CN113106067A - Construction and application of chimeric antigen receptor-monocyte/macrophage (CAR-M) - Google Patents
Construction and application of chimeric antigen receptor-monocyte/macrophage (CAR-M) Download PDFInfo
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Abstract
The invention relates to a construction and an application of a chimeric antigen receptor-monocyte/macrophage (CAR-M). A genetic engineering receptor molecule suitable for introducing mononuclear/macrophage cell membrane organization is designed and optimized by using a genetic editing means and serves as a chimeric antigen receptor, and the chimeric antigen receptor comprises a leader peptide, a recognition region, a hinge region and a transmembrane/intracellular signal region, and specifically comprises a sequence shown as SEQ ID NO.1-SEQ ID NO. 18. The invention also relates to the application of the chimeric antigen receptor-monocyte/macrophage (CAR-M). the invention adopts the monocyte/macrophage as a carrier cell for cellular immunotherapy, and can solve the problem that CAR-T cannot enter the interior of solid tumor by utilizing good tissue infiltration and cytokine chemotaxis of the monocyte/macrophage. The invention uses the chimeric antigen receptor to endow the mononuclear/macrophage with the targeting property to the tumor cells, enhances the phagocytosis to the tumor cells, further can present the tumor antigen to the T cells, and enhances the anti-tumor immunity. The chimeric antigen receptor-monocyte/macrophage (CAR-M) constructed by the invention can be used for the cellular immunotherapy of solid tumors such as liver cancer, gastric cancer, lung cancer, breast cancer and the like.
Description
The technical field is as follows:
the invention belongs to the technical field of immunotherapy, and particularly relates to construction and application of a chimeric antigen receptor-monocyte/macrophage (CAR-M).
Background art:
cells currently used internationally for cellular immunotherapy are mainly divided into two main categories: the first category includes lymphokine activated killer cells (LAK), natural killer cells (NK), cytokine induced killer Cells (CIK), and the like; the second category is T lymphocytes with tumor antigen specificity, including Tumor Infiltrating Lymphocytes (TIL), cytotoxic T Cells (CTL). The first technical system is simple, particularly the treatment of CIK cells is popular in China, the second technical system is relatively complex, the cell culture period is long, the culture success rate is relatively low, but the curative effect is more obvious because of high specificity to kill tumor cells. CAR-T is a novel therapeutic cell obtained based on the two types of researches, CAR is the abbreviation of Chimeric antigen receptor, and CAR-T cell immunotherapy is that T cells of a patient are modified through genetic engineering, added with a Chimeric antigen receptor, and then amplified in vitro and then transfused into the body of the patient. The CAR-T cells have achieved remarkable curative effects on treatment of blood system tumors such as acute leukemia, non-Hodgkin lymphoma and the like. CAR-T cell immunotherapy, however, encounters an unprecedented challenge during clinical treatment of solid tumors: 1) the solid tumor extracellular matrix hinders CAR-T cell infiltration; 2) solid tumor immunosuppressive microenvironments (heavily infiltrated immunosuppressive cells, tregs, TAMs, MDSCs, etc.; immunosuppressive cytokines, TGF-. beta., IL-10, etc.; arrestin expression, PD-1/PD-L1, CTLA-4, etc.) inhibits CAR-T tumor killing activity; 3) the high heterogeneity of solid tumors makes CAR-T cell therapy deficient in effective tumor antigens.
The macrophage has strong phagocytic function, and can be cleared by about 2 × 10 every day11Each red blood cell is equivalent to 3kg of hemoglobin which can be recycled by the organism every year. In addition, macrophage cell-mediated immunityCells play an important role in phagocytosis, antigen presentation, immune balance regulation and the like, and play an important monitoring function for maintaining the immune homeostasis of an organism. The macrophage can detect tissue damage signals or microorganism invasion signals in the surrounding environment through a surface receptor, and once abnormality is found, phagocytosis and antigen presentation of the macrophage can be induced, so that specific immune response of an organism is promoted. For example, alveolar macrophages are effective in clearing lung allergens; kupffer cells help clear circulating pathogens and toxins from the blood. Macrophages can also phagocytose immunogenic dead tumor cells and present tumor antigens to T cells, exerting anti-tumor immunity.
However, in recent years, it has been found that tumor cells or other cells in tumor tissues recruit macrophage infiltration by releasing chemokines such as CCL2, CXCL12, CSF1, and macrophages are immune cells with the highest infiltration rate in the microenvironment of solid tumors. Macrophages infiltrating tumor tissues are also called Tumor Associated Macrophages (TAMs), and play an important role in the generation, development and metastasis of tumors. In the tumor microenvironment, tumor cells can bind to receptors on macrophages through CD47 and MHCI molecules, and phagocytosis of the tumor cells by the macrophages is inhibited. Meanwhile, lipid metabolism in TAMs is regulated and controlled, lipid drop accumulation is promoted, and phagocytic ability of macrophages is damaged. Therefore, although macrophages infiltrated in tumor tissues have the potential of killing tumor cells, the molecules such as CD47 on the surface of the tumor cells inhibit the TAMs, so that the TAMs do not kill the tumor cells, and can promote the growth of tumors by promoting the angiogenesis and other effects.
Based on this, the mononuclear/macrophage is taken as a carrier cell of cellular immunotherapy, the mononuclear/macrophage is modified from a molecular level by combining the advantages of the vigorous development of the existing chimeric antigen receptor technology and the gene optimization technology which is mature day by day, and the chimeric antigen receptor-macrophage (CAR-M) is prepared and obtained, and the high-efficiency infiltration property of the chimeric antigen receptor-macrophage is utilized to enhance the phagocytosis capacity and the antigen presentation capacity of the chimeric antigen receptor-macrophage, so that the CAR-M is expected to become a new generation of the cellular immunotherapy.
Disclosure of Invention
The invention aims to design and optimize a genetic engineering receptor molecule suitable for introducing a mononuclear/macrophage cell membrane structure by using a gene editing means to obtain a chimeric antigen receptor mononuclear/macrophage capable of specifically recognizing and phagocytosing tumor cells. Therefore, a high-efficiency mononuclear/macrophage immunotherapy system for adoptive therapy of tumors is established.
In order to achieve the aim of the invention, the technical scheme adopted by the invention is that
The present invention provides chimeric antigen receptors for enhancing phagocytic capacity of macrophages. Including leader peptides, extracellular recognition regions, hinge regions, and transmembrane/intracellular signaling regions.
The leader peptide is the leader peptide of colony stimulating factor 2 receptor alpha (CSF2R alpha), and the amino acid sequence of the CSF2R alpha leader peptide is shown as SEQ ID NO. 1.
The extracellular recognition region is a single-chain antibody scFv of an anti-tumor specific antigen, in a specific embodiment, HER2 is selected as a tumor antigen, and the amino acid sequence of the single-chain antibody scFv of the anti-HER 2 is shown as SEQ ID NO. 2.
The hinge region is a CD28hinge region, and further, the amino acid sequence of the CD28hinge region is shown as SEQ ID NO. 3.
The transmembrane/intracellular signaling region is selected from the corresponding transmembrane/intracellular sequences of CD16(Fc γ RIIIA), CD32(Fc γ RIIA), CD64(Fc γ RIA), FCER1G or CD36 molecules. CD16, CD32 and CD64 belong to Fc gamma R family (Fc-gamma receptor family), can recognize and combine Fc fragments of Ig, can effectively remove IgG-Ag complex, and have very important functions in both natural immunity and acquired immunity. FCER1G contains a tyrosine-based immunoreceptor activation motif (ITAM) that transmits activation signals from a variety of immunoreceptors, and mediates allergic inflammatory signaling in mast cells as part of the high affinity immunoglobulin e (ige) receptor. CD36 belongs to scavenger receptor superfamily, can discern oxidation low density lipoprotein, collagen, long chain fatty acid etc. and mainly express on monocyte/macrophage membrane surface, and this receptor not only can mediate phagocytosis to take place, can promote the formation of TLR4, TLR6 heterodimer to activation aseptic inflammatory response. Activation of the TLR signaling pathway can, in turn, promote macrophage polarization to anti-tumor macrophages of type M1. Therefore, the present invention selects the intracellular region of the above receptor molecule as an internal signal sequence to activate phagocytic ability of macrophages. The amino acid sequence of the transmembrane/intracellular region of the CD16 is shown as SEQ ID NO.4, the amino acid sequence of the transmembrane/intracellular region of the CD32 is shown as SEQ ID NO.5, the amino acid sequence of the transmembrane/intracellular region of the CD64 is shown as SEQ ID NO.6, the amino acid sequence of the transmembrane/intracellular region of the FCER1G is shown as SEQ ID NO.7, and the amino acid sequence of the transmembrane/intracellular region of the CD36 is shown as SEQ ID NO. 8.
The chimeric antigen receptors are specifically CAR-16, CAR-32, CAR-64, CAR-R1G and CAR-36. The amino acid sequence of the chimeric antigen receptor CAR-16 is shown as SEQ ID NO. 9. The amino acid sequence of the chimeric antigen receptor CAR-32 is shown as SEQ ID NO. 10. The amino acid sequence of the chimeric antigen receptor CAR-64 is shown as SEQ ID NO. 11. The amino acid sequence of the chimeric antigen receptor CAR-RIG is shown as SEQ ID NO. 12. The amino acid sequence of the chimeric antigen receptor CAR-36 is shown as SEQ ID NO. 13.
The nucleotide sequence of the chimeric antigen receptor CAR-16 is shown as SEQ ID NO. 14. The nucleotide sequence of the chimeric antigen receptor CAR-32 is shown as SEQ ID NO. 15. The nucleotide sequence of the chimeric antigen receptor CAR-64 is shown as SEQ ID NO. 16. The nucleotide sequence of the chimeric antigen receptor CAR-RIG is shown as SEQ ID NO. 17. The nucleotide sequence of the chimeric antigen receptor CAR-36 is shown as SEQ ID NO. 18.
The invention provides a recombinant expression vector containing the nucleic acid fragment. The recombinant expression vector comprises lentivirus, retrovirus, adenovirus, adeno-associated virus or plasmid and the like; further, the original recombinant expression vector is a lentivirus. In a specific embodiment, the vector used is a lentiviral vector. The CAR lentiviral vector plasmid was co-transfected into HEK293T cells in the presence of helper packaging plasmids psPAX2 and pmd2.g, and packaged as a lentivirus with a CAR molecule.
The present invention provides a host cell containing the above recombinant expression vector.
The host cells are monocytes and macrophages.
The invention provides a method for constructing the host cell, which comprises a step of constructing the recombinant expression vector, a step of packaging the recombinant expression vector and a step of transducing the recombinant expression vector into the host cell.
The invention provides the chimeric antigen receptor, the nucleic acid segment, the recombinant expression vector and the application of the host cell in preparing human tumor treatment medicines.
Further, the human tumor is mainly referred to as a solid tumor.
Further, the application is a tumor antigen specific CAR-M cell (CAR-monoclonal/macrophage) that confers tumor antigen specific CAR on monocytes/macrophages through lentivirus mediated transduction, thereby conferring the ability of monocytes/macrophages to recognize specific tumor antigens and phagocytize tumor cells.
In a specific embodiment, the tumor antigen selected is HER2, the chimeric antigen receptor used comprises a single chain antibody targeting HER2, and the tumor cell lines selected are the HER2 positive breast cancer cell line MDA-MB-453 and the HER2 positive gastric cancer cell line MKN 45.
Compared with the prior art, the technical scheme provided by the invention has the following beneficial effects:
1. the host cell of choice in the present invention is a monocyte/macrophage. It is currently accepted that CAR-T, which has a definite therapeutic effect in the treatment of hematological cancers, cannot enter the interior of solid tumors, whereas monocytes/macrophages have good tissue infiltration and cytokine chemotaxis.
2. According to the immune cell biological characteristics of the mononuclear cell/macrophage, the mononuclear cell/macrophage is endowed with tumor targeting property through gene modification, the phagocytic capacity of the mononuclear cell/macrophage is enhanced, the tumor antigen is further presented, the tumor immune microenvironment is improved, and the anti-tumor immunity is enhanced, so that the mononuclear cell/macrophage immune cell has the remarkable technical innovation characteristic.
Description of the drawings:
FIG. 1 is a schematic diagram of a chimeric antigen receptor.
FIG. 2 flow assay of chimeric antigen receptor expression in macrophages.
FIG. 3 flow assay of the phagocytic capacity of CAR-36 macrophages on MDA-MB-453 breast cancer cells.
FIG. 4 flow assay of the phagocytic capacity of CAR-36 macrophages on MKN45 gastric cancer cells.
FIG. 5(A) construction of MKN45 gastric cancer mouse subcutaneous model, intravenous injection of control macrophages and CAR-36 macrophages, and measurement of tumor growth every 3 days; (B) the mice were sacrificed and the tumors were removed and weighed for re-analysis; (C) the proportion of GFP positive cells in the tumor is detected by flow to analyze the infiltration condition of the feedback macrophages.
Figure 6 in vivo imaging detection of tumor size in MKN45 abdominal cavity dissemination model of gastric cancer. Mice were sacrificed and the abdominal cavity was dissected to photograph the abdominal cavity neoplasia.
The specific implementation mode is as follows:
the present invention will be described in detail with reference to the following specific examples: the present invention will be better understood from the following examples. However, the specific material ratios, process conditions and results thereof described in the examples are merely illustrative of the present invention and should not, nor should they limit the invention as detailed in the claims.
Example one
This example is the assembly of a chimeric antigen receptor molecule targeting HER2, comprising a leader peptide, an extracellular recognition region, a hinge region, a transmembrane region/intracellular signal region, in order from N-terminus to C-terminus the leader peptide is CSF2R α leader peptide (SEQ ID No.1), the extracellular recognition region is an anti-HER 2 antibody sequence (scFv amino acid sequence, SEQ ID No.2), the hinge region is a CD28hinge region (SEQ ID No.3), and the transmembrane region/intracellular signal region is CD16(SEQ ID No.4), CD32(SEQ ID No.5), CD64(SEQ ID No.6), fcr 1G (SEQ ID No.7) or CD36(SEQ ID No. 8). The specific nucleic acid sequences CAR-16(SEQ ID NO.14), CAR-32(SEQ ID NO.15) and CAR-64(SEQ ID NO.16), CAR-R1G (SEQ ID NO.17) and CAR-36(SEQ ID NO.18) are artificially synthesized in a living organism to obtain the chimeric antigen receptor sequence. FIG. 1 is a schematic diagram of a chimeric antigen receptor.
Specifically, the chimeric antigen receptors of the invention are specifically CAR-16, CAR-32, CAR-64, CAR-R1G and CAR-36. The base sequence of the obtained CAR sequence is integrated into a lentiviral plasmid vector pLenti6/v5 by an enzyme digestion method, and the method comprises the following steps: the Spe I/Xho I double enzyme digestion vector is linked with a CAR base sequence and T4 ligase to obtain target plasmids of pLenti-CAR-16, pLenti-CAR-32, pLenti-CAR-64, pLenti-CAR-R1G and pLenti-CAR-36.
Example two
This example is the preparation of lentiviruses with a chimeric antigen receptor selected as CAR-36.
Experimental materials: OPTI-MEM, Lipofectamine 2000(lipo2000) purchased from Invitrogen; HEK 293T.
The experimental steps are as follows:
1) the first day, passage 293T, was performed overnight in 10cm x 10cm dishes with DMEM containing 10% FBS to achieve a density of 80%.
2) The next day, cells were transfected, old medium was aspirated, 5mL of preheated OPTI-MEM was added quickly, and then returned to the incubator.
3) Preparation of DNA/lipo2000 complexes:
a) to an EP tube, 1.5mL of OPTI-MEM was added, followed by the following plasmids and mixing.
pLenti-CAR-36 15μg
psPAX2 7.5μg
pMD2.G 2.5μg
b) 1.5mL of OPTI-MEM was added to an EP tube, followed by 75. mu.L of lipo2000, mixed well and left at room temperature for 5 min.
c) Mixing a) and b), and standing at room temperature for 20 min.
4) The DNA/lipo2000 complex is added into a cell culture dish drop by drop, is shaken up gently and is placed back into an incubator for continuous culture.
5) After 6h, the solution was changed, the DNA/lipo2000 complex was removed, 8mL of preheated DMEM containing 5% FBS was added, and the culture was continued.
6) On the third day, 5mL of the medium was added to make the total volume 10-20 mL.
7) On day four, the culture supernatant was collected (special attention: the medium cannot be yellowed because the half-life of the virus is only 10min at pH values between 6 and 8).
8) Centrifugation was carried out at 1500 Xrpm for 5min to remove cell debris, and the resulting solution was filtered through a 0.4 μm filter.
9) The virus concentrate and the filtered virus supernatant were added at a ratio of 1: 4, mixed well and incubated overnight (over 12 hours) at 4 ℃.
10) The virus mixture was centrifuged (2000g, 30min) and a white precipitate appeared at the bottom of the tube, the supernatant was discarded and centrifuged again (2000g, 5min) to aspirate all residual liquid without destroying the precipitated virus particles.
11) The viral pellet was resuspended in 1/10 volume of DMEM medium and used immediately or stored in aliquots at-80 ℃.
EXAMPLE III
This example is the construction of chimeric antigen receptor monocytes/macrophages
Experimental materials:
polybrene purchased from Sigma; the flow cytometric sorter was BD FACS Aria.
The experimental steps are as follows:
1) THP-1 cells in logarithmic growth phase were plated in 6-well plates (1mL), 1X 106cell/well。
2) 1mL of viral supernatant and ploybrene (8. mu.g/mL) were added slowly and shaken gently.
3) Centrifuge at 1900rpm for 60min (32 ℃).
4) Placing in a cell culture box for culturing for 1 h.
5) Repeat 3) once.
6) After 24h, the solution was changed and infected once with the lentivirus prepared in example two, as described above.
7) After 48h, the expression of intracellular GFP was observed using a fluorescence microscope.
8) The above cells were subjected to flow sorting to obtain GFP positive cells.
9) And performing amplification culture on the obtained cells to obtain the THP-1 cell line (CAR-36-THP-1) expressing the chimeric antigen receptor CAR-36.
Example four
This example is the identification of chimeric antigen receptor macrophages
1) CAR-36-THP-1 monocytes constructed in example three were induced with 50nM PMA for 24h to become THP-1 macrophages.
2) By using an in vitro co-culture model, HER2+ tumor cells (breast cancer cells MDA-MB-453 and gastric cancer cells MKN45) and CAR-36-THP-1 macrophages are directly contacted and co-cultured in a ratio of 1: 1, and the phagocytic effect of CAR-36 on HER2 positive cells is directly observed by a fluorescence confocal microscope and a flow type. The CAR expression vector is provided with EGFP, and green fluorescence represents chimeric antigen receptor macrophage; tumor cells were stained with the live cell marker dye DiR and showed red color. As shown in fig. 3 and 4, the constructed chimeric antigen receptor macrophages have significant tumor cell phagocytic activity.
EXAMPLE five
This example demonstrates the ability of CAR-36 macrophages to inhibit HER2 positive gastric cancer in a mouse subcutaneous tumor model.
Selecting 18-22g Balb/c male nude mice, planting MKN45 gastric cancer cells in hind leg with cell amount of 5 × 106Tumor growth was recorded by observation and 1X 10 intravenous injection on days 7 and 14 post-inoculation6Control THP-1 macrophages or 1X 106Individual CAR-positive CAR-36-THP-1 macrophages. Tumor size was measured every three days from day 7 of inoculation using calipers. The results are shown in FIG. 5A, where the tumor growth was significantly inhibited in the group of injected CAR-CD36-THP-1 macrophages. On day 30 of inoculation, mice were euthanized, tumors were removed and weighed (fig. 5B), single cell suspensions were prepared, and infiltration of reinfused macrophages was analyzed by flow. Results figure 5C shows that CAR-36 is able to promote infiltration of THP-1 macrophages in the tumor.
EXAMPLE six
This example demonstrates the ability of CAR-36 macrophages to inhibit HER2 positive gastric cancer in a mouse peritoneal dissemination model.
Selecting a gastric cancer cell line MKN45 with luciferase, collecting cells after trypsinization, and resuspending the cells to 10 ℃ with physiological saline7Per ml; selecting male nude mice (SPF grade) with weight of 18-22g, and inoculating abdominal tumor in 1ml sterile syringe with cell amount of about 106One/one; intraperitoneal injection at 1X 10 after 14 and 21 days after inoculation6Control THP-1 macrophages or 1X 106Individual CAR-positive CAR-36-THP-1 macrophages. On day 40 post inoculation, mice were examined for abdominal tumor size by in vivo imaging. The results are shown in FIG. 6, where the mice injected with the CAR-36-THP-1 macrophage group had complete tumor regression.
The foregoing examples further illustrate the present invention but are not to be construed as limiting thereof. It will be apparent to those skilled in the art that modifications and substitutions to methods, steps or conditions of the invention can be made without departing from the spirit and substance of the invention.
Claims (13)
1. A chimeric antigen receptor-monocyte/macrophage (CAR-M), which is characterized in that the chimeric antigen receptor suitable for being introduced into the cell membrane structure of the monocyte/macrophage to enhance the phagocytic capacity of the monocyte/macrophage is designed and optimized by using a gene editing means.
2. The chimeric antigen receptor for enhancing phagocytic capacity of monocytes/macrophages of claim 1 comprising a leader peptide, a recognition region, a hinge region and a transmembrane/intracellular signal region.
3. The chimeric antigen receptor for enhancing phagocytic capacity of monocytes/macrophages according to claim 1, wherein said leader peptide is the leader peptide of colony stimulating factor 2 receptor alpha (CSF2R a), and the amino acid sequence of CSF2R a leader peptide is shown in SEQ ID No. 1.
4. The chimeric antigen receptor for enhancing phagocytic capacity of monocytes/macrophages according to claim 1, wherein said recognition region is a single chain antibody scFv that binds to a tumor antigen.
5. The chimeric antigen receptor for enhancing phagocytic capacity of monocytes/macrophages of claim 1, wherein said hinge region is a CD28 hingge region, and the amino acid sequence thereof is represented by SEQ ID No. 3.
6. The chimeric antigen receptor for enhancing phagocytic capacity of monocytes/macrophages according to claim 1, wherein said transmembrane/intracellular signaling region is the transmembrane/intracellular domain of CD16(SEQ ID No.4), CD32(SEQ ID No.5), CD64(SEQ ID No.6), FCER1G (SEQ ID No.7) or CD36(SEQ ID No.8) molecule capable of activating phagocytic signal of macrophages.
7. The chimeric antigen receptor for enhancing phagocytic capacity of monocytes/macrophages according to claim 1, wherein said chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID No.9-SEQ ID No. 13.
8. A nucleic acid encoding the chimeric antigen receptor of any one of claims 1-7 for use in enhancing phagocytic capacity of monocytes/macrophages.
9. The nucleic acid according to claim 8, wherein the nucleotide sequence of the nucleic acid comprises the nucleotide sequence shown in SEQ ID No.14-SEQ ID No. 18.
10. A recombinant expression vector comprising the nucleic acid of any one of claims 8-9.
11. A monocyte/macrophage cell comprising the recombinant expression vector of claim 10.
12. The method of constructing a monocyte/macrophage as claimed in claim 11, which comprises the steps of constructing a recombinant expression vector, packaging the recombinant expression vector, and transducing the recombinant expression vector into the monocyte/macrophage.
13. The chimeric antigen receptor of any one of claims 1 to 7 for enhancing phagocytic capacity of monocytes/macrophages, the nucleic acid of any one of claims 8 to 9, the recombinant expression vector of claim 10 and the use of monocytes/macrophages of claim 11 in the treatment of human solid tumors.
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