WO2019233281A1 - Chimeric antigen receptor with truncated or non-truncated natural cytotoxic receptor signal structure, and application thereof - Google Patents

Abstract

Disclosed in the present invention is a chimeric antigen receptor (CAR), comprising: an antigen binding structural domain (scfv) and a signal transduction structural domain, the signal transduction structural domain comprising a first transduction structural domain and a second transduction structural domain, the antigen binding structural domain being connected in tandem between the first transduction structural domain and the second transduction structural domain. When stimulated by antigens, the CAR structure disclosed in the present invention has a very low level of secreted cytokines, better ensuring the safety of clinical application, i.e. having high clinical application safety; the capacity for killing in vitro antigen-positive tumour cells is stronger, and anti-tumour activity is better.

Description

Chimeric antigen receptor carrying truncated or untruncated natural cytotoxic receptor signal structure and application thereof Technical field

The invention relates to the technical field of tumor immunotherapy, in particular to a chimeric antigen receptor carrying a truncated or untruncated natural cytotoxicity receptor signal structure and its application.

Background technique

Chimeric antigen receptor (CAR) is the core component of CAR-T. Using the characteristics of the ligand binding domain, CAR can redirect the specificity and reactivity of selected immune cells, thus conferring T cells an HLA-independent manner The ability to recognize tumor antigens allows CAR-engineered T cells to recognize a wider range of targets than the native T-cell surface receptor TCR. The basic design of CAR includes a tumor-associated antigen (TAA) binding region (usually derived from the scFV segment of the monoclonal antibody antigen-binding region), an extracellular hinge region, a transmembrane region, and an intracellular signal. Area.

At present, the traditional CAR-T is effective for hematological tumors, but it is not obvious for solid tumors, which limits its clinical application. From a safety perspective, cytokine release syndrome (CRS) is a common complication of CAR-T cell therapy and can even be life-threatening. After CAR-T cells are infused into the body, because the chimeric antigen receptor specifically binds to the corresponding tumor-associated antigen, CAR-T cells are activated and begin to proliferate, triggering the release of cytokine cascades and mediating multiple types of immune responses. As a result, clinical manifestations such as fever, hypotension, dyspnea, coagulopathy, and terminal organ disorders, that is, CRS, the traditional CAR-T is directly affected by the antigen-stimulated secretion of cytokines to the severity of CRS. From the perspective of effectiveness, first, the tumor matrix composed of solid tumor-associated fibroblasts (CAFs) provides a physical barrier for CAR-T cell infiltration. CAFs also secrete extracellular matrix proteins to transport T cells from cancer cells. Second, the metabolic environment of the solid tumor tumor microenvironment is not conducive to the persistence of traditional CAR-T cells, because once tumor formation is activated, tumor cells stop producing ATP through oxidative phosphorylation and convert to aerobic glycolysis Solution, causing the tumor environment to become acidic, which is the so-called "Wattock effect". The pH dropped from 7.4 to 6.5; finally, the tumor microenvironment caused an hypoxic state to further produce immunosuppression, and tumor cells produced a HIF1 in a hypoxic environment. -Plutonium molecule, which weakens the anti-tumor function of traditional CAR-T cells by attracting regulatory T cells (Tregs) to the tumor site. Tregs suppress the immune response, thus limiting the therapeutic effect of traditional CAR-T on solid tumors.

Summary of the Invention

An object of the present invention is to provide a chimeric antigen receptor (CAR) carrying a signal structure of a natural cytotoxic receptor NKp44, which has better safety and more remarkable curative effect.

Another object of the present invention is to provide the application of the chimeric antigen receptor.

A third object of the present invention is to provide a signaling domain of a chimeric antigen receptor.

A fourth object of the present invention is to provide an application of the signal transduction domain.

The specific technical solution of the above objective of the present invention is detailed as follows:

A chimeric antigen receptor (CAR) comprising: an antigen binding domain (scfv) and a signaling domain, wherein the signaling domain includes a first conductive domain and a second conductive domain, and the first conductive domain And a second conducting domain in tandem with an antigen binding domain.

The first conductive domain, antigen binding domain and second conductive domain in tandem in the chimeric antigen receptor according to the present invention become a multi-chain form capable of transmitting an activation signal after the antigen binding domain specifically binds the antigen. , The activation signal is transmitted to the immune cells expressing it, to achieve the role of immunotherapy.

The chimeric antigen receptor is a multi-chain structure chimeric antigen receptor. It uses a first conductive domain and a second conductive domain to form a signal transduction domain of the CAR. The antigen binding domain can specifically bind to the target. And induces the activation of immune cells to produce an immune response.

The second conductive domain of the present invention is DAP12, and the second conductive domain is connected in series with the antigen-binding domain through T2A.

The DAP12 in the present invention is a transmembrane domain, which is widely present on the surface of natural killer cells, granulocytes, monocytes / macrophages, and is used to transmit activation signals. Its nucleotide sequence is as shown in SEQ ID NO.1. The amino acid sequence is shown in SEQ ID NO.2.

The T2A according to the present invention is used to tandem a second conductive domain and an antigen-binding domain. Its nucleotide sequence is shown in SEQ ID NO. 3 and its amino acid sequence is shown in SEQ ID NO. 4.

The first conductive domain according to the present invention may be truncated or untruncated NKp44, NKp46, NKp30 or NKG2D, wherein the full-length nucleotide sequence of the NKp44 gene can be found in NCBI, GenBank: AJ225109.1, amino acid sequence See NCBI, GenBank: CAB39168.1; For the full-length nucleotide sequence of the NKp46 gene, see NCBI, Accession: NM_004829.6, for the amino acid sequence, see NCBI, Accession: NP_004820; For the full-length nucleotide sequence of the NKp30 gene, see NCBI, Accession: NM_147130.2, amino acid sequence see NCBI, Accession: NP_667341.1; NKG2D gene full-length nucleotide sequence see NCBI, Accession: AF461811, amino acid sequence see NCBI, GenBank: AAL65233.1. The truncated NKp44, NKp46, NKp30 or NKG2D represents a C-terminally truncated amino acid sequence of its full-length amino acid sequence.

In order to improve the safety and efficacy of the chimeric antigen receptor according to the present invention, the present invention also provides a preferred first conductive domain, which is selected from the truncated NKp44 amino acid sequence and named as NKp44 _cut . The NKp44 _cut is a polypeptide of 100-160 amino acids at the C-terminus of the full-length NKp44 amino acid sequence, preferably a polypeptide of 120-160 amino acids at the C-terminus of the NKp44 full-length amino acid sequence, and more preferably 140-160 at the C-terminus of the NKp44 full-length amino acid sequence. A polypeptide having an amino acid is either an amino acid sequence having 80% or more identity with the aforementioned polypeptide sequence, or an amino acid sequence having 85% or more identity with the sequence, or an amino acid sequence having 90% or more identity with the sequence, or Or an amino acid sequence having 95% or more identity with the sequence.

The present invention also provides a preferred first conductive domain, which is a 160-terminal polypeptide of the full-length amino acid sequence of NKp44. The amino acid sequence is shown in SEQ ID NO.8, and its nucleotide sequence is shown in SEQ ID NO.7. As shown.

The antigen-binding domain according to the present invention can be routinely selected in the art according to different tumor targets.

More specifically, the chimeric antigen receptor according to the present invention is DAP12, T2A, an antigen-binding domain, and a first transduction domain, which are tandemly connected in sequence through 2-10 arbitrary amino acids. The sequence and number of 2-10 arbitrary amino acids of the present invention have no significant effect on the efficacy of the chimeric antigen receptor described in the present application, and may be any 2-10 amino acid sequence.

The chimeric antigen receptor according to the present invention may use, for example, a retroviral vector to transfer a nucleic acid encoding the chimeric antigen receptor to an immune cell such as a T cell. When the chimeric antigen receptor binds a target antigen, the first conductive structure The domain and antigen-binding domain are separated to form an activation signal that is transmitted to the immune cells that express it.

The invention also provides an immune cell carrying the chimeric antigen receptor.

The invention also provides the application of the chimeric antigen receptor or immune cells with the chimeric antigen receptor in tumor immunotherapy.

The present invention provides a signal transduction domain including a first transduction domain and a second transduction domain.

The first conductive domain and the second conductive domain of the present invention, after the antigen-binding domain specifically binds to the antigen, becomes a multi-chain form capable of transmitting an activation signal, and transmits the activation signal to the immune cells expressing it, thereby realizing The role of immunotherapy.

The second conductive domain of the present invention is DAP12. The DAP12 in the present invention is a transmembrane domain, which is widely present on the surface of natural killer cells, granulocytes, monocytes / macrophages, and is used to transmit activation signals. Its nucleotide sequence is as shown in SEQ ID NO.1. The amino acid sequence is shown in SEQ ID NO.2.

The first conductive domain according to the present invention may be truncated or untruncated NKp44, NKp46, NKp30 or NKG2D, wherein the full-length nucleotide sequence of the NKp44 gene is described in NCBI, GenBank: AJ225109.1, amino acid sequence See NCBI, GenBank: CAB39168.1; For the full-length nucleotide sequence of the NKp46 gene, see NCBI, Accession: NM_004829.6, for the amino acid sequence, see NCBI, Accession: NP_004820; For the full-length nucleotide sequence of the NKp30 gene, see NCBI, Accession: NM_147130.2, amino acid sequence see NCBI, Accession: NP_667341.1; NKG2D gene full-length nucleotide sequence see NCBI, Accession: AF461811, amino acid sequence see NCBI, GenBank: AAL65233.1. The truncated NKp44, NKp46, NKp30 or NKG2D represents an amino acid sequence truncated from the C-terminus of its full-length amino acid sequence.

In order to improve the safety and efficacy of the chimeric antigen receptor formed by the signal transduction domain of the present invention, the present invention also provides a preferred first transduction domain, which is selected from the truncated NKp44 amino acid sequence and named as NKp44 _cut The NKp44 _cut according to the present invention is a polypeptide of 100-160 amino acids at the C-terminus of the full-length NKp44 amino acid sequence, preferably a polypeptide of 120-160 amino acids at the C-terminus of the NKp44 full-length amino acid sequence, and more preferably the NKp44 full-length amino acid sequence C. A 140-160 amino acid terminal polypeptide, or an amino acid sequence having 80% or more identity with the aforementioned polypeptide sequence, or an amino acid sequence having 85% or more identity with the sequence, or 90% or more identity with the sequence Or an amino acid sequence with 95% or more identity to the sequence.

The present invention also provides a preferred first conduction domain, when the NKp44 full-length amino acid sequence of the 160-terminal polypeptide of 160 amino acids, the amino acid sequence is shown in SEQ ID NO. 8, and its nucleotide sequence is shown in SEQ ID ID NO. 7 is shown.

According to the signaling domain of the present invention, the transmembrane receptor DAP12 is combined with the first conductive domain to form the signaling domain of the CAR. When the CAR specifically binds to a ligand in the target, it can induce the activation of immune cells To produce an immune response.

The invention also provides the application of the signaling domain in a chimeric antigen receptor or tumor immunotherapy.

The beneficial effects of the present invention:

(1) When the CAR structure involved in the present invention is stimulated by an antigen, the secreted cytokine level is extremely low, which can well ensure the safety of clinical application, that is, the safety of clinical application is higher;

(2) The CAR structure involved in the present invention has confirmed its significant effect on solid tumors through in vitro functional experiments. Therefore, the present invention can not only be applied to the treatment of blood tumors, but also expand the application of CAR-T in the treatment of solid tumors;

(1) (3) The CAR structure involved in the present invention has stronger killing ability in vitro and positive antitumor activity.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a lentiviral vector containing different signal domain structures;

Figure 2 shows the positive expression rate of CAR structure that recognizes MSLN antigen on the surface of T cells by flow cytometry after 7 days of MSLN4CAR-T cell infection with lentivirus;

Figure 3 shows the proliferation of CAR-T cells after lentivirus infection;

Figure 4 is the IL-2 secretion of MSLN4CAR-T cells under antigen stimulation;

Figure 5 shows the secretion of IFN-γ by MSLN4CAR-T cells under antigen stimulation;

Figure 6 is the secretion of IL-2 and IFN-γ by CAR-T in different groups under antigen stimulation;

Figure 7 shows the killing effect of CAR-T in different groups on antigen-positive tumor cell lines.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental methods without specific conditions in the examples are generally based on conventional conditions, such as those described in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al.) Or according to the conditions recommended by the manufacturer. Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

Example 1. Construction of CAR Lentivirus Containing 4 Elements DAP12-T2A-scFv-NKp44 _cut

In order to prove that CAR-T cells containing DAP12-NKp44 _cut intracellular signal domain have more advantages than previously reported CAR-T cells containing 4-1BB-CD3ζ, DAP12-KIRS2 and single DAP12 stimulation signals, it is necessary to separately construct Viral vectors with different combinations of stimulus signals. In this embodiment, a single-chain antibody targeting human mesothelin (MSLN) is used as a unified extracellular recognition antigen structure, and the following five chimeric antigen receptors need to be constructed respectively (Figure 1):

MSLN (scfv) -CD8α-4-1BB-CD3ζ (MSLN1)

DAP12-T2A-MSLN (scfv) (MSLN2)

DAP12-T2A-MSLN (scfv) -KIRS2 (MSLN3)

DAP12-T2A-MSLN (scfv) -NKp44 _cut (MSLN4)

DAP12-T2A-MSLN (scfv) -NKp44 _wt (MSLN5)

1. Synthesis of human mesothelin-targeting chimeric antigen receptor gene sequences containing different intracellular stimulation signals

Synthesis of natural killer activated receptor (DAP12), T2A, anti-human mesothelin single chain antibody scfv (MSLN (scfv)), natural cytotoxicity receptor (NKp44 _wt ), truncated natural cytotoxicity The body (referred to as NKp44 _cut ), its structure is shown in Figure 1. The nucleotide sequence of DAP12 is shown in SEQ ID NO.1, the amino acid sequence is shown in SEQ ID NO.2, the nucleotide sequence of T2A is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4 As shown, the nucleotide sequence of the anti-human mesothelin single chain antibody (MSLN) scfv is shown in SEQ ID NO.5, the amino acid sequence is shown in SEQ ID NO.6, and the full-length nucleotide sequence of the NKp44 gene is shown in NCBI, GenBank: AJ225109.1, amino acid sequence see NCBI, GenBank: CAB39168.1, nucleotide sequence of NKp44 _cut is shown in SEQ ID NO.7, amino acid sequence is shown in SEQ ID NO.8, nucleoside of KIRS2 See the patent for acid and amino acid sequences (targeted cytotoxic cells with chimeric receptors for adoptive immunotherapy, publication number: CN107580628 A). For the nucleotide sequence and amino acid sequence of CD8α-4-1BB-CD3ζ, see the patent ( Methods for treatment of cancer, US 20130309258A1).

2. Construction of a lentiviral vector expressing a chimeric antigen receptor

pELNS Dap12-T2A-MSLN-KIRS2 is kept by Nanjing Kati Medical Technology Co., Ltd., or according to the literature (Enxiu Wang et al. Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreceptors. 2015, Cancer Immunology Research, 3 (7): 815) was used to construct. The truncated NKp44 _cut gene synthesis was synthesized by Nanjing Kingsray Biotechnology Company and provided pUC19-NKp44 _cut plasmid. The plasmids pELNS Dap12-T2A-MSLN-KIRS2 and pUC19 -NKp44 _{cut was} double-digested by NheI and SalI (purchased from Takara). The digestion reaction was performed according to the instructions to obtain a pELNS Dap12-T2A-MSLN fragment of about 8900bp, a truncated NKp44 _cut fragment of about 483bp of DNA fragment, and then recovered Kit (Takara company) for DNA fragment recovery, the specific method is described in the instruction manual, the obtained pELNS Dap12-T2A-MSLN and NKp44 _cut genes were recovered, and then the target fragment NKp44 _cut and the vector fragment pELNS Dap12-T2A-MSLN were ligated by T4 ( (Purchased from Takara)), and obtained a lentiviral vector expressing a chimeric antigen receptor, named pELNS Dap12-T2A-MSLN-NKp44 _cut (abbreviated as MSLN4). 5 μL of the lentiviral vector MSLN4 was transformed into E. coli TOP10 competent cells (purchased from Nanjing Anjieyou Biotechnology Co., Ltd.), and monoclonals were picked after 16 hours of culture at 37 ° C. Plasmids were extracted using a plasmid extraction kit (purchased from Takara). The specific method is described in the instructions.

According to the above methods, pELNS MSLN-CD8α-4-1BB-CD3ζ (MSLN1 for short); pELNS Dap12-T2A-MSLN (for short MSLN2); pELNS Dap12-T2A-MSLN-KIRS2 (for short MSLN3); pELNS Dap12-T2A-MSLN -NKp44 _cut (abbreviated MSLN4); pELNS Dap12-T2A-MSLN-NKp44 _wt (abbreviated MSLN5) lentiviral vector.

3.Lentivirus packaging

In this embodiment, the lentivirus is packaged using the calcium phosphate method. The specific steps are as follows:

(1) 293T cells were passaged the next day

5 × 10 ⁶ cells were seeded in each T150 cell flask. After 48 hours, the number of cells should reach 20-25 million / flask.

(2) 293T cell bottle

a) Take a T150 cell culture flask as an example, gently wash the cells twice with about 15ml of 1 × PBS

b) Add 3ml 0.25% trypsin-2.21mM EDTA

c) Wait until the cells fall off, add 12ml of 10% (wt) FBS (purchased from Gibico) in DMEM medium (purchased from corning) to the cells that have fallen off

d) Collect and transfer the cells to a sterile centrifuge tube, centrifuge at 1000 rpm for 10 minutes

e) Aspirate the supernatant and resuspend the pellet in 10 ml of 10% (wt) FBS DMEM medium.

f) Cell count, calculate the volume required for 12 × 10 ⁶ cells based on cell concentration

g) Combine the cells with 25 ml of 10% (wt) FBS DMEM culture solution, put them into a T150 cell flask, and shake gently to make the cells evenly distributed to the bottom of the cell flask at 37 ° C. and culture overnight in a 5% CO 2 incubator.

(3) Cell transfection

Observe the cells, the cell density is about 80% -90%, at this time you can start transfection

a) Gently aspirate the culture medium 30-60 minutes before transfection.

b) Mixing plasmid DNA and calcium chloride solution, taking a T150 bottle as an example, 28ug pRSV.rev (purchased from Invitrogen), 28ug pGAG-Pol (purchased from Invitrogen), 11ug pVSVG (purchased from Invitrogen), 23ug recombinant lentivirus expression plasmid pELNS Dap12-T2A-MSLN-NKp44 _{cut was} added to 1.5 ml of calcium chloride solution and mixed.

c) Add 1.5ml BBS solution to a 15ml sterile centrifuge tube, mix the DNA-calcium chloride solution with a 1ml pipette tip, and then add it dropwise to the BBS solution, quickly mix 15-20 times, and incubate at room temperature 25-30 minute.

d) Using a 5ml pipette, add the DNA-calcium chloride-BBS mixture (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) evenly and dropwise to the T150 bottle. The cells were cultured in a 37 ° C cell incubator containing 5% carbon dioxide, and the solution was changed for 6 hours.

e) Change the liquid after 6h. Gently shake the culture plate several times to fully suspend some calcium phosphate precipitates. Aspirate the medium containing the calcium phosphate precipitates, add 20 ml of fresh 5% (wt) FBS DMEM medium, and continue the culture.

(4) Collect virus supernatant for the first time

a) The 293T cell culture supernatant transfected the previous day was collected into a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, labeled, and temporarily stored in a refrigerator at 4 ° C.

b) 20 ml of 5% (wt) FBS pre-warmed DMEM medium was added to the cell flask, and the cells were incubated at 37 ° C overnight.

(5) The virus supernatant was collected for the second time (48h / day 4).

(6) Filter the supernatant

The two supernatants collected were pooled together and filtered through a 0.45 μm filter to remove cell debris.

(7) Virus concentration

Centrifuge overnight at 4 ℃, 12000-24000rpm

(8) Virus storage

After centrifugation, the entire supernatant was decanted, and fresh 5% (wt) FBS DMEM medium was added to resuspend, virus was aliquoted, and quickly stored in a -80 ° C refrigerator for future use.

(9) Determination of lentivirus titer

a) Virus infected 293T cells

293T cells were plated into 24-well plates before infection, and 200 μL of purified concentrated virus was added to 293T cells. After 24 hours, the medium was exchanged with DMEM medium containing 10% FBS (wt). After 72 hours of infection, the cells were treated at 1200 r / min. Centrifuge for 5 min to collect cells and extract the genome.

b) Extraction of the genome

The genomic extraction kit was purchased from Takara Company and operated according to the kit instructions

c) qPCR for virus titer

The reaction system is as follows: Probe qPCR Mix 12.5 μL (purchased from Takara), upstream primer 0.5 μL (synthesized from Nanjing Kingsray), downstream primer 0.5 μL (synthesized from Nanjing Kingsray), probe 1 μL (from Nanjing Kingsray) Synthesis), template 2 μL, sterilized water 8.5 μL, reaction system is 25 μL, reaction conditions were set according to the instructions, after the reaction, the data was analyzed by analysis software, and the virus titer was calculated according to the standard curve. The calculation results showed that the virus titer was 1 × 10 ⁶ TU / ml.

Example 2. Viral infection of T cells

1. Isolation and activation of T cells and virus infection

(1) Isolation of human peripheral blood mononuclear cells

About 10ml of peripheral blood was collected with an anticoagulant blood collection tube, and the natural settling was performed at room temperature (18-25 ° C) for about 30min. The upper plasma was collected, and the collected upper plasma was centrifuged at 5000r / min for 10min, and the volume ratio was 1: 1. Add to lymphocyte separation solution (purchased from Tianjin Ouyang Biological Products Technology Co., Ltd.), gradient centrifugation, 3000r / min, centrifugation for 30min. After centrifugation, the centrifuge tube is layered from top to bottom: the first layer is the plasma layer ; The second layer is the white blood cell layer of lymphocytes; the third layer is a transparent separating liquid layer; the fourth layer is a red blood cell layer. The white membrane layer of lymphocytes was aspirated, washed twice with PBS, centrifuged twice at 1500 r / min, centrifuged for 10 min, and the cells were resuspended in PBS. 5% autologous plasma + 300 IU / ml recombinant human IL-2 + KBM581 complete medium was cultured Peripheral blood mononuclear cells.

(2) T lymphocytes infected by lentivirus

Newly prepared mononuclear cells PBMC were cultured in complete medium containing 5% autologous plasma + 300IU / ml recombinant human IL-2 + KBM581, IL-2 was purchased from R & D Systems, KBM581 was purchased from Corning, and CD3 / CD28 Dynabeads was added on day 0 for immunization. Magnetic beads (purchased from invitrogen) activated T cells. Lentiviral infection was performed in the first 3 days. Lentiviral vectors corresponding to 0.25MOI were added. Uninfected T lymphocytes were used as blank controls. After 48 hours, the medium was replaced with 5% autologous plasma. + 300IU / ml recombinant human IL-2 + KBM581 complete medium was continued for 7-9 days.

2. Detection of CAR positive rate in T cells

Virus-infected T cells cultured to day 7 were centrifuged at 1200 r / min for 5 min. The supernatant was discarded to collect the cells, and the cells were resuspended with a PBS solution containing 1% FBS in volume fraction, and the cells were adjusted to a density of 1 × 10 ⁵ cells / ml, add biotin-labeled goat anti-mouse F (ab) ₂ (Jackson ImmunoResearch), then add Streptavidin-PE (BD Biosciences), incubate for 15 min at 4 ° C, wash twice with PBS solution, and perform flow cytometry The results showed that after 7 days of culture, the CAR-T cell CAR positive rate was 41% in the MSLN1 virus infection group, 52% in the MSLN2 virus infection group, 59% in the MSLN3 virus infection group, and MSLN4 virus. The positive rate in the infected group was 61% (Figure 2), and the positive rate in the MSLN5 virus infected group was 49%.

Example 3 Effect of virus-infected CAR-T cells on cell proliferation

After the T cells were infected by each group of viruses, the T cells were counted every 1-2 days with 5% autologous plasma + 300 IU / ml recombinant human IL-2 + KBM581 complete medium. Then observe the growth of T lymphocytes, and the results are shown in Figure 3. The results show that cells infected with CAR-expressing virus can still form typical proliferating clones. By counting the cells and plotting the cell proliferation curve, we can see that the proliferation of infected MSLN4CAR-T cells is similar to that of MSLN1, MSLN2, MSLN3, and MSLN5CAR-T. Slightly less proliferative capacity than non-infected T cells (NTD in Figure 3).

Example 4. Detection of cytokine secretion by CAR-T cells infected by a virus

(1) The cytokine detection was performed using Elisa's method using R & D's kit.

(2) Dilution of the standard: prepare 7 1ml centrifuge tubes, serially number them, add 500 μL of the standard dilution to each centrifuge tube, and then add 500 μL of the original standard to a centrifuge tube that has been numbered In the centrifuge tube, add 500μL to the second centrifuge tube and mix thoroughly; then add 500μL to the third centrifuge tube and mix thoroughly; then in the centrifuge Take 500μL from the tube and add it to the fourth centrifuge tube, mix thoroughly; then take 500μL from this tube and add it to the fifth centrifuge tube, mix thoroughly; then take 500μL from this tube and add it to the sixth centrifuge tube Mix thoroughly; add 500 μL to this centrifuge tube and add it to the seventh centrifuge tube, mix thoroughly.

(3) Set standard wells on the enzyme-labeled coating plate, and add 100 μL of standards of different concentrations in sequence, with 2-3 parallel wells of each concentration.

(4) Sample loading: set blank wells (the blank control wells are replaced with water, and the enzyme-labeled reagents and biotin-labeled antibodies are handled as usual) and the sample wells to be tested. Add 100 μL of samples to the sample wells on the enzyme-labeled coating plate. Add the sample to the bottom of the well of the microtiter plate, try not to touch the wall of the well, and shake gently to mix

(5) Incubation: incubate at room temperature for 2h

(6) Washing: Discard the liquid, shake it, add 200 μL of washing solution to each well, and discard it after standing for 30 seconds. Repeat this 3 times and pat dry

(7) Add antibody: add 100 μL detection antibody to the enzyme-coated plate

(8) Incubation: same operation as (5)

(9) Washing: same operation as (6)

(10) Labeling: Add 100 μL horseradish peroxidase-labeled streptavidin to each well

(11) Incubation: Incubate at room temperature for 20 min in the dark

(12) Washing: same operation as (6)

(13) Color development: Add 100 μL of color development solution to each well, mix gently by shaking, and incubate at room temperature for 20 min in the dark

(14) Stop: add 50 μL of stop solution to each well to stop the reaction

(15) Measurement: Zero the blank value and sequentially measure the absorbance (OD value) of each well at 450nm. The measurement should be performed within 15min after adding the stop solution.

Select target cells with different antigen expression levels to co-culture with MSLN4CAR-T, detect the levels of IL-2 and IFN-γ secreted by MSLN4CAR-T in response to antigen stimulation, and select OVCAR3 (MSLN high expression) and SKOV3 (MSLN low) Expression), 293T (MSLN negative), to show that MSLN4CAR-T specifically secretes IL-2 and IFN-γ when stimulated by MSLN antigen, the results reflect that MSLN4CAR produces target cells with different levels of antigen expression Gave a different response. CAR-T of MSLN4 significantly secreted IFN-γ and IL-2 when co-cultured with MSLN-highly expressed target cells OVCAR3 (Figures 4 and 5), indicating that MSLN4CAR has a response to antigen-positive tumor cells.

On the other hand, when MSLN1, MSLN2, MSLN3, MSLN4, MSLN5CAR-T were co-cultured with OVCAR3, the levels of IL-2 and IFN-γ cytokines secreted by each group were compared. The results are shown in Figure 6. Compared with the MSLN2 and MSLN5CAR-T groups, the secretion level of MSLN4CAR-T was significantly increased (since the antitumor effect of MSLN2CAR-T is very weak, so there is no secretion of cytokines), and the secretion of MSLN1 and MSLN3 is to some extent Decline. In vitro cytokine secretion results show that MSLN4CAR-T and MSLN5CAR-T produce lower levels of cytokines, which is likely to improve clinical application safety.

Example 5 Evaluation of killing effect of virus-infected CAR-T cells in vitro

(1) Culture target cells OVCAR3 cells (MSLN overexpressing cell line), SKOV3 cells (MSLN underexpressing cell line), 293T (MSLN negative cell line) and effector cells MSLN1, MSLN2, MSLN3, MSLN4, and MSLN5 respectively. cell.

(2) Collect target cells and effector cells, centrifuge at 1500 rpm / min for 5 min, and discard the supernatant

(3) Resuspend target cells and effector cells with 10% FBS + 1640 complete medium

(4) Using real-time cell analysis system (RTCA), add 50 μL 1640 medium in the air of E-Plate16

(5) Use RTCA to detect the baseline and confirm that the selected wells are in normal contact

(6) Set the effect target ratio to 0: 1, 1: 1, 5: 1, 10: 1

(7) Take out E-Plate16 and add 100 μL of the target cell suspension uniformly into each well according to the effect target ratio, so that the number of seed cells in each well is 10 ⁴ cells / 100 μL.

(8) Put E-Plate16 in an incubator and leave it at 37 ° C, 5% CO2 overnight.

(9) On the second day, remove E-Plate16, add 50 μL of corresponding effector cells, and calculate the killing rate after adding effector cells for 8 hours.

(10)

The test results are shown in Figure 7. The killing effect of CAR-T of MSLN4 on MSLN antigen-positive tumor cells was significantly better than the other four groups, especially significantly higher than the MSLN2 and MSLN5 groups. The results of Figures 6 and 7 show that the results of in vitro killing experiments show that the truncated natural cytotoxic receptor, that is, the NKp44 _cut as the first signaling domain, has strong antitumor activity on the basis of improving the safety of CAR. CAR signal area design is conducive to clinical application.

Finally, it is explained that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that Various changes are made in details without departing from the scope defined by the claims of the present invention.

Claims (13)

1. A chimeric antigen receptor, comprising: an antigen-binding domain and a signaling domain, wherein the signaling domain includes a first conducting domain and a second conducting domain, a first conducting domain and a second conducting domain Tandem antigen-binding domains.
2. The chimeric antigen receptor according to claim 1, wherein the first conductive domain, the antigen-binding domain, and the second conductive domain in tandem in the chimeric antigen receptor are in the antigen-binding domain. After specifically binding the antigen, it becomes a multi-chain form capable of transmitting the activation signal, and transmits the activation signal to the immune cells that express it, realizing the role of immunotherapy.
3. The chimeric antigen receptor according to claim 1, wherein the second conductive domain is DAP12, the second conductive domain is connected in series with the antigen-binding domain through T2A; and the first conductive domain is Truncated or un-truncated NKp44, NKp46, NKp30 or NKG2D; preferably, the DAP12 nucleotide sequence is shown in SEQ ID NO.1, the amino acid sequence is shown in SEQ ID NO.2; the T2A core The nucleotide sequence is shown in SEQ ID No. 3, and the amino acid sequence is shown in SEQ ID No. 4.
4. The chimeric antigen receptor according to claim 3, wherein the first conducting domain is a truncated NKp44 amino acid sequence, preferably a polypeptide having a full-length amino acid sequence of NKp44 near the C-terminus of 100 to 160 amino acids. , More preferably a polypeptide having a full-length amino acid sequence of NKp44 near the C-terminus of 120-160 amino acids, still more preferably a polypeptide having a full-length amino acid sequence of NKp44 near the C-terminus of 140-160 amino acids, or 80% or more identical to the polypeptide sequence Nucleotide sequence, or a nucleotide sequence having 85% or more identity with the sequence, a nucleotide sequence having 90% or more identity with the sequence, or 95% or more with the sequence Identity nucleotide sequence.
5. The chimeric antigen receptor according to claim 4, wherein the amino acid sequence of the first conduction domain is shown in SEQ ID No. 8 and its nucleotide sequence is shown in SEQ ID NO. 7.
6. The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor is DAP12, T2A, an antigen-binding domain, and a first transduction domain in sequence through 2-10 arbitrary amino acids Tandem.
7. An immune cell bearing the chimeric antigen receptor of claim 1.
8. Use of the chimeric antigen receptor according to any one of claims 1 to 6 or the immune cell according to claim 7 in tumor immunotherapy.
9. A signaling domain includes a first conducting domain and a second conducting domain.
10. The signal transduction domain according to claim 9, wherein the second transduction domain is DAP12, the DAP12 nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2 Shown; the first conductive domain is truncated or untruncated NKp44, NKp46, NKp30 or NKG2D.
11. The signal transduction domain according to claim 9, wherein the first transduction domain is a truncated NKp44 amino acid sequence, preferably a polypeptide having 100 to 160 amino acids at the C-terminus of the full-length amino acid sequence of NKp44, more It is preferably a polypeptide having 120 to 160 amino acids at the C-terminus of the full-length amino acid sequence of NKp44, more preferably a polypeptide having 140 to 160 amino acids at the C-terminus of the full-length amino acid sequence of NKp44, or a nucleoside having 80% identity to the aforementioned polypeptide sequence The acid sequence is either a nucleotide sequence that is more than 85% identical to the sequence, or a nucleotide sequence that is more than 90% identical to the sequence, or a core that is more than 95% identical to the sequence. Nucleotide sequence.
12. The signal transduction domain according to claim 9, characterized in that the amino acid sequence of the first transduction domain is shown in SEQ ID NO.8, and the nucleotide sequence is shown in SEQ ID NO.7.
13. Use of the signalling domain according to any one of claims 9 to 12 in the preparation of a chimeric antigen receptor or tumor immunotherapy.

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