阅读说明：本技术 一种靶向CD19的人源化scFv嵌合抗原受体T细胞及制备方法、应用 (humanized scFv chimeric antigen receptor T cells targeting CD19, and preparation method and application thereof ) 是由 谭毅 孔群芳 张慧慧 韩镇 于 2019-11-18 设计创作，主要内容包括：本发明属于基因工程和细胞生物学技术领域,具体涉及一种靶向CD19的人源化scFv嵌合抗原受体T细胞及制备方法、应用。具体为构建含有靶向CD19的人源化scFv嵌合抗原受体(anti-hCD19CAR)基因结构的慢病毒表达载体,通过慢病毒介导的方式将含有anti-hCD19CAR表达载体转导至T细胞,anti-hCD19CAR修饰的T细胞能够特异性杀伤CD19阳性的肿瘤细胞,能够有效避免鼠源CD19基因导致的人抗鼠抗体的产生,降低鼠源CAR的免疫原性,能够改善CAR-T细胞的存活时间,高特异性的与人源CD19蛋白结合,增强CAR-T的治疗效果,提高CAR-T治疗的安全性和有效性。(The invention belongs to the technical field of genetic engineering and cytobiology, and particularly relates to humanized scFv chimeric antigen receptor T cells targeting CD19, a preparation method and application thereof, wherein a lentivirus expression vector containing a gene structure of a humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) targeting CD19 is constructed, the anti-hCD19CAR expression vector is transduced to the T cells in a lentivirus mediated mode, the anti-hCD19CAR modified T cells can specifically kill CD19 positive tumor cells, the generation of human anti-mouse antibodies caused by murine CD19 genes can be effectively avoided, the immunogenicity of murine CAR can be reduced, the survival time of the CAR-T cells can be improved, the high-specificity is combined with human CD19 protein, the treatment effect of CAR-T is enhanced, and the safety and the effectiveness of treatment of the CD-T are improved.)

lentivirus expression vectors, which is characterized in that the vectors comprise a humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) gene structure targeting CD19, and the nucleotide sequence of the vectors is SEQ ID NO.1, SEQ ID NO. 2 or SEQ ID NO. 3.

2. The lentiviral expression vector of claim 1, wherein the chimeric antigen receptor gene construct comprises, in serial order, a CD8 transmembrane signal peptide, anti-hCD19scFv, a CD8 transmembrane region, a 4-1BB co-stimulatory signal region, and a CD3Zeta TCR activation region.

3. The lentiviral expression vector of claim 2, wherein the CD8 transmembrane signal peptide has the amino acid sequence shown in SEQ ID No.4, the anti-hCD19scFv comprises or is selected from any amino acid sequence selected from the group consisting of SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, the CD8 transmembrane region has the amino acid sequence shown in SEQ ID No.8, the 4-1BB co-stimulatory signal region has the amino acid sequence shown in SEQ ID No.9, and the CD3Zeta TCR activation region has the amino acid sequence shown in SEQ ID No. 10.

4, A method for preparing CD19 targeted humanized scFv chimeric antigen receptor T cells comprising the lentiviral expression vector of claim 1, wherein the method comprises the steps of:

(1) constructing a plasmid vector: after the anti-CD19 scFv fragment is subjected to humanized transformation, obtaining anti-hCD19scFv, inserting an Mlu I enzyme cutting site and a CD8 transmembrane signal peptide into the fragment, inserting a BamH I enzyme cutting site into the fragment, synthesizing a pUC57-Amp plasmid containing Mlu I + CD8a-hCD19scFv + BamH I, performing double enzyme cutting by Mlu I and BamH I, identifying the enzyme cutting effect by agarose gel electrophoresis, and recovering gel to obtain a modified gene fragment; meanwhile, carrying out double enzyme digestion on the lentiviral backbone plasmid pHR containing the CD8 transmembrane region, the 4-1BB costimulatory signal region and the CD3Zeta TCR activation region by Mlu I and BamH I, and recovering long fragments from gel after agarose gel electrophoresis identification; connecting the modified anti-hCD19scFv fragment to a lentiviral backbone plasmid pHR, extracting the plasmid, and obtaining a pHR-anti-hCD19CAR plasmid after determining that the sequencing is correct;

(2) and (3) preparing lentivirus: mixing the pHR-anti-hCD19CAR plasmid carrying the target gene, a pCMV vector and a pMD.2G vector, transfecting into 293FT cells, replacing with a DMEM complete culture medium containing 10% FBS and 1% glutamine for culture after 6-8 h after transfection, collecting a culture solution after 48h, centrifuging, retaining a supernatant, filtering the supernatant by using a 0.45 mu m filter, retaining a filtrate, wherein the filtrate is a recombinant lentivirus solution, and concentrating for later use;

(3) anti-hCD19CAR-T preparation: 50mL of fresh blood was taken and passed through lymphocytesSubjecting the separation solution to density gradient centrifugation to separate mononuclear cells, wherein the single mononuclear cells are subjected to density gradient centrifugation according to the ratio of 1-2 × 10⁶/mL resuspended CTS^TMAIM V^TM5% ICS, 50ng/mL of CD3 monoclonal antibody and 50ng/mL of CD28 monoclonal antibody are added into an SFM culture medium at the same time to activate T lymphocytes, and the cells are cultured for 48 hours at 37 ℃ with 5% CO 2; after 2 days of culture, cells were harvested and resuspended to 1X10⁶Adding the concentrated recombinant lentivirus solution according to MOI = 5, simultaneously adding IL-2 and polybrene with final concentration of 500U/mL, uniformly mixing, and 5% CO at 37 DEG C₂Culturing for 6-8 hours, centrifuging at 300g for 5min, and changing the liquid into fresh CTS^TMAIM V^TMSFM medium; adding fresh CTS every 2-3 days^TMAIM V^TMSFM medium, maintaining cell density at 1X10⁶about/mL, and performing amplification culture for 10-12 days.

5. The method according to claim 4, wherein, in the step (3), the CTS is^TMAIM V^TMSFM medium contained 500U/mL IL-2.

Use of humanized scFv chimeric antigen receptor T-cells targeting CD19 according to any of claims 1-3 to for the preparation of a medicament for the prevention and/or treatment and/or adjuvant treatment of malignancies.

7. The use of claim 7, wherein said malignant cells comprise a CD19 positive B-lymphocyte leukemia or B-lymphoma.

Technical Field

The invention belongs to the technical field of genetic engineering and cell biology, and particularly relates to humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) T cells targeting CD19, and a preparation method and application thereof.

Background

In view of the poor curative effect, i.e. low Complete response rate (CR) or high recurrence rate, of the traditional therapies such as chemotherapy, radiotherapy and hematopoietic stem cell transplantation on hematological malignancies, for example, in Acute Lymphoblastic Leukemia (ALL), although the CR rate after chemotherapy can reach 80% -90%, the recurrence rate of about 70% and the Disease free survival rate (DFS) of about 30% in 3 years, researchers still need to search for better therapeutic methods.

With the development of tumor immunology theory and technology, cellular immunotherapy has advanced greatly in recent years, and is listed as the first ten scientific breakthroughs in 2013 by the Science journal. The target immunotherapy of tumor represented by T cells modified by Chimeric Antigen Receptor (CAR) is particularly outstanding in achievement, shows good targeting, killing and durability in vitro and clinical tests, and shows huge application potential and development prospect.

CAR-T cell therapy expresses a Single chain antibody fragment (scFv) for recognizing tumor-associated specific antigen and a fusion protein of a T cell activation sequence to the surface of a T cell by an exogenous gene transduction technology, so that the scFv is coupled with an activation proliferation signal domain in the T cell through a transmembrane region, is greatly amplified after being back-transfused into a patient body, and can show a strong anti-tumor effect in an antigen-dependent and non-MHC-restricted mode.

CD19 is transmembrane glycoproteins belonging to Ig superfamily members, expressed on B lineage cells (including pre-B cells, immature B cells, mature B cells and activated B cells) and follicular dendritic cells, and is an important membrane antigen involved in B lymphocyte differentiation, activation, proliferation and antibody production.most B cell malignant tumor cells express CD19 molecules, and hematopoietic stem cells and non-hematopoietic cells do not express CD19, so CD19 is the best marker for diagnosing B lymphocyte lineage tumors and identifying B lymphocytes, and is the most potential target for treating B lineage tumors.

In 8 months 2017, the U.S. FDA officially approved the first CAR-T drug kymeriah (CTL 019) for clinical treatment of relapsed or refractory (r/r) childhood and young adult B-cell acute lymphoblastic leukemia, which is a human historical milestone event in the same year 10 months the U.S. FDA approved the 2 nd CAR-T drug yescata (KTE-C10) for treatment of certain types of adult large B-cell lymphoma patients who either failed to respond to other therapies or relapsed after receiving at least 2 treatment regimens, including diffuse large B-cell lymphoma, transformed follicular lymphoma, primary mediastinal B-cell lymphoma.

The current data show a Complete Remission (CR) rate of 70-90% in patients with refractory relapse B-ALL to anti-CD19 CAR-T therapy. The regression of hematologic malignant cells is closely related to the level of proliferation of CAR-T cells and their survival time in the blood. CAR-T cell depletion in ALL patients was accompanied by recovery of normal B cells, and some patients developed a relapse of CD19+ leukemia. The CD19 antigen receptor used in CAR-T technology at present adopts murine gene, such as murine monoclonal antibody FMC63, but such murine gene fragment has possibility of causing the generation of Human anti-mouse antibody (HAMA) in the process of treating Human diseases. There are studies showing that the immunogenicity of murine CAR sequences may lead to the inability of CAR-T cells to activate and persist.

Disclosure of Invention

In response to the problems of the prior art, the present invention provides humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) T cells targeting CD 19.

The invention also provides a preparation method of humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) T cells targeting CD 19.

The invention also provides application of the humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) T cell targeting CD 19.

The technical scheme adopted by the invention for realizing the purpose is as follows:

the invention provides lentivirus expression vectors, which are characterized by comprising a humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) gene structure targeting CD19, and the nucleotide sequences of the humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) gene structure are SEQ ID NO.1, SEQ ID NO. 2 or SEQ ID NO. 3.

Further , the chimeric antigen receptor gene structure comprises in serial order a CD8 transmembrane signal peptide, anti-hCD19scFv, CD8 transmembrane region, 4-1BB costimulatory signal region, and CD3Zeta TCR activation region.

The amino acid sequence of the CD8 transmembrane signal peptide is shown as SEQ ID NO.4, the anti-hCD19scFv comprises or is selected from any amino acid sequence shown as SEQ ID NO. 5 or SEQ ID NO. 6 or SEQ ID NO. 7, wherein the scFv shown as (b), (c) and (a) have the same function, and the scFv amino acid sequences shown as (b), (c) and (a) have more than 90% of consistency, (b), (c) and (a) all consist of a light chain variable region, a light chain connecting region and a heavy chain connecting region, the amino acid sequence of the CD8 transmembrane region is shown as SEQ ID NO.8, the amino acid sequence of the 4-1BB co-stimulation signal region is shown as SEQ ID NO.9, and the amino acid sequence of the CD3 ZeTCR activation region is shown as SEQ ID NO. 10.

The invention also provides a preparation method of humanized scFv chimeric antigen receptor T cells which contain the lentivirus expression vector and target CD19, and the humanized scFv chimeric antigen receptor T cells are prepared by the following method:

(1) constructing a plasmid vector: after the anti-CD19 scFv fragment is subjected to humanized transformation, obtaining anti-hCD19scFv, inserting an Mlu I enzyme cutting site and a CD8 transmembrane signal peptide into the fragment, inserting a BamH I enzyme cutting site into the fragment, synthesizing a pUC57-Amp plasmid containing Mlu I + CD8a-hCD19scFv + BamH I, performing double enzyme cutting by Mlu I and BamH I, identifying the enzyme cutting effect by agarose gel electrophoresis, and recovering gel to obtain a modified gene fragment; meanwhile, carrying out double enzyme digestion on the lentiviral backbone plasmid pHR containing the CD8 transmembrane region, the 4-1BB costimulatory signal region and the CD3Zeta TCR activation region by Mlu I and BamH I, and recovering long fragments from gel after agarose gel electrophoresis identification; connecting the modified anti-hCD19scFv fragment to a lentiviral backbone plasmid pHR, extracting the plasmid, and obtaining a pHR-anti-hCD19CAR plasmid after determining that the sequencing is correct;

(2) and (3) preparing lentivirus: mixing the pHR-anti-hCD19CAR plasmid carrying the target gene, a pCMV vector and a pMD.2G vector, transfecting into 293FT cells, replacing with a DMEM complete culture medium containing 10% FBS and 1% glutamine for culture after 6-8 h after transfection, collecting a culture solution after 48h, centrifuging, retaining a supernatant, filtering the supernatant by using a 0.45 mu m filter, retaining a filtrate, wherein the filtrate is a recombinant lentivirus solution, and concentrating for later use;

(3) anti-hCD19CAR-T preparation: taking 50mL of fresh blood, separating mononuclear cells by density gradient centrifugation of lymphocyte separating medium, and performing 1-2 × 10 centrifugation on the mononuclear cells⁶/mL resuspended CTS^TMAIM V^TM5% ICS, 50ng/mL of CD3 monoclonal antibody and 50ng/mL of CD28 monoclonal antibody are added into an SFM culture medium at the same time to activate T lymphocytes, and the cells are cultured for 48 hours at 37 ℃ with 5% CO 2; after 2 days of culture, cells were harvested and resuspended to 1X10⁶Adding the concentrated recombinant lentivirus solution according to MOI = 5, simultaneously adding IL-2 and polybrene with final concentration of 500U/mL, uniformly mixing, and 5% CO at 37 DEG C₂Culturing for 6-8 hours, centrifuging at 300g for 5min, and changing the liquid into fresh CTS^TMAIM V^TMSFM medium; adding fresh CTS every 2-3 days^TMAIM V^TMSFM medium, maintaining cell density at 1X10⁶about/mL, and the amplification culture is carried out for 10-12 days, step , in step (3), the CTS^TMAIM V^TMSFM medium contained 500U/mL IL-2.

The invention also provides application of humanized scFv chimeric antigen receptor T cells targeting CD19 in preparation of medicines for preventing and/or treating and/or adjunctively treating malignant tumors.

The malignant tumor cells include CD19 positive B lymphocyte leukemia or B lymphocyte tumor.

some common terms used in the present invention are described below:

CAR: chimeric antigen receptors

scFv Single chain antibody fragment

anti-hCD19 CAR: humanized scFv chimeric antigen receptor targeting CD19

anti-mCD19CAR targeting murine CD19scFv chimeric antigen receptor

T lymphocytes

The invention provides a preparation method of humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) T cells of targeting CD19 and specifically killing CD19 malignant B cell tumors, wherein the chimeric antigen receptor can be combined with human CD19 protein with high specificity, a humanized antibody fragment of the targeting CD19 integrated in the CAR is engineered and expressed in the T cells by utilizing a genetic engineering technology, and the obtained CAR-T cells can be used for treating hematological malignancies expressing CD 19.

The anti-hCD19CAR T cell provided by the invention can specifically kill CD19 positive tumor cells, in-vitro tests prove that the anti-hCD19CAR T killing effect is achieved, the killing efficiency is over 95% when the ratio of effector cells to target cells is 3:1, and cytokines such as IL-2 and IFN-gamma are generated in the killing process.

The anti-hCD19CAR T cell provided by the invention can effectively protect B cell lymphoma loaded mice, and after 60 days of observation, the survival rate of the mice reaches 75 percent and is obviously higher than that of a tumor loaded mouse group transplanted with the anti-mCD19CAR-T cell.

The invention has the following beneficial effects:

(1) the anti-hCD19CAR T cell provided by the invention can effectively avoid the generation of human anti-mouse antibodies caused by murine CD19 genes, reduce the immunogenicity of murine CAR, improve the survival time of CAR-T cells, combine with human CD19 protein with high specificity, enhance the therapeutic effect of CAR-T, and improve the safety and effectiveness of CAR-T therapy.

Drawings

FIG. 1 is a schematic diagram of the modification of anti-hCD19scFv fragment according to the present invention.

FIG. 2 shows the construction of pHR-anti-hCD19CAR plasmid according to the present invention.

FIG. 3 is a schematic diagram showing the flow assay results of lentivirus titer according to the present invention.

FIG. 4 flow cytometry described herein detects anti-hCD19CAR T cell CAR protein expression.

FIG. 5 shows the specific killing results of anti-hCD19CAR T cells of the present invention in vitro killing CD19 positive tumor cells at different target ratios.

FIG. 6 is a schematic diagram showing the results of the content of IL-2 and IFN-gamma secreted to the outside of cells when anti-hCD19CAR T cells kill CD19 positive tumor cells in vitro.

FIG. 7 is a schematic diagram of tumor-bearing mouse survival curves after the anti-hCD19CAR T cells of the present invention are transplanted into a human B-cell lymphoma transplanted tumor mouse model.

The present invention is further illustrated in and will not be limited to the details of the embodiments illustrated in the drawings and described in the following description, it is understood that the embodiments of the invention are not limited to the examples disclosed herein, but are intended to cover modifications within the scope of the invention as expressed in the claims.

Example 1: plasmid vector construction

after the anti-CD19 scFv fragment is subjected to humanized transformation, the anti-hCD19scFv is obtained, the Mlu I enzyme cutting site and the CD8 transmembrane signal peptide are inserted in the front of the fragment, the BamH I enzyme cutting site is inserted in the back of the fragment (shown in figure 1), and the fragment is handed to a gene company (Jinwei Zhi) for synthesis. A pUC57-Amp plasmid containing Mlu I + CD8a-hCD19scFv + BamH I is synthesized by a gene company, and is subjected to double enzyme digestion of Mlu I and BamH I, agarose gel electrophoresis is performed to identify the enzyme digestion effect, and gel is recovered to obtain a modified gene fragment, which is shown in figure 1. Meanwhile, the slow virus skeleton plasmid pHR (see patent CN 108753774A) which is already in a laboratory and contains a CD8 transmembrane region, a 4-1BB costimulatory signal region and a CD3Zeta TCR activation region is subjected to double enzyme digestion of Mlu I and BamH I, and the gel is recovered into a long fragment after agarose gel electrophoresis identification. The modified anti-hCD19scFv fragment was ligated into the lentiviral backbone plasmid pHR, and the pHR-anti-hCD19CAR plasmid was obtained after plasmid extraction to confirm correct sequencing (as shown in FIG. 2).