CN109265563B

CN109265563B - human chimeric antigen receptor for treating blood tumor and its application

Info

Publication number: CN109265563B
Application number: CN201811125546.4A
Authority: CN
Inventors: 张同存; 顾潮江; 吴寒; 李杰珍; 舒冲; 牛敏杰; 许婧; 王芳
Original assignee: Wuhan Ruida Biotechnology Co Ltd
Current assignee: Wuhan Ruida Biotechnology Co Ltd
Priority date: 2018-09-26
Filing date: 2018-09-26
Publication date: 2020-01-31
Anticipated expiration: 2038-09-26
Also published as: CN109265563A

Abstract

The invention relates to human chimeric antigen receptors for treating hematological tumors and application thereof, wherein the human chimeric antigen receptor comprises a human anti-CD 30 single-chain antibody, an extracellular hinge region, a transmembrane domain and an intracellular signal domain, and the amino acid sequence of the human anti-CD 30 single-chain antibody is shown in SEQ ID NO: 3. the invention also relates to nucleic acid for encoding the chimeric antigen receptor, CAR-T cells can be obtained by transducing T cells with the lentiviral vector loaded with the chimeric antigen receptor, so that the CAR-T cells can be obtained, and the CD30 positive tumor cells can be efficiently and specifically killed, thereby providing a more efficient method with fewer side effects and adverse reactions for treating CD30 surface antigen positive malignant tumors, such as Hodgkin lymphoma and the like.

Description

human chimeric antigen receptor for treating blood tumor and its application

Technical Field

The invention relates to the field of cellular immunotherapy, in particular to human chimeric antigen receptors for treating hematological tumors, and a coding sequence and application thereof.

Background

CD30, a member of Tumor Necrosis Factor Receptor Superfamily (TNFRSF), belongs to type I transmembrane glycoprotein, is highly expressed on the surface of Hodgkin Lymphoma (HL) and Anaplastic Large Cell Lymphoma (ALCL), is lowly expressed on the surface of T cells and B cells which are activated in a non-pathological state, but is not expressed in normal cells, early studies show that CD30 is involved in cell activation and differentiation, transduction of signals such as NF-kB and T cell immune activation, and the like, and the current studies show that CD30 is closely related to proliferation and death of cells, and the intracellular part of the CD can interact with a plurality of members of TNFR associated factor (TNFR associated factor) family, thereby mediating apoptosis of T cells through JNK and p38 pathways, and mediating apoptosis of cells through NF-kB pathways, CD30 is rapidly activated by TNFR associated factor, and is formed in serum, and is used as a high content of soluble protein which can be used as a diagnostic index of CD 678663 in a human body under a low content of serum, and serum content of CD 8678, and serum.

Monoclonal antibodies have been used extensively in the field of tumor therapy over the last 20 years, and since the advent of the th generation anti-CD 30 monoclonal antibody, MDX-060 second generation anti-CD 30 monoclonal antibody, MDX-1403, and XmAb third generation anti-CD 30 monoclonal antibody, SGN-35, there have been major breakthroughs in hodgkin lymphoma and anaplastic large cell lymphoma therapy, changing traditional therapies, increasing the survival of CD30 positive patients, but there remain many clinical problems, such as FDA-approved SGN-35 only for current HL patients who have failed autologous stem cell transplantation or failed previous chemotherapy twice and ALCL patients who have failed previous HL transplantation, while tumor patients need to receive chemotherapy earlier, generating toxic and side effects and developing drug resistance, and CAR-T (cardiac gene therapy) is a constant problem, but the development of CD30 and the development of CD-light therapy approaches for these patients has been greatly improved.

The complete CAR structure includes an antigen binding region (scFv, single chain antibody, antibody fragment recognizing tumor-associated antigens), a transmembrane connecting region, an intracellular signaling region (T cell activation motif, CD3 intracellular signaling domain ITAM) [ eleano j.cheadle, et al, CAR T cells: drivingthe road from the laboratory to the clinical.immunological reviews2014.vol.257: 91-106 ] CAR-T cell therapy targeting CD30 by isolating the patient's own T cells, programming with lentiviral vectors encoding CAR to specifically target CD30, thereby recognizing and eliminating CD30 positive malignant tumor cells CD30 is a target for which T therapy is significant, studies have become a model for gene modification T cell therapy studies in most clinical institutions, whereas the targeting of CD30 Receptor antibodies, which easily cause recurrence of murine antigens, in vivo, and human immune Receptor recurrence.

Disclosure of Invention

To solve the above problems, the present invention provides humanized chimeric antigen receptors targeting CD30, the humanized chimeric antigen receptor comprising a human anti-CD 30 single-chain antibody, an extracellular hinge region, a transmembrane domain and an intracellular signaling domain, the amino acid sequence of the human anti-CD 30 single-chain antibody is shown in SEQ ID No. 3.

Preferably, the extracellular hinge region is derived from the hinge region of CD8, and the amino acid sequence of the extracellular hinge region is shown as SEQ ID NO. 4.

Preferably, the transmembrane domain is derived from CD28TM, and the amino acid sequence is shown in SEQ ID NO. 5.

Preferably, the intracellular signaling domain consists of CD28ICD, 4-1BB and CD3zeta, and the amino acid sequence thereof is shown in SEQ ID NO 6.

The invention also provides nucleic acids for coding the chimeric antigen receptor, and the nucleotide sequence of the nucleic acids is shown as SEQ ID NO. 7.

The chimeric antigen receptor of the invention is expressed in T cells, so that the obtained CAR-T cells can efficiently and specifically kill CD30 positive malignant tumor cells, and a method which is efficient and has few side effects and adverse reactions is provided for treating malignant tumors expressing CD30 surface antigens, such as Hodgkin lymphoma and the like.

Drawings

FIG. 1 is a schematic diagram of the structure of a chimeric antigen receptor encoding hCD30 according to an embodiment of the present invention;

FIG. 2 is a schematic plasmid map of the hCD30 lentiviral expression vector BRD-PTK-hCD30 in an example of the present invention;

FIG. 3 shows the results of the measurement of transduction efficiency of PTK-hCD30CAR-T cells in the examples of the present invention;

FIG. 4 is a graph of the in vitro killing efficiency of positive target cells L428 by PTK-hCD30CAR-T cells and T cells in an example of the invention;

FIG. 5 is a graph showing the in vitro killing efficiency of negative target cells Raji by PTK-hCD30CAR-T cells and T cells in the present example;

FIG. 6 shows the results of flow cytometry detection of the construction of L428-luc-GFP in the examples of the present invention;

FIG. 7 is an evaluation of the killing effect of CAR-T cells on tumor cells in vivo as measured by fluorescence intensity in the examples of the present invention.

Detailed Description

The principles and features of this invention are described in conjunction with examples and the accompanying drawings, which are set forth to illustrate the invention and are not intended to limit the scope of the invention.

1. Construction of chimeric antigen receptor expression plasmid of human origin targeting CD30

A DNA fragment (nucleic acid sequence of hCD30 CAR) with the length of 1614bp shown in SEQ ID NO:7 is artificially synthesized, wherein the nucleotides at the 1 st to 69 th positions encode SP (the amino acid sequence of which is shown in SEQ ID NO: 2), the nucleotides at the 70 th to 798 th positions encode SCFV targeting CD30 (the amino acid sequence of which is shown in SEQ ID NO: 3), the nucleotides at the 799 th and 945 th positions encode CD8 hinge region (the amino acid sequence of which is shown in SEQ ID NO: 4), the nucleotides at the 946 and 1026 th positions encode CD28TM (the amino acid sequence of which is shown in SEQ ID NO: 5), the nucleotides at the 1027 th and 1149 th positions encode CD28ICD, the nucleotides at the 1150 th and 1215 positions encode 4-1BB, and the nucleotides at the 1216 th and 1614 th positions encode CD3zeta, and the specific structural diagram is shown in FIG. 1. Wherein, the amino acid sequence of the hCD30CAR is shown as SEQ ID NO. 1, and the latter three regions form an intracellular signal region (the amino acid sequence is shown as SEQ ID NO. 6).

The DNA fragment is inserted into the downstream of EF1alpha promoter of a lentiviral expression vector BRD-PTK to obtain a chimeric antigen receptor expression plasmid BRD-PTK-hCD30 of human origin and targeting CD30, and the plasmid map is shown in figure 2.

2. T cell transfected by chimeric antigen receptor expression plasmid of human origin and targeting CD30

1) Packaging preparation of lentiviruses

In a multilayer cell culture flask (Hyperflash) (Corning) 130.0-140.0X 10⁶A total of 560mL DMEM complete medium (50mL fetal bovine serum, 5mL of antimicrobial-antimicrobial 100X) containing 5% CO at 37 ℃ was prepared from 293T cells (Takara)₂The culture was carried out in an incubator for 24 hours. DMEM complete medium mixed with 320. mu.g of plasmid (PTK-hCD 30: pMDLg: pRSV: pMD2. G: 12:10:5:6) was added to a tube containing 960. mu.g of PEI, vortexed, mixed well, the volume of the mixture was 35mL, and the mixture was allowed to equilibrate at room temperature for 10 min. The mixture of 35ml of the above plasmid and 35ml of the above plasmid was mixed with 525ml of the complete medium of ldmem, and the mixture was transferred to the above multilayer cell culture flask. Placing the multi-layered cell culture flask at 37 deg.C with 5% (v/v) CO₂After 3 days in the incubator, cell culture supernatant was collected.

After centrifugation of the supernatant at 4000rpm (or 3000g) for 30min, the lentiviral supernatant was suction filtered using a 0.22 μm filter and centrifuged at 30000g for 2.5h at 4 ℃. The supernatant was removed and 1mLT cell culture medium (OpTsizer) was added^TMCTS^TMT-CellExpansion Basal Medium，Gibco，A3021201；OpTmizer^TMCTS T-Cell expansion supplement, Gibco, A3021601), 500IU/mL of IL-2 (double Lut pharmaceutical) heavy suspension pellet. After resuspension, 20. mu.L of the suspension was retained for virus activity titer detection, and the remaining lentivirus concentrate was aliquoted as Lenti3-hCD30 lentivirus and stored at-80 ℃ for future use.

And (3) detecting the active titer of the lentivirus:

the principle is as follows: goat anti-Human IgGANtibody, FITC conjugate is labeled with fluorescein, and Goatant-Human IgGANtibody can be specifically combined with a single-chain antibody of CAR, and the expression of CAR in 293T cells is indirectly reflected by a fluorescence signal detected by a flow cytometer.

The method comprises the following steps: the 5.0 x 10 of the wells are connected into a 6-well plate⁵One well of 293T cells and one well of lentivirus concentrate were added with 0.1. mu.L, 0.5. mu.L and 1. mu.L, respectively, and 1 negative control was set. Placing at 37 ℃ with 5% (v/v) CO₂Culturing in an incubator. Three days later, 293T cells were harvested using Versene solution (Gibco), sent to flow cytometry to examine the proportion of CAR-positive 293T cells, and converted to the activity titer of Lenti3-hCD30 lentivirus concentrate. The detection and analysis results are shown in Table 1, and the obtained virus has high activity titer, which reaches 1.5E + 09.

TABLE 1 results of the Activity titer assay for Lenti3-hCD30 lentivirus

Sample (I)	Virus addition amount (μ L)	Efficiency of transfection	Active titer
				CK	-	0.31％	/
Lenti3-hCD30-0.1	0.1	30.3％	1.5E+09

2) Preparation of T cells

Taking 10ml of fresh blood of healthy people, and treating with stranguriaThe peripheral blood mononuclear cells are separated by a lymphocyte separation solution (Mediatech), and the specific method is shown in the specification. Complete culture with T cells (OpTsizer)^TMCTS^TMT-Cell Expansion BasalMedium，Gibco，A3021201；OpTmizer^TMCTS T-Cell Expansion Supplement, Gibco, A3021601), 500IU/mL of IL-2 (double Lut pharmaceutical) while culturing T cells at 25. mu.L/10⁶Dynabeads Human T-Activator CD3/CD28(Gibco, 11132D) was added to each cell to obtain T cells.

3) Lentiviral infected T cells and expansion culture of infected T cells

After 24 hours, PTK-hCD30CAR lentivirus was added at MOI of 2 for transduction, mixed well and then placed in CO₂Incubating in incubator, supplementing complete culture medium for T cells after 4 hr, and regulating cell density to 1.0 × 10⁶The culture was carried out in a volume of/mL.

After 24 hours of lentivirus transduction, the cells were replaced with fresh T cell complete medium and the viable cell density was adjusted to 1.0X 10⁶and/mL, continuously culturing and amplifying for 10-20 days, observing and counting every day, performing fluid replacement amplification culture according to the counted cell number, and always keeping the cell culture density at 1.0 multiplied by 10⁶/mL。

Transduction efficiency assay of PTK-hCD30CAR-T cells

Take 1.0X 10⁶After each transduction of T cells, they were incubated with Goat anti-Human IgG Antibody, FITC conjugate for 30 minutes at room temperature, washed twice with physiological saline, and FITC fluorescent signal was detected by flow cytometry to measure the ratio of FITC positive cells, reflecting the ratio of CAR-T cells in total cells. The results of the measurements are shown in FIG. 3 and Table 2. Indicating that PTK-hCD30CAR-T cells were successfully prepared.

TABLE 2 transduction efficiency assay results for PTK-hCD30CAR-T cells

Transduction type	Efficiency of transduction
		PTK-hCD30CAR-T	37.4％

Specific killing activity of PTK-hCD30CAR-T cells against malignant cells positive for CD30

PTK-hCD30CAR-T cells were tested for tumor killing function in vitro using calcein assay.

Taking appropriate amount of L428 cells as target cells at 1X 10⁶Cell suspension/mL (PBS, 5% fetal calf serum) was added Calcein-acetohydroxymethyl ester (Calcein-AM) to a final concentration of 25. mu.M and incubated in an incubator for 30 min. After washing twice at room temperature, the cells were resuspended to 1.5X 10⁵and/mL. PTK-hCD30CAR-T cells are added according to different effective target ratios, 200g of the PTK-hCD30CAR-T cells are centrifuged for 30 seconds, and the PTK-hCD30CAR-T cells are incubated for 2-3 hours at 37 ℃. After the incubation, the supernatant was taken, the fluorescence intensity of calcein therein was measured, and the percentage of target cell lysis was calculated from the spontaneous release control and the maximum release control.

Tumor killing experimental data: before application, functional detection such as killing of a tumor cell line by lentivirus-transduced T cells is carried out, and a calcein detection method is used. See fig. 4 and 5 and tables 3 and 4 for results. The results show that PTK-hCD30CAR-T cells have specific killing activity on tumor cells highly expressing CD 30.

TABLE 3 killing efficiency of PTK-hCD30CAR-T cells and T cells on positive target cell L428 in vitro

TABLE 4 in vitro killing efficiency of PTK-hCD30CAR-T cells and T cells against negative target cells Raji

Construction of L-428-luc-GFP Stable Transit cell line

To examine the proliferation of L428 cells in animals, Luc-Puro-GFP triple-standard lentivirus (Henan organism, ZH-LP01) was selected to transduce L428 cells to construct L428-Luc-GFP cells, which was used for the subsequent drug effect experiment of PTK-hCD30CAR-T cells. Puromycin (puromycin, Gibco, A1113803) was used to screen for highly pure L428-luc-GFP stable cell lines after successful transfection. The specific test steps are as follows:

1) transfection, using serum-free 1640 basic medium (Gibco, C11875500BT) to resuspend 2 x 106 cells, adding polybrene (Henan, HB-PB-05) and Luc-puro-GFP three standard lentivirus 37 ℃ incubator incubation.

2) And (5) incubating for 4h, 24h and 48h for fluid infusion, observing the state of the transduced L428 under an inverted microscope, and observing the GFP expression condition after the transduction is carried out for 48h by a fluorescence microscope.

3) Screening, after 72h incubation, after centrifugation, transduced L428 cells were incubated with 1640 complete medium containing 2.5. mu.g/ml puromycin, the same number of untransduced L428 cells were taken and the cell pellet was resuspended in 1640 complete medium containing the same 2.5. mu.g/ml puromycin as a control;

(a) l428control group, 2.5. mu.g/ml puromycin, 1640 complete Medium culture 2 x 10⁶L428 cells;

(b) L428-luc-GFP group, 2.5. mu.g/ml puromycin, 1640 complete Medium culture 2 x 10⁶L428-luc-GFP cells.

4) The cells were cultured in 1640 complete medium for 6 days with 2.5. mu.g/ml puromycin, during which the cells were periodically changed and the screening was checked on day 6 by flow cytometry.

The flow cytometry results showed that the cells in the L428control group were dead in large numbers after the addition of 2.5. mu.g/ml puromycin, and the control group cells were all dead at day 6. After the L428-luc-GFP group is screened for 6 days, the GFP positive cells account for 95.1% by flow cytometry detection (see figure 6), so far, an L428-luc-GFP group stable cell line is successfully constructed, and preparation is made for a subsequent PTK-hCD30CAR-T cell efficacy experiment.

Anti-tumor Effect of PTK-hCD30CAR-T cells in mouse tumor-bearing models

To examine the antitumor effect of PTK-hCD30CAR-T cells in vivo, immunodeficient B-NSG mice (jataso genebio ltd) and L428-luc-GFP cells were selected for tumor modeling in this example, grouped and injected with PTK-hCD30CAR-T cells and normal T cells, respectively, in the caudal vein after successful modeling, and live imaging was performed at different times using IVIS Spectrum small animal live imaging system (Xenogen, Hopkinton, USA) to analyze the imaging test results. The specific test steps are as follows:

1) modeling, injecting (i.v.)1.5 multiplied by 10 tail vein into B-NSG mice with 6-8 weeks of age⁶L428-luc-GFP cells/cell.

2) After 7 days, animals are imaged, D-luciferin (Molecular Imaging Products, Bend, USA) is intraperitoneally injected at a dose of 100-150 mg/kg and 1% pentobarbital sodium injection is intraperitoneally injected at a dose of 50-75 mg/kg to anaesthetize mice, and after 10-15 minutes, an IVIS small animal living body Imaging system (Xenogen, Hopkinton, USA) is used for collecting optical signals.

3) After successful modeling, grouping was performed on the same day by tail vein injection of PTK-hCD30CAR-T cells and normal T cells, respectively, as follows:

(a) PTK-hCD30CAR-T cell injection group, PTK-hCD30CAR-T cell injection group in tail vein, 1 x 10⁷One/one;

(b) the group of normal T cells was injected, and the tail vein was injected with normal T cells at 1X 10⁷One/only.

4) In vivo imaging of tumor-bearing mice was performed 7, 10, 14, 18 days after intravenous injection of T cells, and the results of the in vivo imaging assay were analyzed, see fig. 7, where CD30: PTK-hCD30CAR-T cell injection group; NT: general T cell injection group. The in vivo imaging result of the tumor-bearing mice shows that the tumors of the mice in the common T cell injection group are gradually increased until the mice die; compared with the common T cell injection group, the tumor in the tumor-bearing mice of the PTK-hCD30CAR-T cell injection group gradually disappears. The result shows that the common T cells have no anti-tumor effect on tumor cells in a tumor-bearing mouse, and the PTK-hCD30CAR-T cells have a good anti-tumor effect in the tumor-bearing mouse, so that a theoretical basis is provided for clinical medication.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

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<120> human chimeric antigen receptors for treating hematological tumors and application thereof

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gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg ctaa 1614

Claims

The human chimeric antigen receptor targeting the CD30 surface antigen is characterized by consisting of a human single-chain antibody resisting CD30, an extracellular hinge region, a transmembrane domain and an intracellular signal domain, wherein the amino acid sequence of the human single-chain antibody resisting CD30 is shown in SEQ ID NO. 3, the extracellular hinge region is from the hinge region of CD8 and is shown in SEQ ID NO. 4, the transmembrane domain is from CD28TM and is shown in SEQ ID NO. 5, and the intracellular signal domain is composed of CD28ICD, 4-1BB and CD3zeta and is shown in SEQ ID NO. 6.
2, nucleic acid encoding the chimeric antigen receptor of claim 1, wherein the nucleotide sequence of said nucleic acid is set forth in SEQ ID No. 7.