CN110917365B - CAR-T living body tracing method - Google Patents

CAR-T living body tracing method Download PDF

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CN110917365B
CN110917365B CN201911324560.1A CN201911324560A CN110917365B CN 110917365 B CN110917365 B CN 110917365B CN 201911324560 A CN201911324560 A CN 201911324560A CN 110917365 B CN110917365 B CN 110917365B
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霍力
方鹏
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Abstract

The invention relates to the technical field of in-vivo tracing, in particular to a CAR-T in-vivo tracing method, which has the technical key points that: s1, combining the pre-target molecule A with an amino group on the surface of the CAR-T cell to obtain the pre-target CAR-T cell with an A group; wherein R is2‑NH2Is CAR-T cell, -NH2Is an amino group on the surface of a CAR-T cell; s2, mixing18F is combined with the targeting molecule B to obtain the peptide with18R of F1‑N3(ii) a S3, injecting the pre-targeted CAR-T cells obtained in S1 into a living body, and at a desired imaging time point, injecting R1‑N3Injected into the body and binds to pre-targeted CAR-T; s4, the distribution of the CAR-T cells in vivo can be observed by PET/CT scanning. The invention increases the operation safety of experimenters and also avoids the possible damage of radioactivity to CAR-T cells and living bodies.

Description

CAR-T living body tracing method
Technical Field
The invention relates to the technical field of in-vivo tracing, in particular to a CAR-T in-vivo tracing method.
Background
In recent years, although chemotherapy and bone marrow transplantation techniques are applied to common malignant tumors of the blood system, such as leukemia, myeloma and lymphoma, great progress is made, the treatment failure rate is still high, and the problems of drug resistance and relapse are still difficult to overcome. Therefore, it is urgent to develop a new way and search for a safer and more effective treatment means.
The therapy of chimeric antigen receptor T cells (Car-T cells for short) is one of the most fire-hot fields of anti-tumor immunity at present, and is also a major breakthrough of the immune medicine in the last decade. The effect of the Car-T cells on treating the hematological malignancy is remarkable, and various large pharmaceutical companies and innovative companies occupy the market of the Car-T cells. Novartis and Gilead are at the forefront of the field, products are approved by the FDA of the United states to be on the market, and various products enter clinical trials by companies such as Celgene and Merck. In 8 months 2017, CAR-T cells Kymriah of Novartis company are approved by the US FDA to be marketed, are used for treating relapsed or refractory patients with Acute Lymphoblastic Leukemia (ALL) under 25 years old, become the first autologous cell CAR-T therapy on the market worldwide, and are a milestone for the development history of Car-T cell therapy.
In CAR-T cell therapy, T cells extracted from the patient themselves, after genetic engineering, are transplanted back into the patient to discover and kill the cancer. This class of immunotherapy has revolutionized the treatment of certain cancers, but once CAR-T cells enter the patient, where will they go? How are physicians aware that they have successfully reached their destination and continue to fight the disease after weeks, months or even years?
In the prior art, In order to realize the tracking of CAR-T cells, nuclides such as Zr-89, In-111 and the like are labeled In advance In the CAR-T cells and then injected into a human body, and the distribution condition of the CAR-T cells In the human body can be observed through PET/CT scanning; however, there is a need for improvement in that radioactive nuclides enter a living body (animal or human) along with CAR-T cells, and damage is caused to CAR-T cells and the living body.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defects that when practical nuclides are used for CAR-T cell tracing in the prior art, the nuclide has long half-life period, long irradiation time on living bodies, high irradiation dose and easy damage to the living bodies.
The technical purpose of the invention is realized by the following technical scheme:
a CAR-T in vivo tracking method comprising the steps of:
s1, combining the pre-target molecule A with an amino group on the surface of the CAR-T cell to obtain the pre-target CAR-T cell with an A group;
Figure BDA0002328035230000021
wherein R is2-NH2Is CAR-T cell, -NH2Is an amino group on the surface of a CAR-T cell;
s2, mixing18F is combined with the targeting molecule B to obtain the peptide with18R of F1-N3
Figure BDA0002328035230000031
S3, injecting the pre-targeted CAR-T cells obtained in S1 into a living body, and at a desired imaging time point, injecting R1-N3Injected into the body and binds to pre-targeted CAR-T;
Figure BDA0002328035230000032
s4, the distribution of the CAR-T cells in vivo can be observed by PET/CT scanning.
Alternatively, in step S3, R is added1-N3Intravenously into the body and through the internal circulation into the body.
Optionally, in step S3, R that is not bound to CAR-T cells1-N3Is discharged to the outside of the body.
Optionally, the method of pre-target molecule a binding to an amino group on the surface of CAR-T cell comprises the steps of: and (2) after the T cells are sorted and activated in vitro, the slow virus vector transduction is carried out to obtain the CAR-T cells, the obtained CAR-T cells are cultured and amplified for 7-10 days, the pre-targeting molecule A is added into the culture solution from the next day of culture and amplification, then the culture solution containing the pre-targeting molecule A is supplemented into a cell culture bottle, and the cell amplification is periodically supplemented until the cell amplification is completed.
Optionally, the amount of pre-targeting molecule a added is 1-10 micrograms per 1 × 10E +6 cells.
Alternatively, the medium containing the pre-homing molecule a is supplemented every 2 days.
Optionally, the cell culture medium is a lymphocyte blood-free medium.
Optionally, the CAR-T cells are cultured in an environment at a constant temperature of 37 deg.C, a carbon dioxide concentration of 5%, a relative saturation humidity of 95%, and a cell culture medium pH of 7.2-7.4.
The CAR-T living body tracing method provided by the invention adopts a pre-targeting mode to pre-treat CAR-T cells which do not enter a living body, and only injects a targeting molecule with nuclide into the body when imaging is needed, on the one hand, before the targeting molecule with nuclide is injected, the targeting molecule with nuclide is injectedThe CAR-T cells pre-loaded with the target molecule are not radioactive, do not cause damage to the living body when they are diffused into the body, and are injected into the body at a time point when imaging is desired18Targeting molecules of the F nuclide due to short development time, and18the half-life period of the F nuclide is short, the irradiation time of the living body is short, the irradiation dose is low, the operation safety of experimenters is improved, and the possible damage of radioactivity to CAR-T cells and the living body is also avoided.
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FIG. 1 is a graph showing the results of Micro PET scanning in example 4 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1:
a CAR-T in vivo tracking method comprising the steps of:
s1, combining the pre-target molecule A with an amino group on the surface of the CAR-T cell to obtain the pre-target CAR-T cell with an A group;
Figure BDA0002328035230000051
wherein R is2-NH2In, R2I.e. representing the CAR-T cell body, -NH2Represents an amino group on the surface of a CAR-T cell. The reaction formula is a process of combining the pre-targeting molecule A with amino on the surface of the cell membrane of CAR-T, the pre-targeting molecule A with an isothiocyanate group can be combined with the amino on the surface of the cell membrane under mild conditions to form a stable covalent bond, so that the pre-targeting molecule can be firmly combined on the surface of the cell membraneThe formula is consistent with the conventional cell culture mode, and the activity of cells is not influenced; the second is that the pre-attached target molecules which are not bound to the cells are consumed by the proteinaceous material in the culture medium and are removed when the medium is changed. The pretargeted CAR-T cells can be administered to mice in animal models, or in human experiments, following the normal use procedure for CAR-T.
S2, mixing18F is combined with the targeting molecule B to obtain the peptide with18R of F1-N3；
Figure BDA0002328035230000061
The molecular structure has stably combined positron nuclide18F, so that the tissue distribution of the molecule can be observed by a PET/CT living body after the molecule is injected into an animal body.
S3, injecting the pre-targeted CAR-T cells obtained in S1 into a living body, and at a desired imaging time point, injecting R1-N3Injected into the body and binds to pre-targeted CAR-T;
Figure BDA0002328035230000062
wherein, in step S3, R is added1-N3Intravenously injected into the body and enters all parts of the body through the internal circulation;
wherein, in step S3, R that is not bound to CAR-T cells1-N3Is discharged out of the body;
89the half-life period of Zr is 78.4h, the radioactive irradiation time to the living body is about 32 days,111the In half-life is 67.9h, and the radioactive irradiation time to the living body is about 28 days.
CAR-T cells that have been pretargeted are first injected into the animal and, at a point in time when imaging is desired, will18R marked by F1-N3Intravenously injected into the animal, R1-N3Enters all parts of the body through internal circulation, and is rapidly combined when meeting pre-target moleculesForming a strong covalent bond, indirectly effecting18R labelled to CAR-T cells by F, not bound to CAR-T cells1-N3Then it is cleared rapidly and excluded from the body, and the CAR-T cell distribution in vivo can be observed by PET/CT scanning at appropriate time points. The CAR-T activity tracing method based on the pre-targeting strategy has the advantages that: firstly, the pre-targeting and CAR-T injection process into living bodies (animals or human bodies) do not have radioactivity, so that the operation safety of experimenters is improved, and possible damage of radioactivity to CAR-T cells and living bodies is avoided; secondly, when tracing is needed, the tracing is carried out18R marked by F1-N3And when the targeting molecule is injected into a living body, the targeting molecule is specifically combined with the pre-adhesion targeting molecule, and then CAR-T cell imaging is realized.18F vs. used in conventional CAR-T tracer method89Zr、111Short half-life of In and other nuclides, short irradiation time for living body radioactivity, low irradiation dose, and89Zr、111the method for imaging In vivo by injecting In nuclide pre-labeled cells is limited by the half-life of the nuclide, imaging can be realized only within a period of time, and the pre-targeting strategy adopts injection at the time point needing imaging18F is marked with a targeting molecule, so that the imaging time is not influenced by the half-life of the nuclide.
Example 2:
a CAR-T in vivo tracking method comprising the steps of:
s1, combining the pre-target molecule A with an amino group on the surface of the CAR-T cell to obtain the pre-target CAR-T cell with an A group;
Figure BDA0002328035230000071
wherein R is2-NH2In, R2I.e. representing the CAR-T cell body, -NH2Represents an amino group on the surface of a CAR-T cell. The reaction formula is a process of combining the pre-adhesion target molecule A with amino on the surface of CAR-T cell membrane, and the pre-adhesion target molecule A with an isothiocyanate group can be combined with the surface of the cell membrane under mild conditionsThe method has the advantages that the pre-targeting molecules can be directly added into the conventional cell culture solution, the addition amount is low in microgram level, and the pre-targeting molecules are continuously combined with the amino on the surface of the cell membrane along with the long-time proliferation culture of the cells so as to realize the pre-targeting, and firstly, the pre-targeting mode is consistent with the conventional cell culture mode and cannot influence the activity of the cells; the second is that the pre-attached target molecules which are not bound to the cells are consumed by the proteinaceous material in the culture medium and are removed when the medium is changed. The pretargeted CAR-T cells can be administered to mice in animal models, or in human experiments, following the normal use procedure for CAR-T.
S2, mixing18F is combined with the targeting molecule B to obtain the peptide with18R of F1-N3；
Figure BDA0002328035230000081
The molecular structure has stably combined positron nuclide18F, so that the tissue distribution of the molecule can be observed by a PET/CT living body after the molecule is injected into an animal body.
S3, injecting the pre-targeted CAR-T cells obtained in S1 into a living body, and at a desired imaging time point, injecting R1-N3Injected into the body and binds to pre-targeted CAR-T;
Figure BDA0002328035230000091
wherein, in step S3, R is added1-N3Intravenously injected into the body and enters all parts of the body through the internal circulation;
wherein, in step S3, R that is not bound to CAR-T cells1-N3Is discharged out of the body;
CAR-T cells that have been pretargeted are first injected into the animal and, at a point in time when imaging is desired, will18R marked by F1-N3Intravenously injected into the animal, R1-N3Enters all parts of the whole body through internal circulation, and when meeting pre-target molecules, the molecules are quickly combined to form a firm covalent bond, so that the aim of directly combining the molecules is fulfilled18R labelled to CAR-T cells by F, not bound to CAR-T cells1-N3Then it is cleared rapidly and excluded from the body, and the CAR-T cell distribution in vivo can be observed by PET/CT scanning at appropriate time points. The CAR-T activity tracing method based on the pre-targeting strategy has the advantages that: firstly, the pre-targeting and CAR-T injection process into living bodies (animals or human bodies) do not have radioactivity, so that the operation safety of experimenters is improved, and possible damage of radioactivity to CAR-T cells and living bodies is avoided; secondly, when tracing is needed, the tracing is carried out18R marked by F1-N3And when the targeting molecule is injected into a living body, the targeting molecule is specifically combined with the pre-adhesion targeting molecule, and then CAR-T cell imaging is realized.18F vs. used in conventional CAR-T tracer method89Zr、111Short half-life of In and other nuclides, short irradiation time for living body radioactivity, low irradiation dose, and89Zr、111the method for imaging In vivo by injecting In nuclide pre-labeled cells is limited by the half-life of the nuclide, imaging can be realized only within a period of time, and the pre-targeting strategy adopts injection at the time point needing imaging18F is marked with a targeting molecule, so that the imaging time is not influenced by the half-life of the nuclide.
The method for binding the pre-adhesion target molecule A to the amino group on the surface of the CAR-T cell comprises the following steps: and (2) after the T cells are sorted and activated in vitro, the slow virus vector transduction is carried out to obtain the CAR-T cells, the obtained CAR-T cells are cultured and amplified for 7-10 days, the pre-targeting molecule A is added into the culture solution from the next day of culture and amplification, then the culture solution containing the pre-targeting molecule A is supplemented into a cell culture bottle, and the cell amplification is periodically supplemented until the cell amplification is completed.
Example 3:
a CAR-T in vivo tracking method comprising the steps of:
s1, combining the pre-target molecule A with an amino group on the surface of the CAR-T cell to obtain the pre-target CAR-T cell with an A group;
Figure BDA0002328035230000101
wherein R is2-NH2In, R2I.e. representing the CAR-T cell body, -NH2Represents an amino group on the surface of a CAR-T cell. The reaction formula is a process of combining the pre-adhesion target molecule A with amino on the surface of a CAR-T cell membrane, the pre-adhesion target molecule A with an isothiocyanate group can be combined with the amino on the surface of the cell membrane under a mild condition to form a stable covalent bond, so that the pre-adhesion target molecule can be firmly combined on the surface of the cell membrane, and the method has the advantages that the pre-adhesion target molecule can be directly added into a conventional cell culture solution, the addition amount is a microgram level, and the pre-adhesion target molecule is continuously combined with the amino on the surface of the cell membrane along with the long-time proliferation culture of cells to realize the pre-adhesion target, and firstly, the pre-adhesion target mode is consistent with the conventional cell culture mode and cannot influence the activity of the cells; the second is that the pre-attached target molecules which are not bound to the cells are consumed by the proteinaceous material in the culture medium and are removed when the medium is changed. The pretargeted CAR-T cells can be administered to mice in animal models, or in human experiments, following the normal use procedure for CAR-T.
S2, mixing18F is combined with the targeting molecule B to obtain the peptide with18R of F1-N3；
Figure BDA0002328035230000111
The molecular structure has stably combined positron nuclide18F, so that the tissue distribution of the molecule can be observed by a PET/CT living body after the molecule is injected into an animal body.
S3, injecting the pre-targeted CAR-T cells obtained in S1 into a living body, and at a desired imaging time point, injecting R1-N3Injected into the body and binds to pre-targeted CAR-T;
Figure BDA0002328035230000112
wherein, in step S3, R is added1-N3Intravenously injected into the body and enters all parts of the body through the internal circulation;
wherein, in step S3, R that is not bound to CAR-T cells1-N3Is discharged out of the body;
CAR-T cells that have been pretargeted are first injected into the animal and, at a point in time when imaging is desired, will18R marked by F1-N3Intravenously injected into the animal, R1-N3Enters all parts of the whole body through internal circulation, and when meeting pre-target molecules, the molecules are quickly combined to form a firm covalent bond, so that the aim of directly combining the molecules is fulfilled18R labelled to CAR-T cells by F, not bound to CAR-T cells1-N3Then it is cleared rapidly and excluded from the body, and the CAR-T cell distribution in vivo can be observed by PET/CT scanning at appropriate time points. The CAR-T activity tracing method based on the pre-targeting strategy has the advantages that: firstly, the pre-targeting and CAR-T injection process into living bodies (animals or human bodies) do not have radioactivity, so that the operation safety of experimenters is improved, and possible damage of radioactivity to CAR-T cells and living bodies is avoided; secondly, when tracing is needed, the tracing is carried out18R marked by F1-N3And when the targeting molecule is injected into a living body, the targeting molecule is specifically combined with the pre-adhesion targeting molecule, and then CAR-T cell imaging is realized.18F vs. used in conventional CAR-T tracer method89Zr、111Short half-life of In and other nuclides, short irradiation time for living body radioactivity, low irradiation dose, and89Zr、111the method for imaging In vivo by injecting In nuclide pre-labeled cells is limited by the half-life of the nuclide, imaging can be realized only within a period of time, and the pre-targeting strategy adopts injection at the time point needing imaging18F is marked with a targeting molecule, so that the imaging time is not influenced by the half-life of the nuclide.
The method for binding the pre-adhesion target molecule A to the amino group on the surface of the CAR-T cell comprises the following steps: after the T cells are sorted and activated in vitro, the CAR-T cells are obtained after lentiviral vector transduction, the obtained CAR-T cells are cultured and amplified for 7-10 days, the pre-targeting molecule A is added into the culture solution from the second day of culture and amplification, then the culture solution containing the pre-targeting molecule A is supplemented into a cell culture bottle, and the cell amplification is periodically supplemented until the cell amplification is completed;
wherein the addition amount of the pre-targeting molecule A is 1-10 micrograms per 1 × 10E +6 cells;
wherein the replenishing period of the culture solution containing the pre-targeting molecule A is once every 2 days;
wherein the cell culture medium is a lymphocyte blood-free medium;
wherein the CAR-T cell culture environment is constant temperature of 37 deg.C, carbon dioxide concentration of 5%, relative saturation humidity of 95%, and cell culture medium pH of 7.2-7.4.
Example 4: a CAR-T living body tracing method, referring to figure 1, when the test object is a tumor-bearing mouse, the scanning steps are as follows:
a1: anesthesia: anaesthetizing the animals by using a small animal anesthetic, wherein the anesthetic is isoflurane with the concentration of about 1-2 percent, and the gas flow rate is 400-800 mL/min; placing the animal subjected to induction anesthesia on a small animal PET/CT bed, and continuing isoflurane to maintain anesthesia;
A2：18injection of F-R1-N3: firstly, establishing a tail vein injection channel to prepare for injection18F-R1-N3, recording information such as injection time, dosage, residual dosage measurement, residual time measurement and the like, and performing the next injection (the specific time is determined according to the reperfusion time) about 15min after one injection is finished;
a3: fixing and scanning: carrying out static scanning for 10min after injection for 1h, lightly putting the anesthetized tumor-bearing mouse on a scanning bed, fixing the four limbs of the tumor-bearing mouse by using a medical adhesive tape, scanning and collecting data;
a4: and performing data reconstruction in an OSEM3D mode after scanning, delineating an ROI, and calculating the uptake value (SUV and% ID/g) of the radioactive substance of each organ.
In FIG. 1, the a region is a tumor region, which has a high uptake and the% ID/gmean value of the uptake is 4.5.
In conclusion, the CAR-T cells which do not enter the living body are pretreated by adopting a pre-targeting way, and the targeting molecule with the nuclide is injected into the body when imaging is needed, on one hand, before the targeting molecule with the nuclide is not injected, the CAR-T cells with the pre-targeting molecule do not have radioactivity, and do not damage the living body when the CAR-T cells diffuse to various places in the body, and on the other hand, at the time point needing imaging, the CAR-T cells with the pre-targeting molecule are injected18Targeting molecules of the F nuclide due to short development time, and18the half-life period of the F nuclide, the irradiation time to the living body and the irradiation dose are short, the operation safety of experimenters is improved, and the possible damage of radioactivity to CAR-T cells and the living body is also avoided.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims (8)

1. Use of a pre-targeting molecule a and a homing molecule B for the preparation of a medicament for use in a CAR-T in vivo tracking method, said method comprising the steps of:
s1, combining the pre-target molecule A with an amino group on the surface of the CAR-T cell to obtain the pre-target CAR-T cell with an A group;
Figure FDA0003299300450000011
wherein R is2-NH2Is CAR-T cell, -NH2Is an amino group on the surface of a CAR-T cell;
s2, mixing18F is combined with the targeting molecule B to obtain the peptide with18R of F1-N3；
Figure FDA0003299300450000012
S3, injecting the pre-targeted CAR-T cells obtained in S1 into a living body, and at a desired imaging time point, injecting R1-N3Injected into the body and binds to pre-targeted CAR-T;
Figure FDA0003299300450000021
s4, the distribution of the CAR-T cells in vivo can be observed by PET/CT scanning.
2. The use according to claim 1, wherein in step S3, R is1-N3Intravenously into the body and through the internal circulation into the body.
3. The use according to claim 1, wherein in step S3, R that is not bound to CAR-T cells1-N3Is discharged to the outside of the body.
4. Use according to claim 1, characterized in that the method of binding the pre-target molecule a to the amino groups of the CAR-T cell surface comprises the following steps: and (3) after the T cells are sorted and activated in vitro, the slow virus vector transduction is carried out to obtain the CAR-T cells, the obtained CAR-T cells are cultured and amplified for 7-10 days, the pre-attached target molecule A is added into the culture solution from the next day of culture and amplification, then the culture solution containing the pre-attached target molecule A is supplemented into a cell culture bottle, and the periodic supplementation is carried out until the cell amplification is completed.
5. The use according to claim 4, wherein the amount of preincubation target molecule A is 1-10 micrograms per 1 × 10E +6 cells.
6. The use according to claim 5, wherein the culture medium containing the pre-target A is supplemented every 2 days.
7. The use according to claim 4, wherein the cell culture medium is a lymphocyte blood-free medium.
8. The use according to claim 4, wherein the CAR-T cells are cultured in an environment at a constant temperature of 37 ℃, at a carbon dioxide concentration of 5%, at a relative saturation humidity of 95%, and at a cell culture medium pH of 7.2-7.4.
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