer (phosphate buffer PBS + 2% fetal bovine serum), washing the cells to be detected with FACE buffer for 1 time, then resuspending the cells with the antibody diluent, and incubating the cells in dark at 4 ℃ for 25 minutes; after washing 3 times with FACE buffer, it was resuspended in FACE buffer and tested on the machine.

Staining of markers within cells: resuspending the cells in 4% paraformaldehyde (Meilunbio # MA0192), fixing for 15 minutes at room temperature, and centrifuging to remove paraformaldehyde; slowly dropwise adding precooled methanol, breaking the membrane for 50 minutes on ice, and centrifuging to remove the methanol; adding antibody diluent (same with surface staining) for resuspension, incubating for 60 minutes at room temperature in dark environment, washing for 3 times with FACE buffer solution, resuspending with FACE buffer solution, and detecting on a machine. The cells may be labeled for viability prior to cell fixation using the Zombie Violet Fixable viability kit (Biolegend #423114) following the product instructions.

Flow cytometry data were obtained via a BD lsrportessa device (BD bioscience) and data analysis was performed using FlowJo software (Tree Star). The information on the antibodies used in flow cytometry can be found in the following table.

Fourth, CAR-T cell in vitro killing function detection

TRBC1+ Jurkat target cells and non-target cells (TRBC2+ Jurkat) which overexpress TRBC2+ exogenous TCR after endogenous TCR knockout, are respectively transferred into firefly Luciferase (Luciferase) genes. After replacing each of the two cells with a different CAR-T cell, or a control T cell not transfected with CAR, with fresh, IL-2-free medium, according to different effector cells: the ratio of target cells (E: T) was incubated for 12 to 16 hours. And taking the cell culture medium for detecting the secretion level of the cell factor. The cells were resuspended in 100. mu.l of firefly luciferase substrate D-fluorescein (1.5mg/ml, Perkinelmer #122799) and the 460-540nm fluorescence was detected using a microplate reader. The decrease of the fluorescence value (Xe) of each sample well was calculated with reference to the fluorescence value (X0) of the sample well having only the same number of target cells, i.e., the ratio of the target cells to be killed. The killing efficiency of T cells is (X0-Xe)/X0 × 100%.

Fifthly, detecting the secretion level of the cytokine under the condition of CAR-T cell in vitro stimulation

And (3) detecting the secretion level of the cell factor by using a human IL-2 and IFN-gamma ELISA detection kit (BD biosciences #550611 and #550612) according to the operation of the instruction, wherein the cell factor secretion level is detected on the culture medium supernatant obtained by incubating the CAR-T cells and the target cells in the fourth step. And calculating the content of the corresponding cytokine in the sample according to a standard curve established by the standard substance.

Sixth, mouse tumor model and CAR-T function detection

All used in vivo experiments are 5-8 weeks old severe combined immunodeficiency (NOD/SCID/IL2R gamma) ^null NSG) mice. To compare the in vivo anti-tumor effects of CAR-T, mice were inoculated with 1X10 injections via tail vein ⁶ TRBC1+ Jurkat target cells expressing the firefly luciferase gene; after 4 days, 2X10 of the drug was administered to tumor-bearing mice by tail vein injection ⁶ Individual CAR-T cells, or control T cell treatment without CAR transfer. The development condition of the tumor is tracked by detecting the intensity of a fluorescence signal generated by the target cell expressing the firefly luciferase through a small animal living body imaging technology. The specific operation comprises the following steps: d-luciferin (Perkinelmer #122799), a firefly luciferin substrate, was administered to tumor-bearing mice by intraperitoneal injection at a dose of 1.5mg/g, based on the weight of the mice; after 8 minutes, the substrate is fully circulated to the whole body of the mouse, and 2.5-3 percent of the substrate is usedImaging was performed 2 minutes after mice were anesthetized with 5% isoflurane gas. Bioluminescence imaging was performed and quantified by IVIS spectral imaging system (Perkin Elmer) and fluorescence quantification data was obtained by in vivo imaging software (Perkin Elmer) analysis.

The experimental results are as follows:

(1) FIG. 1 shows the sorting of T cell samples from human Peripheral Blood Mononuclear Cells (PBMC) using TRBC1 specific antibody coupled magnetic beads and flow cytometry analysis to evaluate the sorting effect. Taking wild type Jurkat T cells (TRBC1+), knocking out endogenous TCR out, overexpressing Jurkat T cells containing TCR of TRBC2, and taking T cell samples before and after enrichment by magnetic beads, wherein each cell is 1x10 ^6 cells; centrifuging at 300g for 3 min, discarding supernatant, resuspending the cell sample with anti-TRBC1 antibody (diluted to 0.02mg/ml) diluted in PBS, and incubating on ice for 20 min in the dark; washing the cell sample with a proper amount of PBS for 2 times, then re-suspending the cell sample with anti-human IgG APC secondary antibody diluted in PBS, and incubating for 20 minutes on ice under the condition of keeping out of the sun; after washing the cell sample with an appropriate amount of PBS for 2 times, the cell sample was resuspended with DAPI-containing PBS to an appropriate cell concentration, and the APC fluorescence signal intensity on each cell sample was detected by flow cytometry. TRBC1+ and TRBC2+ Jurkat T cells were used as positive and negative controls, respectively; the proportion of TRBC1+ T cells in human T cells prior to magnetic bead sorting was about 30-35%; after magnetic bead sorting, the proportion of TRBC1+ T cells is remarkably reduced, and is about 0-5%.

(2) Figure 2 is a test scenario specific for TRBC1 CAR function. A murine antibody JOVI.1 specifically recognizing TRBC1 and an engineered humanized antibody (H101, H102, H103 and H104) are respectively constructed into a single chain antibody (scFv), and are connected with a 41-BB costimulation domain and a CD3 zeta chain signal domain through a CD8 Hinge and a transmembrane region to construct a second-generation CAR targeting TRBC1, and the second-generation CAR is expressed in a TCR-knocked-out Jurkat T cell line. Wild type CAR (M001) and modified CAR (H101, H102, H103, H104) were tested for CAT-T cell activation (up-regulated level of the T cell activation indicator protein CD69 in response to T cell activation and changes therein) in resting (rest) state and 12-hour stimulation of target cells (TRBC1+ T cell), non-target cells (TRBC2+ T cell, TCR-KO T cell), respectively. As shown, all 4 CAR-T cells were activated and up-regulated in CD69 expression only upon stimulation by TRBC1 expressing target cells, indicating good specificity, and sequence humanization of the antibody did not alter its specific recognition of the antigen.

(3) FIG. 3 is a graph of the detection of the TRBC1 CAR background activation level. Under the condition that the background activation level of the T cells is higher due to CAR, the T cells can enter a depletion state (T cell expansion) because of being continuously in an activation state, so that the proliferation capacity of the T cells is reduced, and the killing function is weakened; on CAR-T cells, the cell line is further characterized by difficult in-vitro amplification, weakened in-vivo continuous proliferation and target cell killing capacity and insufficient anti-tumor treatment effect. The wild type CAR (M001) and the humanized and modified CAR (H101, H102, H103, H104) are respectively expressed in a TCR knockout Jurkat T cell line, the expression condition of an indicator protein CD69 activated by T cells under the condition of no antigen stimulation is detected, and the influence of different CAR on the background activation level of the T cells is observed. As shown in fig. 3, the sequence of the TRBC1 antibody is humanized and modified, so that on one hand, the immunogenicity of the murine antibody is eliminated, and the risk of possible immunological rejection is reduced; on the other hand, the condition that the background activation level of the T cells is higher due to the CAR is reduced through the optimization of the humanized antibody sequence, the proliferation capacity of the CAR-T cells is improved, and the continuous anti-tumor function of the CAR-T cells is improved. Since the humanized antibody H104 constructed CAR has the lowest background activation level and the activation level after encountering antigen is higher, subsequent experiments are completed based on H104 constructed CAR.

(4) FIGS. 4 to 6 show that TRBC 1-targeted M001 CAR constructed by expressing a murine antibody on human primary T cells and H104 CAR constructed by an antibody optimized by sequence humanization are used for verifying the functions of recognizing and killing target cells, cytokine secretion and the like. As shown in fig. 4, the background activation level of H104 was low, indicating a low level of T cell activation at rest.

Referring to fig. 5 (a), the lower background activation level ensures that the H104 CAR-T cells can kill TRBC1+ tumor target cells more effectively without changing their specificity, and referring to fig. 5 (b), the non-specific killing level for non-target cells (TRBC2+ T cells) is low; after 24 hours of incubation with target cells, media supernatants were collected and tested for levels of cytokine IL-2 secreted by CAR-T cells and IFN- γ by enzyme-linked immunosorbent assay (ELISA), as shown in fig. 6, H104 was able to more efficiently secrete more IL-2 and IFN- γ in response to TRBC1+ tumor target cell stimulation.

(5) FIG. 7 is a graph showing the effect of H104 CAR-T cells on killing TRBC1+ tumor target cells in a tumor-bearing mouse animal model. By inoculating NSG (NOD/SCID/IL2R gamma null) into the tail vein of severe combined immunodeficiency mice by intravenous injection with 1x10 ⁶ Forming a TRBC + T cell lymphoma mouse model by using TRBC1+ Jurkat-T cells; after 3 days, they were given 2x10 ⁶ (ii) an H104 CAR-T cell or peripheral mononuclear cell (PBMC) treatment that does not express a CAR; the occurrence and development of tumors were followed by weekly measurements of the fluorescence intensity produced by the oxidation of the substrate by firefly Luciferase (Luciferase) expressed by T tumor cells. As shown in fig. 7 and fig. 8 (a), the PBMC cells inoculated to the control mice failed to effectively control the tumor development, and all the test mice died within 35 days or due to tumor burden, or euthanized because their disease progression reached the euthanization standard ethically required for the experimental animals. As shown in figure 7 and figure 8 (b), the tumor of the mice receiving the H104 CAR-T cell treatment is effectively controlled, the tumor clearance reaches over 75 percent, and the survival rate and the survival state of the tumor-bearing mice are obviously improved.

In conclusion, the present invention provides CAR-T products in which the antibody sequences humanize, specifically recognize and kill TRBC1+ T cells. In the humanized antibody provided by the invention, the background activation level of the CAR constructed by H104 is lowest, the activation level after encountering an antigen is higher, the humanized antibody can effectively and specifically recognize and kill TRBC1+ tumor target cells, the self-activation level of the CAR-T cells is reduced, the condition of function exhaustion of the CAR-T cells is relieved, and the humanized antibody has more advantages in the aspects of continuous anti-tumor effect and the like.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be limited only by the attached claims.

Sequence listing

<110> first-person hospital in Shanghai City

<120> TRBC 1-targeted humanized chimeric antigen receptor, T cell and application

<150> 2020108114627

<151> 2020-08-13

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1524

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagcaga agctgatcag cgaggaggac ctggaggtga ggctgcagca gagcggcccc 120

gacctgatca agcccggcgc cagcgtgaag atgagctgca aggccagcgg ctacaccttc 180

accggctacg tgatgcactg ggtgaagcag aggcccggcc agggcctgga gtggatcggc 240

ttcatcaacc cctacaacga cgacatccag agcaacgaga ggttcagggg caaggccacc 300

ctgaccagcg acaagagcag caccaccgcc tacatggagc tgagcagcct gaccagcgag 360

gacagcgccg tgtactactg cgccaggggc gccggctaca acttcgacgg cgcctacagg 420

ttcttcgact tctggggcca gggcaccacc ctgaccgtga gcagcggcgg cggcggcagc 480

ggcggcggcg gcagcggcgg cggcggcagc gacgtggtga tgacccagag ccccctgagc 540

ctgcccgtga gcctgggcga ccaggccagc atcagctgca ggagcagcca gaggctggtg 600

cacagcaacg gcaacaccta cctgcactgg tacctgcaga agcccggcca gagccccaag 660

ctgctgatct acagggtgag caacaggttc cccggcgtgc ccgacaggtt cagcggcagc 720

ggcagcggca ccgacttcac cctgaagatc agcagggtgg aggccgagga cctgggcatc 780

tacttctgca gccagagcac ccacgtgccc tacaccttcg gcggcggcac caagctggag 840

atcaagagga ccaccactcc cgcaccccgc cctcctactc ctgcccctac cattgctagc 900

caaccgctta gtctgagacc tgaggcctgt aggcccgctg ctggtggcgc tgtgcacacc 960

cgaggattgg acttcgcttg cgacatctac atctgggcac ctctggctgg gacctgcggc 1020

gtgttgttgt tgagcctggt gattacgctg tactgtaaac ggggcagaaa gaaactcctg 1080

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1140

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1200

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1260

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1320

ggaaagccgc agagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1380

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1440

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1500

atgcaggccc tgcctcctcg ctga 1524

<210> 2

<211> 507

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Glu

20 25 30

Val Arg Leu Gln Gln Ser Gly Pro Asp Leu Ile Lys Pro Gly Ala Ser

35 40 45

Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Val

50 55 60

Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

65 70 75 80

Phe Ile Asn Pro Tyr Asn Asp Asp Ile Gln Ser Asn Glu Arg Phe Arg

85 90 95

Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr Thr Ala Tyr Met

100 105 110

Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala

115 120 125

Arg Gly Ala Gly Tyr Asn Phe Asp Gly Ala Tyr Arg Phe Phe Asp Phe

130 135 140

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser

145 150 155 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln

165 170 175

Ser Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser

180 185 190

Cys Arg Ser Ser Gln Arg Leu Val His Ser Asn Gly Asn Thr Tyr Leu

195 200 205

His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr

210 215 220

Arg Val Ser Asn Arg Phe Pro Gly Val Pro Asp Arg Phe Ser Gly Ser

225 230 235 240

Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu

245 250 255

Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr

260 265 270

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala

275 280 285

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

290 295 300

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

305 310 315 320

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

325 330 335

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

340 345 350

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

355 360 365

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

370 375 380

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

385 390 395 400

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

405 410 415

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

420 425 430

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn

435 440 445

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

450 455 460

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

465 470 475 480

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

485 490 495

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

<210> 3

<211> 1521

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagcaga agctgatcag cgaggaggac ctgcaggtgc agctggtgca gagcggcgcc 120

gaggtgaaga agcccggcgc cagcgtgaag gtgagctgca aggccagcgg ctacaccttc 180

accggctacg tgatgcactg ggtgaggcag gcccccggcc agggcctgga gtggatgggc 240

ttcatcaacc cctacaacga cgacatccag agcaacgaga ggttcagggg cagggtgacc 300

atgaccaggg acaccagcat cagcaccgcc tacatggagc tgagcaggct gaggagcgac 360

gacaccgccg tgtactactg cgccaggggc gccggctaca acttcgacgg cgcctacagg 420

ttcttcgact tctggggcca gggcaccacc gtgaccgtga gcagcggcgg cggcggcagc 480

ggcggcggcg gcagcggcgg cggcggcagc gacgtggtga tgacccagag ccccctgagc 540

ctgcccgtga ccctgggcca gcccgccagc atcagctgca ggagcagcca gaggctggtg 600

cacagcaacg gcaacaccta cctgcactgg ttccagcaga ggcccggcca gagccccagg 660

aggctgatct acagggtgag caacaggttc cccggcgtgc ccgacaggtt cagcggcagc 720

ggcagcggca ccgacttcac cctgaagatc agcagggtgg aggccgagga cgtgggcgtg 780

tactactgca gccagagcac ccacgtgccc tacaccttcg gcggcggcac caaggtggag 840

atcaagacca ccactcccgc accccgccct cctactcctg cccctaccat tgctagccaa 900

ccgcttagtc tgagacctga ggcctgtagg cccgctgctg gtggcgctgt gcacacccga 960

ggattggact tcgcttgcga catctacatc tgggcacctc tggctgggac ctgcggcgtg 1020

ttgttgttga gcctggtgat tacgctgtac tgtaaacggg gcagaaagaa actcctgtat 1080

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1140

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1200

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1260

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1320

aagccgcaga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1380

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1440

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1500

caggccctgc ctcctcgctg a 1521

<210> 4

<211> 506

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gln

20 25 30

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser

35 40 45

Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Val

50 55 60

Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

65 70 75 80

Phe Ile Asn Pro Tyr Asn Asp Asp Ile Gln Ser Asn Glu Arg Phe Arg

85 90 95

Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met

100 105 110

Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala

115 120 125

Arg Gly Ala Gly Tyr Asn Phe Asp Gly Ala Tyr Arg Phe Phe Asp Phe

130 135 140

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser

145 150 155 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln

165 170 175

Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser

180 185 190

Cys Arg Ser Ser Gln Arg Leu Val His Ser Asn Gly Asn Thr Tyr Leu

195 200 205

His Trp Phe Gln Gln Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile Tyr

210 215 220

Arg Val Ser Asn Arg Phe Pro Gly Val Pro Asp Arg Phe Ser Gly Ser

225 230 235 240

Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu

245 250 255

Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser Thr His Val Pro Tyr Thr

260 265 270

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

325 330 335

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys

340 345 350

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

355 360 365

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro

370 375 380

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

385 390 395 400

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

405 410 415

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

420 425 430

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro

435 440 445

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

450 455 460

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

465 470 475 480

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

485 490 495

Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

<210> 5

<211> 1521

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagcaga agctgatcag cgaggaggac ctgcaggtgc agctggtgca gagcggcgcc 120

gaggtgaaga agcccggcgc cagcgtgaag gtgagctgca aggccagcgg ctacaccttc 180

accggctacg tgatgcactg ggtgaggcag gcccccggcc agggcctgga gtggatgggc 240

ttcatcaacc cctacaacga cgacatccag agcaacgaga ggttcagggg cagggtgacc 300

atgaccaggg acaccagcat cagcaccgcc tacatggagc tgagcaggct gaggagcgac 360

gacaccgccg tgtactactg cgccaggggc gccggctaca acttcgacgg cgcctacagg 420

ttcttcgact tctggggcca gggcaccacc gtgaccgtga gcagcggcgg aggtggcagc 480

ggaggtggag gaagcggtgg aggcggaagc gacgtggtga tgacccagag ccccctgagc 540

ctgcccgtga ccctgggcca gcccgccagc atcagctgca ggagcagcca gaggctggtg 600

cacagcaacg gcaacaccta cctgcactgg taccagcaga ggcccggcca gagccccagg 660

ctgctgatct acagggtgag caacaggttc cccggcgtgc ccgacaggtt cagcggcagc 720

ggcagcggca ccgacttcac cctgaagatc agcagggtgg aggccgagga cgtgggcgtg 780

tacttctgca gccagagcac ccacgtgccc tacaccttcg gcggcggcac caagctggag 840

atcaagagga ccaccactcc cgcaccccgc cctcctactc ctgcccctac cattgctagc 900

caaccgctta gtctgagacc tgaggcctgt aggcccgctg ctggtggcgc tgtgcacacc 960

cgaggattgg acttcgcttg cgacatctac atctgggcac ctctggctgg gacctgcggc 1020

gtgttgttgt tgagcctggt gattacgctg tactgtaaac ggggcagaaa gaaactcctg 1080

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1140

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1200

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1260

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1320

ggaaagccgc agagaaggaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1380

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1440

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1500

caggccctgc ctcctcgctg a 1521

<210> 6

<211> 506

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gln

20 25 30

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser

35 40 45

Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Val

50 55 60

Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

65 70 75 80

Phe Ile Asn Pro Tyr Asn Asp Asp Ile Gln Ser Asn Glu Arg Phe Arg

85 90 95

Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met

100 105 110

Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala

115 120 125

Arg Gly Ala Gly Tyr Asn Phe Asp Gly Ala Tyr Arg Phe Phe Asp Phe

130 135 140

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser

145 150 155 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln

165 170 175

Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser

180 185 190

Cys Arg Ser Ser Gln Arg Leu Val His Ser Asn Gly Asn Thr Tyr Leu

195 200 205

His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr

210 215 220

Arg Val Ser Asn Arg Phe Pro Gly Val Pro Asp Arg Phe Ser Gly Ser

225 230 235 240

Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu

245 250 255

Asp Val Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr

260 265 270

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala

275 280 285

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

290 295 300

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

305 310 315 320

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

325 330 335

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

340 345 350

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

355 360 365

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

370 375 380

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

385 390 395 400

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

405 410 415

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

420 425 430

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Asn Pro

435 440 445

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

450 455 460

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

465 470 475 480

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

485 490 495

Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

<210> 7

<211> 1524

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagcaga agctgatcag cgaggaggac ctgcaggtgc agctggtgca gagcggcgcc 120

gaggtgaaga agcccggcgc cagcgtgaag gtgagctgca aggccagcgg ctacaccttc 180

accggctacg tgatgcactg ggtgaggcag gcccccggcc agggcctgga gtggatgggc 240

ttcatcaacc cctacaacga cgacatccag agcaacgaga ggttcagggg cagggtgacc 300

atgaccagcg acaagagcat caccaccgcc tacatggagc tgagcaggct gaggagcgac 360

gacaccgccg tgtactactg cgccaggggc gccggctaca acttcgacgg cgcctacagg 420

ttcttcgact tctggggcca gggcaccacc gtgaccgtga gcagcggcgg aggtggcagc 480

ggaggtggag gaagcggtgg aggcggaagc gacgtggtga tgacccagag ccccctgagc 540

ctgcccgtga ccctgggcca gcccgccagc atcagctgca ggagcagcca gaggctggtg 600

cacagcaacg gcaacaccta cctgcactgg ttccagcaga ggcccggcca gagccccagg 660

aggctgatct acagggtgag caacaggttc cccggcgtgc ccgacaggtt cagcggcagc 720

ggcagcggca ccgacttcac cctgaagatc agcagggtgg aggccgagga cgtgggcgtg 780

tactactgca gccagagcac ccacgtgccc tacaccttcg gcggcggcac caagctggag 840

atcaagagga ccaccactcc cgcaccccgc cctcctactc ctgcccctac cattgctagc 900

caaccgctta gtctgagacc tgaggcctgt aggcccgctg ctggtggcgc tgtgcacacc 960

cgaggattgg acttcgcttg cgacatctac atctgggcac ctctggctgg gacctgcggc 1020

gtgttgttgt tgagcctggt gattacgctg tactgtaaac ggggcagaaa gaaactcctg 1080

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1140

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1200

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1260

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1320

ggaaagccgc agagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1380

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1440

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1500

atgcaggccc tgcctcctcg ctga 1524

<210> 8

<211> 507

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gln

20 25 30

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser

35 40 45

Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Val

50 55 60

Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

65 70 75 80

Phe Ile Asn Pro Tyr Asn Asp Asp Ile Gln Ser Asn Glu Arg Phe Arg

85 90 95

Gly Arg Val Thr Met Thr Ser Asp Lys Ser Ile Thr Thr Ala Tyr Met

100 105 110

Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala

115 120 125

Arg Gly Ala Gly Tyr Asn Phe Asp Gly Ala Tyr Arg Phe Phe Asp Phe

130 135 140

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser

145 150 155 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln

165 170 175

Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser

180 185 190

Cys Arg Ser Ser Gln Arg Leu Val His Ser Asn Gly Asn Thr Tyr Leu

195 200 205

His Trp Phe Gln Gln Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile Tyr

210 215 220

Arg Val Ser Asn Arg Phe Pro Gly Val Pro Asp Arg Phe Ser Gly Ser

225 230 235 240

Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu

245 250 255

Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser Thr His Val Pro Tyr Thr

260 265 270

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala

275 280 285

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

290 295 300

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

305 310 315 320

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

325 330 335

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

340 345 350

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

355 360 365

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

370 375 380

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

385 390 395 400

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

405 410 415

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

420 425 430

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn

435 440 445

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

450 455 460

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

465 470 475 480

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

485 490 495

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

<210> 9

<211> 1524

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagcaga agctgatcag cgaggaggac ctgcaggtgc agctggtgca gagcggcgcc 120

gaggtgaaga agcccggcgc cagcgtgaag gtgagctgca aggccagcgg ctacaccttc 180

accggctacg tgatgcactg ggtgaggcag gcccccggcc agggcctgga gtggatgggc 240

ttcatcaacc cctacaacga cgacatccag agcaacgaga ggttcagggg cagggtgacc 300

atgaccagcg acaagagcat caccaccgcc tacatggagc tgagcaggct gaggagcgac 360

gacaccgccg tgtactactg cgccaggggc gccggctaca acttcgacgg cgcctacagg 420

ttcttcgact tctggggcca gggcaccacc gtgaccgtga gcagcggcgg aggtggcagc 480

ggaggtggag gaagcggtgg aggcggaagc gacgtggtga tgacccagag ccccctgagc 540

ctgcccgtga ccctgggcca gcccgccagc atcagctgca ggagcagcca gaggctggtg 600

cacagcaacg gcaacaccta cctgcactgg taccagcaga ggcccggcca gagccccagg 660

ctgctgatct acagggtgag caacaggttc cccggcgtgc ccgacaggtt cagcggcagc 720

ggcagcggca ccgacttcac cctgaagatc agcagggtgg aggccgagga cgtgggcgtg 780

tacttctgca gccagagcac ccacgtgccc tacaccttcg gcggcggcac caagctggag 840

atcaagagga ccaccactcc cgcaccccgc cctcctactc ctgcccctac cattgctagc 900

caaccgctta gtctgagacc tgaggcctgt aggcccgctg ctggtggcgc tgtgcacacc 960

cgaggattgg acttcgcttg cgacatctac atctgggcac ctctggctgg gacctgcggc 1020

gtgttgttgt tgagcctggt gattacgctg tactgtaaac ggggcagaaa gaaactcctg 1080

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1140

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1200

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1260

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1320

ggaaagccgc agagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1380

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1440

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1500

atgcaggccc tgcctcctcg ctga 1524

<210> 10

<211> 507

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gln

20 25 30

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser

35 40 45

Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Val

50 55 60

Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

65 70 75 80

Phe Ile Asn Pro Tyr Asn Asp Asp Ile Gln Ser Asn Glu Arg Phe Arg

85 90 95

Gly Arg Val Thr Met Thr Ser Asp Lys Ser Ile Thr Thr Ala Tyr Met

100 105 110

Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala

115 120 125

Arg Gly Ala Gly Tyr Asn Phe Asp Gly Ala Tyr Arg Phe Phe Asp Phe

130 135 140

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser

145 150 155 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln

165 170 175

Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser

180 185 190

Cys Arg Ser Ser Gln Arg Leu Val His Ser Asn Gly Asn Thr Tyr Leu

195 200 205

His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr

210 215 220

Arg Val Ser Asn Arg Phe Pro Gly Val Pro Asp Arg Phe Ser Gly Ser

225 230 235 240

Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu

245 250 255

Asp Val Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr

260 265 270

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala

275 280 285

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

290 295 300

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

305 310 315 320

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

325 330 335

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

340 345 350

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

355 360 365

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

370 375 380

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

385 390 395 400

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

405 410 415

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

420 425 430

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn

435 440 445

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

450 455 460

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

465 470 475 480

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

485 490 495

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

<210> 11

<211> 124

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Phe Ile Asn Pro Tyr Asn Asp Asp Ile Gln Ser Asn Glu Arg Phe

50 55 60

Arg Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ala Gly Tyr Asn Phe Asp Gly Ala Tyr Arg Phe Phe Asp

100 105 110

Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 12

<211> 124

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Phe Ile Asn Pro Tyr Asn Asp Asp Ile Gln Ser Asn Glu Arg Phe

50 55 60

Arg Gly Arg Val Thr Met Thr Ser Asp Lys Ser Ile Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ala Gly Tyr Asn Phe Asp Gly Ala Tyr Arg Phe Phe Asp

100 105 110

Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 13

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Arg Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Arg Val Ser Asn Arg Phe Pro Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 14

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Arg Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Pro Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 15

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 16

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 17

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 18

<211> 69

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 19

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 20

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

Claims (6)

1. A TRBC 1-targeting humanized chimeric antigen receptor comprising scFv sequences derived from a murine antibody jovi.1, CDR regions of the murine antibody jovi.1 being grafted to human antibody frameworks VH1_1-02 and VK2_ a 17; wherein, the scFv comprises a heavy chain and a light chain, and the sequences of the heavy chain and the light chain of the scFv are respectively shown as SEQ ID NO.12 and SEQ ID NO. 14; the chimeric antigen receptor comprises: an extracellular domain structure formed by serially connecting a CD8 alpha signal peptide sequence, a tag sequence, the scFv sequence and a hinge sequence of a CD8 alpha chain in sequence, a transmembrane region sequence of a CD8 alpha chain, and an intracellular domain structure formed by serially connecting an intracellular domain sequence of a human 41BB molecule and an intracellular domain sequence of a human CD3 zeta chain.

2. The TRBC 1-targeting humanized chimeric antigen receptor according to claim 1, wherein a nucleic acid sequence encoding the chimeric antigen receptor is set forth in SEQ ID number 9; the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 10.

3. A T cell, comprising: the humanized chimeric antigen receptor targeting TRBC1 of claim 1 or 2.

4. The T-cell of claim 3, wherein the T-cell is a T-cell purified with magnetic beads; the magnetic beads are coated with TRBC1 specific antibodies.

5. The T-cell of claim 4, wherein said method of preparing said T-cell comprises the steps of:

(1) binding TRBC1 antibody to magnetic beads;

(2) mixing T cells to be purified with magnetic beads combined with TRBC1 antibody;

(3) and standing on a magnetic frame, and taking the supernatant to obtain the T cells.

6. The use of the humanized chimeric antigen receptor targeting TRBC1 according to claim 1 or 2, wherein the chimeric antigen receptor is used for the preparation of a targeted anti-tumor medicament; the tumor is T lymphocyte tumor with TRBC1 positive tumor T cells.

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