Engineered dendritic cell and use thereof

The present application belongs to the technical field of biomedicine, and discloses a chimeric antigen receptor (CAR), an engineered dendritic cell (DC), and a use thereof. The CAR of the present application includes an extracellular domain, a CD8a hinge domain, a CD8a transmembrane domain, and an intracellular domain. The extracellular domain includes a guide sequence and a single-chain antibody sequentially. The intracellular domain includes a Dectin-1 intracellular domain and an intracellular domain of FcR gamma. Chimeric antigen receptor-modified dendritic cells (CAR-DCs) prepared with the CAR of the present application can efficiently recognize a tumor antigen. The combined administration of the CAR-DC and radiotherapy for treating a solid tumor can effectively overcome the immunosuppression of the tumor microenvironment and improve the clinical treatment effect. Therefore, the present application provides an effective immunotherapy strategy for clinical tumor patients, and provides a new idea and method for tumor immunotherapy.

1. 11. A chimeric antigen receptor (CAR), wherein the CAR has an amino acid sequence set forth in SEQ ID NO: 1.
2. 22. A nucleic acid encoding the CAR according to claim 1, wherein the nucleic acid has a nucleotide sequence set forth in SEQ ID NO: 2.
3. 33. An engineered dendritic cell (DC) comprising the CAR according to claim 1.
4. 44. An engineered DC comprising a nucleic acid encoding the CAR according to claim 2.
5. 55. The engineered DC according to claim 3, wherein the engineered DC is derived from at least one selected from the group consisting of a peripheral blood mononuclear cell, a hematopoietic stem cell, an induced pluripotent stem cell, and an embryonic stem cell.
6. 66. A preparation method of an engineered DC, comprising the following steps: transforming DNA or mRNA encoding the CAR according to claim 1 into a DC, and allowing for expression.
7. 77. The preparation method of an engineered DC according to claim 6, wherein the DNA or mRNA encoding the CAR is comprised in any one selected from the group consisting of an expression plasmid, a lentivirus, and a liposome.
8. 88. A preparation comprising an engineered DC comprising the CAR according to claim 1.
9. 99. A drug or preparation comprising the engineered DC according to claim 3, a pharmaceutically acceptable adjuvant, and/or a carrier or an excipient acceptable in a preparation process.
10. 1010. A method for treating a tumor, comprising: administering a population of engineered DCs modified with the CAR according to claim 1 to a tumor patient in combination with radiotherapy.

TECHNICAL FIELD
The present application relates to the technical field of biomedicine, and specifically to an engineered dendritic cell (DC) and a use thereof.
REFERENCE TO SEQUENCE LISTING
The Sequence Listing XML file submitted via the USPTO Patent Center, with a file name of “Sequence listing_SCH-25009-USPT.xml”, a creation date of Jan. 18, 2025, and a size of 22,777 bytes, is part of the specification and is incorporated in its entirety by reference herein.
BACKGROUND
Dendritic cells (DCs), as important immune cells, play a key role in the activation of T cells and the enhancement of anti-tumor immune responses. In the tumor therapy research, DCs are often used in the form of DC-based tumor vaccines for the prevention and treatment of tumors. In 2010, the first therapeutic cancer vaccine worldwide, Provenge (Sipuleucel-T), was approved for the treatment of prostate cancer, marking the important development of therapeutic cancer vaccines. However, despite the early success of Provenge and the generally accepted safety of the conventional DC-based cancer vaccines, these cancer vaccines often exhibit undesired clinical manifestations, and only 5% to 15% of patients acquire an objective immune response. The limited efficacy of cancer vaccines may be mainly attributed to the presence of various immunosuppressive factors in the tumor microenvironment. These immunosuppressive factors act as immunomodulators to inhibit the responses of anti-tumor T cells, thereby inhibiting the clinical effects of cancer vaccines as a whole.
Therefore, the new generation of DC vaccines must overcome multiple immunosuppressive mechanisms in the tumor microenvironment to improve the anti-tumor response mediated by effector T cells. How to improve the activity and anti-tumor effect of DCs in the tumor microenvironment is one of the major challenges for the current research.
SUMMARY
An objective of the present application is to overcome the deficiencies of the prior art and provide an engineered DC and a use thereof.
In order to achieve the above objective, the present application adopts the following technical solutions:
In a first aspect, the present application provides a chimeric antigen receptor (CAR), including an extracellular domain, a CD8a hinge domain, a CD8a transmembrane domain, and an intracellular domain.
The extracellular domain includes a guide sequence and a single-chain antibody sequentially (for example, from N-terminus to C-terminus).
The guide sequence has an amino acid sequence set forth in SEQ ID NO: 3, or has an amino acid sequence that has a homology of 98% or more and preferably 99% or more with the amino acid sequence set forth in SEQ ID NO: 3.
The single-chain antibody includes amino acid sequences set forth in SEQ ID NOs: 5, 7, and 9 sequentially (for example, from N-terminus to C-terminus), or includes amino acid sequences having a homology of 98% or more and preferably 99% or more with the amino acid sequences set forth in SEQ ID NOs: 5, 7, and 9 sequentially.
The intracellular domain includes a Dectin-1 intracellular domain and an intracellular domain of FcR gamma.
In the present application, a CAR is designed by a genetic engineering technology and used for modifying DCs to enhance a function of the DCs. The prepared CAR-DCs can efficiently recognize tumor antigens, transmit an extracellular signal inside cells, activate an intracellular signaling pathway, and further activate DCs. Moreover, the CAR-DCs can reverse the tumor microenvironment and improve the immune response, and can induce a specific immune response in vivo by acquiring a tumor antigen and presenting the tumor antigen to T cells. The radiotherapy can enhance an immune response in the body, and provides a basis for the activation and function improvement of DCs by directly killing tumor cells and releasing antigens. The combined use of the radiotherapy with the CAR-DC of the present application can improve an activation effect for DCs, increase the infiltration of immune cells through the local inflammation caused by the radiotherapy, and improve the efficacy of the radiotherapy by enhancing an anti-tumor immune effect of DCs, thereby improving the overall anti-tumor effect.
As a preferred embodiment of the CAR of the present application, the CD8a hinge domain has an amino acid sequence set forth in SEQ ID NO: 11, or has an amino acid sequence that has a homology of 98% or more and preferably 99% or more with the amino acid sequence set forth in SEQ ID NO: 11; and the CD8a transmembrane domain has an amino acid sequence set forth in SEQ ID NO: 13, or has an amino acid sequence that has a homology of 98% or more and preferably 99% or more with the amino acid sequence set forth in SEQ ID NO: 13.
As a preferred embodiment of the CAR of the present application, the Dectin-1 intracellular domain has an amino acid sequence set forth in SEQ ID NO: 15, or has an amino acid sequence that has a homology of 98% or more and preferably 99% or more with the amino acid sequence set forth in SEQ ID NO: 15; and the intracellular domain of FcR gamma has an amino acid sequence set forth in SEQ ID NO: 17, or has an amino acid sequence that has a homology of 98% or more and preferably 99% or more with the amino acid sequence set forth in SEQ ID NO: 17.
As a preferred embodiment of the CAR of the present application, the CAR has an amino acid sequence set forth in SEQ ID NO: 1, or has an amino acid sequence that has a homology of 98% or more and preferably 99% or more with the amino acid sequence set forth in SEQ ID NO: 1.
In a second aspect, the present application provides a nucleic acid encoding the CAR, where the nucleic acid has a nucleotide sequence set forth in SEQ ID NO: 2, or has a nucleotide sequence that has a homology of 98% or more and preferably 99% or more with the nucleotide sequence set forth in SEQ ID NO: 2.
In a third aspect, the present application provides an engineered DC (CAR-DC) modified with the CAR.
In a fourth aspect, the present application provides an engineered DC (CAR-DC) including a nucleic acid encoding the CAR.
As a preferred embodiment of the engineered DC of the present application, the engineered DC is at least one selected from the group consisting of a peripheral blood mononuclear cell, a hematopoietic stem cell, an induced pluripotent stem cell, and an embryonic stem cell.
In a fifth aspect, the present application provides a preparation method of an engineered DC, including the following step: transforming DNA or mRNA encoding the CAR into a DC, and allowing an expression.
As a preferred embodiment of the preparation method of an engineered DC of the present application, a vector tool for the transforming includes any one selected from the group consisting of an expression plasmid, a lentivirus, and a liposome.
In a sixth aspect, the present application provides a use of a CAR and an engineered DC in preparation of a tumor radiosensitizer administered in combination with radiotherapy, where the CAR is the CAR described above or a CAR having an amino acid sequence set forth in SEQ ID NO: 21; and the engineered DC is the engineered DC described above, or a DC modified with a CAR having an amino acid sequence set forth in SEQ ID NO: 21.
Correspondingly, the present application also provides a preparation for tumor radiosensitization administered in combination with radiotherapy, including a CAR and an engineered DC, where the CAR is the CAR described above or a CAR having an amino acid sequence set forth in SEQ ID NO: 21; and the engineered DC is the engineered DC described above, or a DC modified with a CAR having an amino acid sequence set forth in SEQ ID NO: 21.
In a seventh aspect, the present application provides a drug or preparation for treating a tumor, including the engineered DC, a pharmaceutically acceptable adjuvant, and/or a carrier or an excipient acceptable in a preparation process.
In an eighth aspect, the present application provides a method for treating a tumor, including: administering at least one of the following cells or drug to a tumor patient in combination with radiotherapy:

    
    
        i, the engineered DC described above;
        ii, the tumor therapeutic drug described above; and
        iii, a DC modified with a CAR having an amino acid sequence set forth in SEQ ID NO: 21.
    
    


As a preferred embodiment of the method for treating a tumor of the present application, the radiotherapy includes external beam radiation therapy and/or intracavitary radiotherapy.
As a further preferred embodiment of the method for treating a tumor of the present application, the external beam radiation therapy includes at least one selected from the group consisting of an X-knife, a gamma knife, and a linear accelerator; and the intracavitary radiotherapy includes seed implantation.
As a preferred embodiment of the method for treating a tumor of the present application, the tumor includes any one selected from the group consisting of breast cancer, lung cancer, colorectal cancer, liver cancer, pancreatic cancer, melanoma, glioma, ovarian cancer, and prostate cancer.
As a preferred embodiment of the method for treating a tumor of the present application, a process of the administering includes: on day 1 of the treating, intravenously injecting the engineered DC or the tumor therapeutic drug; on day 2 of the treating, applying the radiotherapy; and on day 7 of the treating, intravenously injecting the engineered DC or the tumor therapeutic drug into the tumor patient.
Compared with the prior art, the present application has the following beneficial effects:
In the present application, CAR-DC is used in combination with radiotherapy (including local radiotherapy and systemic radiotherapy) to treat solid tumors (including solid tumors that are sensitive or insensitive to the radiotherapy). The CAR-DC can enhance the immune effect of the radiotherapy in the tumor microenvironment, effectively overcome the immunosuppression of the tumor microenvironment, and improve the clinical treatment effect. Therefore, the present application provides an effective immunotherapy strategy for clinical tumor patients, and provides a new idea and method for tumor immunotherapy.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of the structure of CAR-DC;
FIG. 2 shows the expression of CAR on a surface of mouse bone marrow-derived DCs that is detected by flow cytometry;
FIG. 3 is a flow chart of an animal experiment in which colorectal cancer MC38 in mice is treated with a combination of CAR-DC and radiotherapy;
FIG. 4 shows tumor growth curves when colorectal cancer MC38 in mice is treated with a combination of CAR-DC and radiotherapy;
FIG. 5 shows pictures of tumors (Day 9) when colorectal cancer MC38 in mice is treated with a combination of CAR-DC and radiotherapy;
FIG. 6 shows tumor weights when colorectal cancer MC38 in mice is treated with a combination of CAR-DC and radiotherapy;
FIG. 7 shows flow cytometry results of tumor single cells when colorectal cancer MC38 in mice is treated with a combination of CAR-DC and radiotherapy;
FIG. 8 shows statistical charts of flow cytometry results of tumor single cells when colorectal cancer MC38 in mice is treated with a combination of CAR-DC and radiotherapy;
FIG. 9 shows the expression of CAR on a surface of mouse bone marrow-derived DCs that is detected by flow cytometry;
FIG. 10 is a flow chart of an animal experiment in which lung cancer LLC in mice is treated with a combination of CAR-DC and radiotherapy;
FIG. 11 shows tumor growth curves when lung cancer LLC in mice is treated with a combination of CAR-DC and radiotherapy;
FIG. 12 shows pictures of tumors (Day 7) when lung cancer LLC in mice is treated with a combination of CAR-DC and radiotherapy;
FIG. 13 shows tumor weights when lung cancer LLC8 in mice is treated with a combination of CAR-DC and radiotherapy;
FIG. 14 shows the expression of CAR on a surface of mouse bone marrow-derived DCs that is detected by flow cytometry;
FIG. 15 is a flow chart of an animal experiment in which breast cancer 4T1 in mice is treated with a combination of CAR-DC and radiotherapy;
FIG. 16 shows tumor growth curves when breast cancer 4T1 in mice is treated with a combination of CAR-DC and radiotherapy;
FIG. 17 shows the expression of CAR on a surface of humanized mouse bone marrow-derived DCs that is detected by flow cytometry;
FIG. 18 shows tumor growth curves when human breast cancer SKRBR3 is treated with a combination of CAR-DC and radiotherapy.



DETAILED DESCRIPTION
To well explain the objective, technical solutions, and advantages of the present application, the present application will be further explained below with reference to specific examples. It should be understood by those skilled in the art that the specific examples described here are merely intended to explain the present application, rather than to limit the present application.
In the examples, unless otherwise specified, the experimental methods used are conventional, and the materials and reagents used are commercially available.
In the following examples, a method of plotting a tumor growth curve is as follows: A tumor is periodically measured for a length and a width by an electronic vernier caliper. A volume of the tumor is calculated based on the length and the width of the tumor according to the following calculation formula: (width{circumflex over ( )}2*length)/2. The tumor growth curve is plotted with the Prism Graphad software.
Example 1: Engineered DC (CAR-DC)
1. Design of an Engineered DC (CAR-DC)
Functional elements of CAR are shown in FIG. 1. The structure of the CAR includes an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular domain (an amino acid sequence of mouse Epha2 CAR is set forth in SEQ ID NO: 1).
The extracellular domain includes a guide sequence (the amino acid sequence of a mouse Epha2 CAR guide sequence is set forth in SEQ ID NO: 3), a single-chain antibody (scFv) (the amino acid sequence of anti-mouse Epha2 VH is set forth in SEQ ID NO: 5, the amino acid sequence of anti-mouse Epha2 VL is set forth in SEQ ID NO: 7, and the amino acid sequence for a mouse Epha2 CAR linker is set forth in SEQ ID NO: 9). The hinge domain (the amino acid sequence of a mouse CD8 hinge domain is set forth in SEQ ID NO: 11) and the transmembrane domain (the amino acid sequence of a mouse CD8a transmembrane domain is set forth in SEQ ID NO: 13) are a hinge domain and a transmembrane domain of CD8a, respectively. The intracellular domain employs human or mouse Dectin-1 (the amino acid sequence of a mouse Dectin1 intracellular domain is set forth in SEQ ID NO: 15) and an intracellular domain of FcR gamma (the amino acid sequence of an intracellular domain of mouse FcR gamma is set forth in SEQ ID NO: 17). After being combined, the amino acid sequences of the elements were optimized by a codon optimization technology. A DNA sequence for mouse CAR-DC was cloned into a pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector.
2. In Vitro Transcription
A gene for the CAR-DC was amplified by PCR. A PCR system (100 μL system) was as follows: DNA (amplified with a codon-optimized base sequence set forth in SEQ ID NO: 2 as a template): 100 ng, 2×Taq mix: 50 μL, primer mix (5 μM): 5 μL, and H2O: to 100 μL. Primer sequences were set forth in SEQ ID NOs: 19 and 20, respectively. Amplification conditions were as follows: 1. 94° C.: 5 min; 2. 94° C.: 20 s, 55° C.: 30 s, and 72° C.: 60 s, with 30 cycles; 3. 72° C.: 5 min; and 4. 4° C.: heat preservation.
CAR-DC mRNA carrying a T7 promoter and a PolyA structure was produced through the transcription with a High Yield T7 RNA Synthesis Kit (HONGENE BIOTECH, Shanghai). A transcription system was as follows: template DNA (4 μg), ATP (8 μL), GTP (8 μL), CTP (8 μL), UTP (8 μL), GAG (8 μL), Reaction buffer (20 μL), Enzyme (7.5 μL), ultrapure water (to 100 μL). The transcription system was incubated at 37° C. for 4 h, then 7.5 μL of DNase was added, and a resulting mixture was incubated at 37° C. for 15 min. After a reaction was completed, mRNA was precipitated with lithium chloride. 150 μL of ultrapure water and 150 μL of a lithium chloride solution were added to a reaction system to precipitate RNA. A resulting mixed system was thoroughly mixed, refrigerated at −20° C. for 60 min, and centrifuged at 4° C. and a maximum rotational speed for 5 min to produce a supernatant and a precipitate. The supernatant was carefully removed. The precipitate was washed with 1 mL of about 70% ethanol, and then the 70% ethanol was carefully removed to produce a washed precipitate. The washed precipitate was air-dried and then resuspended with an appropriate amount of nuclease-free water.
3. Preparation of CAR-DCs
Tibias and fibulas of hind legs of 6-8 week-old C57BL6 or Balbc mice were collected. The bone marrow was flushed out by a 1 ml syringe, properly ground into a single-cell suspension, and centrifuged at 1,500 rpm for 5 min, and a resulting supernatant was removed. Red blood cells were lysed with a 1×ACK buffer. Centrifugation was conducted at 1,500 rpm for 5 min, and a resulting supernatant was removed. Cells were washed twice with phosphate buffered saline (PBS), counted, and inoculated in a 6-well plate at a density of 1×106/mL. 100 ng/ml of GMCSF and IL4 were added to allow the differentiation of DCs. A medium was supplemented every 2 d to 3 d. After 8 d of differentiation, mouse DCs were harvested, counted, and electrotransformed with the above mRNA by a LONZA 4D system under the following conditions: 1E6-3E6/test, 100 μL P3 solution, 5 μg/test, and program CM150. The electrotransformed cells were inoculated in a 6-well plate and cultured for 24 h. Then an expression efficiency of CAR was measured with protein L. Resulting cells were used for downstream applications.
Example 2 Treatment of Colorectal Cancer with a Combination of CAR-DCs and Radiotherapy
Tibias and fibulas of hind legs of 6-8 week-old C57BL6 mice were collected by the method in Example 1 to prepare CAR-DCs. An expression efficiency of CAR was detected with protein L. Results were shown in FIG. 2. A transfection efficiency of CAR in DCs was 72.8%.
Mouse colorectal cancer cells MC38 were transplanted into C57BL6 mice (6 weeks to 8 weeks). One week after the tumor transplantation, the mice were randomly divided into the following four groups: a control group (CTL), a CAR-DC group, a radiotherapy group (IR), and a radiotherapy+CAR-DC group (IR+CAR-DC). On day 1, mice in the CAR-DC group and the radiotherapy+CAR-DC group each were infused with CAR-DCs through the tail vein at a dose of 3×106 cells/mouse. On day 2, mice in the radiotherapy group and the radiotherapy+CAR-DC group each were subjected to X-ray irradiation at an irradiation dose of 10 Gy. On day 4, CAR-DCs were infused through the tail vein for the second time at a dose of 3×106 cells/mouse. The growth and survival of mouse tumors were continuously observed. A specific flow chart was shown in FIG. 3.
At the end of the experiment, the mouse tumors were collected for analysis. The tumors were grouped and arranged, photographed, and weighed by a balance. Data was collected. Tumor tissues were digested with a collagenase to produce single cells. The single cells were labeled with CD45 (BD Pharmingen™, 564279), CD3 (BD Pharmingen™, 560591), CD8 (BD Pharmingen™, 553031), CTLA4 (BD Pharmingen™, 553720), and PDI (BD Pharmingen™, 749422) antibodies, incubated for 30 min on ice in the dark, washed to remove non-specifically bound antibodies, resuspended with 400 μL of PBS, and analyzed by flow cytometry for a cell subtype and cell status.
A therapeutic effect of the combination of the CAR-DC and the radiotherapy for tumors was evaluated. It could be seen from tumor growth curves (FIG. 4), tumor sizes (FIG. 5), and tumor weights (FIG. 6) that the combination of the CAR-DC and the radiotherapy had a significant inhibitory effect on the tumor growth (differences among groups were determined by t-test, **p<0.01 and ***p<0.001). In order to further analyze a phenotype of tumor-infiltrating T cells, single-cell suspensions were prepared from tumors, incubated with CD3, CD8, CTLA4, and PDI antibodies, and tested by flow cytometry for a cell subtype and phenotype (FIG. 7 and FIG. 8). Results showed that the combined use of the CAR-DC and the radiotherapy significantly increased the infiltration of T cells. In particular, the infiltration of effector T cells was significantly increased, and the infiltrating T cells exhibited lower exhausted phenotypes than T cells for animal tumors in other groups (differences among groups were determined by t-test, *p<0.05 and **p<0.01). It further explained a mechanism of the combination of the CAR-DC and the radiotherapy to achieve a prominent anti-tumor effect.
Example 3 Treatment of Lung Cancer with a Combination of CAR-DCs and Radiotherapy
Tibias and fibulas of hind legs of 6-8 week-old Balbc mice were collected by the method in Example 1 to prepare CAR-DCs. An expression efficiency of CAR was detected with protein L. Results were shown in FIG. 9. A transfection efficiency of CAR in DCs was 80.8%.
Mouse lung cancer cells LLC were transplanted into C57BL6 mice (6 weeks to 8 weeks). 9 days after the tumor transplantation, the mice were randomly divided into the following four groups: a control group (CTL), a CAR-DC group, a radiotherapy group (IR), and a radiotherapy+CAR-DC group (IR+CAR-DC). On day 0, mice in the CAR-DC group and the radiotherapy+CAR-DC group each were infused with CAR-DCs through the tail vein at a dose of 3×106 cells/mouse. On day 1, mice in the radiotherapy group and the radiotherapy+CAR-DC group each were subjected to X-ray irradiation at an irradiation dose of 5 Gy. On day 3, CAR-DCs were infused through the tail vein for the second time at a dose of 3×106 cells/mouse. The growth and survival of mouse tumors were continuously observed.
At the end of the experiment, the mouse tumors were collected for analysis. The tumors were grouped and arranged, photographed, and weighed by a balance. Data was collected.
The prepared CAR-DCs were administered in combination with the radiotherapy to LLC tumor-bearing mice (a flow chart was shown in FIG. 10) to evaluate a therapeutic effect of the combination of the CAR-DC and the radiotherapy for the tumor. It could be seen from tumor growth curves (FIG. 11), tumor sizes (FIG. 12), and tumor weights (FIG. 13) that the combination of the CAR-DC and the radiotherapy had a significant inhibitory effect on the growth of lung cancer (differences among groups were determined by t-test, *p<0.05 and **p<0.01).
Example 4 Treatment of Breast Cancer with a Combination of CAR-DCs and Radiotherapy
Tibias of hind legs of 6-8 week-old Balbc mice were collected by the method in Example 1 to prepare CAR-DCs. An expression efficiency of CAR was detected with protein L. Results were shown in FIG. 14. A transfection efficiency of CAR in DCs was 8.86%.
Mouse breast cancer cells 4T1 were transplanted into Balbc mice (6 weeks to 8 weeks). 9 days after the tumor transplantation, the mice were randomly divided into the following four groups: a control group (CTL), a CAR-DC group, a radiotherapy group (IR), and a radiotherapy+CAR-DC group (IR+CAR-DC). On day 0, mice in the CAR-DC group and the radiotherapy+CAR-DC group each were infused with CAR-DCs through the tail vein at a dose of 3×106 cells/mouse. On day 1, mice in the radiotherapy group and the radiotherapy+CAR-DC group each were subjected to X-ray irradiation at an irradiation dose of 5 Gy. On day 3, CAR-DCs were infused through the tail vein for the second time at a dose of 3×106 cells/mouse. The growth and survival of mouse tumors were continuously observed.
At the end of the experiment, the mouse tumors were collected for analysis. The tumors were grouped and arranged, photographed, and weighed by a balance. Data was collected.
The prepared CAR-DCs were administered in combination with the radiotherapy to LLC tumor-bearing mice (a flow chart was shown in FIG. 15) to evaluate a therapeutic effect of the combination of the CAR-DC and the radiotherapy for the tumor. It could be seen from tumor growth curves (FIG. 16) that the combination of the CAR-DC and the radiotherapy had a significant inhibitory effect on the growth of breast cancer.
Example 5 Treatment of Human Breast Cancer with a Combination of CAR-DCs and Radiotherapy
Construction of humanized mice: Immunodeficient mice (purchased from Jiangsu GemPharmatech LLC., NCG, T001475) were irradiated at a sublethal dose. Then a human thymus tissue of about 1 mm3 was transplanted into a renal capsule of each immunodeficient mouse, and a wound was sutured after the surgery. The mice each were injected with CD34+ hematopoietic stem cells through the tail vein after wake. 10 weeks after the surgery, blood was collected and tested to determine the reconstruction of an immune system in mice. 2 to 3 drops of intravenous blood were collected from a hind leg of each mouse, added to an EDTA-PBS buffer, and centrifuged to settle cells. Red blood cells were completely lysed with a 1×ACK lysis buffer until a transparent lysate solution was produced. The transparent lysate solution was centrifuged to produce a supernatant and a precipitate. The supernatant was removed. The precipitate was washed once with Dulbecco's phosphate-buffered saline (DPBS), then incubated with an anti-CAR antibody and an anti-CD11c antibody, then stained, washed once, resuspended in DPBS, and analyzed by flow cytometry to determine the successful reconstruction of humanized mice.
Preparation of humanized CAR-DCs: Tibias and fibulas of hind legs of the humanized mice were collected. The bone marrow was flushed out by a 1 ml syringe, properly ground into a single-cell suspension, and centrifuged at 1,500 rpm for 5 min, and a resulting supernatant was removed. Red blood cells were lysed with a 1×ACK buffer. Centrifugation was conducted at 1,500 rpm for 5 min, and a resulting supernatant was removed. Cells were washed twice with PBS, counted, and inoculated in a 6-well plate at a density of 1×106/mL. 100 ng/ml of human GMCSF and human IL4 were added to allow the differentiation of DCs. A medium was supplemented every 2 d to 3 d. After 8 d of differentiation, DCs were harvested, counted, and transfected with a lentivirus encoding a human CAR-DC receptor (the amino acid sequence of the human Epha2 CAR protein was set forth in SEQ ID NO: 21). 48 h later, a CAR expression efficiency was detected with protein L. As shown in FIG. 17, a transfection efficiency of CAR in DCs was 45.3%.
In vivo treatment: Human breast cancer cells SK-BR3 were transplanted into the humanized mice. One week after the tumor transplantation, the mice were randomly divided into the following five groups: a control group (PBS), a DC group, a CAR-DC group, a radiotherapy+DC group (IR+DC), and a radiotherapy+CAR-DC group (IR+CAR-DC). On day 2, mice in the radiotherapy+DC group and the radiotherapy+CAR-DC group each were subjected to X-ray irradiation at an irradiation dose of 1 Gy. On day 4, DCs and CAR-DCs each were infused through the tail vein at a dose of 3×106 cells/mouse. The growth of mouse tumors were continuously observed. A length and width of a tumor were measured regularly with an electronic vernier caliper. A volume of the tumor was calculated based on the length and width of the tumor according to the following calculation formula: (width{circumflex over ( )}2*length)/2. A tumor volume of each measurement was determined with a measurement on day 0 as a baseline. A baselined tumor growth curve (calculation formula: VolDayn/VolDay0) was plotted with the Prism Graphad software. It could be seen from tumor growth curves (FIG. 18) that the combination of the CAR-DC and the radiotherapy had a significant inhibitory effect on the growth of breast cancer (differences among groups were determined by t-test, *p<0.05).
The sequences involved in the present application include the following:







Amino acid sequence for mouse Epha2 CAR 


(SEQ ID NO: 1):


MASPLTRFLSLNLLLLGESIILGSGEADIQMTQSPSSLSASVGDRVTIT


CRASQYYSYYGVAWYQQKPGKAPKLLIYGASYLYSGVPSRFSGSRSGTD


FTLTISSLQPEDFATYYCQQSFYPITFGQGTKVEIKGGGGSGGGGSGGG


GSEVQLVESGGGLVQPGGSLRLSCAASGFNLSGGGVHWVRQAPGKGLEW


VAGIYSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC


ARSSGGFDYWGQGTLVTVSSTTTKPVLRTPSPVHPTGTSQPQRPEDCRP


RGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHRSRGVAIPR


WPSSPAQSGKESGRPRKHIDQTSFDLQTYGDEDLNEIHSHYKMRLKIQV


RKAAIASREKADAVYTGLNTRSQETYETLKHEKPPQ.


 


Nucleotide sequence for mouse Epha2 CAR 


(SEQ ID NO: 2):


ATGGCCAGCCCTCTGACCAGATTCCTGTCTCTGAACCTGCTCCTGCTGG


GAGAGTCTATCATCCTGGGATCAGGAGAGGCTGACATCCAGATGACCCA


GAGCCCTTCCTCACTGAGCGCTTCCGTGGGTGACAGAGTGACTATTACC


TGCAGAGCCAGCCAGTACTACAGCTACTATGGAGTGGCCTGGTACCAGC


AGAAGCCTGGCAAAGCTCCTAAGCTGCTGATCTATGGAGCTTCTTACCT


GTACTCCGGGGTCCCATCTAGGTTCAGCGGCTCTAGGTCTGGCACCGAC


TTCACTCTGACCATCTCCAGCCTGCAGCCAGAAGACTTCGCCACCTACT


ACTGCCAGCAGAGTTTCTACCCCATCACCTTCGGACAGGGAACCAAGGT


GGAAATCAAAGGCGGCGGCGGGAGCGGGGGCGGCGGCTCTGGAGGCGGC


GGGTCCGAAGTCCAGCTGGTGGAGAGCGGCGGAGGTCTGGTGCAGCCAG


GCGGCTCCCTGAGACTGTCCTGCGCCGCCTCCGGCTTCAACCTGTCCGG


GGGTGGAGTGCACTGGGTGAGGCAGGCTCCCGGCAAGGGACTGGAGTGG


GTGGCTGGAATCTACTCCAGCTCCGGATACACATACTATGCCGACAGCG


TGAAGGGCAGGTTTACCATCAGCGCCGACACCTCTAAAAACACCGCATA


CCTGCAGATGAATAGCCTGCGAGCCGAGGATACAGCCGTGTATTACTGC


GCCAGGAGCTCCGGCGGCTTTGATTACTGGGGGCAGGGCACTCTGGTGA


CTGTGTCCTCTACAACAACTAAGCCTGTGCTGAGGACCCCTTCCCCTGT


GCACCCAACCGGCACCAGCCAGCCCCAGCGACCTGAGGACTGCAGACCC


CGGGGATCTGTGAAGGGCACCGGGCTGGATTTTGCATGTGACATTTATA


TCTGGGCCCCTCTGGCCGGCATCTGCGTGGCCCTGCTGCTGTCTCTGAT


CATTACCCTGATCTGCTATCATAGATCCAGAGGGGTGGCTATCCCCAGA


TGGCCTAGCAGCCCAGCCCAGAGTGGAAAAGAGAGCGGCCGCCCTAGAA


AGCACATCGACCAGACCTCTTTTGATCTGCAAACTTACGGTGACGAGGA


TCTGAATGAGATCCACTCTCACTACAAGATGAGGCTGAAGATACAGGTG


CGGAAGGCAGCCATCGCAAGCAGAGAGAAGGCCGACGCCGTGTACACAG


GCCTGAACACAAGATCTCAGGAGACCTATGAGACCCTGAAGCATGAGAA


GCCCCCCCAGTGA.


 


Amino acid sequence for the guide sequence of 


mouse Epha2 CAR (SEQ ID NO: 3):


MASPLTRFLSLNLLLLGESIILGSGEA.


 


Nucleotide sequence for the guide sequence of 


mouse Epha2 CAR (SEQ ID NO: 4):


ATGGCCAGCCCTCTGACCAGATTCCTGTCTCTGAACCTGCTCCTGCTGG


GAGAGTCTATCATCCTGGGATCAGGAGAGGCT.


 


Amino acid sequence for anti-mouse Epha2 VH 


(SEQ ID NO: 5):


DIQMTQSPSSLSASVGDRVTITCRASQYYSYYGVAWYQQKPGKAPKLLI


YGASYLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSFYPITF


GQGTKVEIK.


 


Nucleotide sequence for anti-mouse Epha2 VH 


(SEQ ID NO: 6):


GACATCCAGATGACCCAGAGCCCTTCCTCACTGAGCGCTTCCGTGGGTG


ACAGAGTGACTATTACCTGCAGAGCCAGCCAGTACTACAGCTACTATGG


AGTGGCCTGGTACCAGCAGAAGCCTGGCAAAGCTCCTAAGCTGCTGATC


TATGGAGCTTCTTACCTGTACTCCGGGGTCCCATCTAGGTTCAGCGGCT


CTAGGTCTGGCACCGACTTCACTCTGACCATCTCCAGCCTGCAGCCAGA


AGACTTCGCCACCTACTACTGCCAGCAGAGTTTCTACCCCATCACCTTC


GGACAGGGAACCAAGGTGGAAATCAAA.


 


Amino acid sequence for anti-mouse Epha2 VL 


(SEQ ID NO: 7):


EVQLVESGGGLVQPGGSLRLSCAASGFNLSGGGVHWVRQAPGKGLEWVA


GIYSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR


SSGGFDYWGQGTLVTVSS.


 


Nucleotide sequence for anti-mouse Epha2 VL 


(SEQ ID NO: 8):


GAAGTCCAGCTGGTGGAGAGCGGCGGAGGTCTGGTGCAGCCAGGCGGCT


CCCTGAGACTGTCCTGCGCCGCCTCCGGCTTCAACCTGTCCGGGGGTGG


AGTGCACTGGGTGAGGCAGGCTCCCGGCAAGGGACTGGAGTGGGTGGCT


GGAATCTACTCCAGCTCCGGATACACATACTATGCCGACAGCGTGAAGG


GCAGGTTTACCATCAGCGCCGACACCTCTAAAAACACCGCATACCTGCA


GATGAATAGCCTGCGAGCCGAGGATACAGCCGTGTATTACTGCGCCAGG


AGCTCCGGCGGCTTTGATTACTGGGGGCAGGGCACTCTGGTGACTGTGT


CCTCT.


 


Amino acid sequence for the linker of mouse Epha2 


CAR (SEQ ID NO: 9):


GGGGSGGGGSGGGGS.


 


Nucleotide sequence for the linker of mouse Epha2 


CAR (SEQ ID NO: 10):


GGCGGCGGCGGGAGCGGGGGCGGCGGCTCTGGAGGCGGCGGGTCC.


 


Amino acid sequence for the CD8a hinge domain of 


the mouse (SEQ ID NO: 11):


TTTKPVLRT PSPVHPTGTS QPQRPEDCRPRGSVKGTGLD FACDIY.


 


Nucleotide sequence for the CD8a hinge domain of 


the mouse (SEQ ID NO: 12):


ACAACAACTAAGCCTGTGCTGAGGACCCCTTCCCCTGTGCACCCAACCG


GCACCAGCCAGCCCCAGCGACCTGAGGACTGCAGACCCCGGGGATCTGT


GAAGGGCACCGGGCTGGATTTTGCATGTGACATTTAT.


 


Amino acid sequence for the CD8a transmembrane 


domain of the mouse (SEQ ID NO: 13):


IWAPLAGICVALLLSLIITLICYHRSR.


 


Nucleotide sequence for the CD8a transmembrane 


domain of the mouse (SEQ ID NO: 14):


ATCTGGGCCCCTCTGGCCGGCATCTGCGTGGCCCTGCTGCTGTCTCTGA


TCATTACCCTGATCTGCTATCATAGATCCAGA.


 


Amino acid sequence for the Dectin1 intracellular 


domain of the mouse (SEQ ID NO: 15):


GVAIPRWPSSPAQSGKESGRPRKHIDQTSFDLQTYGDEDLNEIHSHYKM.


 


Nucleotide sequence for the Dectin1 intracellular 


domain of the mouse (SEQ ID NO: 16):


GGGGTGGCTATCCCCAGATGGCCTAGCAGCCCAGCCCAGAGTGGAAAAG


AGAGCGGCCGCCCTAGAAAGCACATCGACCAGACCTCTTTTGATCTGCA


AACTTACGGTGACGAGGATCTGAATGAGATCCACTCTCACTACAAGATG.


 


Amino acid sequence for the intracellular domain 


of mouse FcR gamma (SEQ ID NO: 17):


RLKIQVRKAAIASREKADAVYTGLNTRSQETYETLKHEKPPQ.


 


Nucleotide sequence for the intracellular domain 


of mouse FcR gamma (SEQ ID NO: 18):


AGGCTGAAGATACAGGTGCGGAAGGCAGCCATCGCAAGCAGAGAGAAGG


CCGACGCCGTGTACACAGGCCTGAACACAAGATCTCAGGAGACCTATGA


GACCCTGAAGCATGAGAAGCCCCCCCAG.


 


Forward strand of the primer for mouse CAR IVT 


(in vitro transcription) (SEQ ID NO: 19):


ATAATACGACTCACTATAGGGAGAGCCACCATGGCCAGCCCTCTGACCA


G.


 


Reverse strand of the primer for mouse CAR IVT 


(SEQ ID NO: 20):


TTTTTTTTTTTTTCTGTCTTTTTATTGCCGTCACTGGGGGGGCTTCTCA


T.


 


Amino acid sequence for human Epha2 CAR 


(SEQ ID NO: 21):


MALPVTALLLPLALLLHAARPQVQLLESGGGLVQPGGSLRLSCAASGFT


FSSYTMSWVRQAPGQALEWMGTISSRGTYTYYPDSVKGRFTISRDNAKN


SLYLQMNSLRAEDTAVYYCAREAIFTHWGRGTLVTVSSGGGGSGGGGSG


GGGSDIQLTQSPSSLSASVGDRVTITCKASQDINNYHSWYQQKPGQAPR


LLIYRANRLVDGVPDRESGSGYGTDFTLTINNIESEDAAYYFCLKYNVF


PYTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH


TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRWPPSAACSGKESVVA


IRTNSQSDFHLQTYGDEDLNELDPHYEMRLKIQVRKAAITSYEKSDGVY


TGLSTRNQETYETLKHEKPPQ.






Finally, it should be noted that the above examples are provided merely to describe the technical solutions of the present application, rather than to limit the protection scope of the present application. Although the present application is described in detail with reference to preferred examples, a person of ordinary skill in the art should understand that modifications or equivalent replacements may be made to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application.