CN113929785B - Chimeric antigen receptor immune cell capable of automatically secreting PD1-TREM2 bispecific antibody and application thereof - Google Patents

Chimeric antigen receptor immune cell capable of automatically secreting PD1-TREM2 bispecific antibody and application thereof Download PDF

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CN113929785B
CN113929785B CN202111063869.7A CN202111063869A CN113929785B CN 113929785 B CN113929785 B CN 113929785B CN 202111063869 A CN202111063869 A CN 202111063869A CN 113929785 B CN113929785 B CN 113929785B
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CN113929785A (en
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黄曦
陈健
李咸君
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Qinyuan Regenerative Medicine Guangdong Co ltd
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Qinyuan Regenerative Medicine Zhuhai Co ltd
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Abstract

The invention belongs to the technical field of biological medicine, and discloses a chimeric antigen receptor immune cell of an autocrine PD1-TREM2 bispecific antibody and application thereof; wherein the chimeric antigen receptor that secretes a PD1-TREM2 bispecific antibody comprises an antigen binding domain, a spreading domain, an intracellular domain capable of recognizing a tumor antibody, and an element encoding a single chain variable fragment of a PD1-TREM2 antibody. The chimeric antigen receptor cells can specifically identify tumor cells, and simultaneously can enhance the anti-tumor capability of dendritic cells and macrophages in a tumor microenvironment by autocrine PD1-TREM2 bispecific antibodies, so as to further enhance the infiltration of the chimeric antigen receptor cells in tumor tissues, thereby enhancing the anti-tumor capability.

Description

Chimeric antigen receptor immune cell capable of automatically secreting PD1-TREM2 bispecific antibody and application thereof
Technical Field
The invention relates to the technical field of biological medicine, in particular to a chimeric antigen receptor immune cell of an autocrine PD1-TREM2 bispecific antibody and application thereof.
Background
Currently, tumors remain one of the important factors threatening human life health worldwide, and there are three main modes for tumors, including two traditional modes of surgery and radiotherapy and chemotherapy, and the other is immunotherapy. The most hot of immunotherapy is chimeric antigen receptor CAR-T therapy, and has achieved significant achievements in a variety of hematological tumors, such as the treatment of acute lymphoblastic leukemia against the target antigen CD 19; also present contemporaneously are bispecific antibodies that target tumor antigens on one end and immune cells on the other end recruit immune cells to kill tumor cells. Treatment of solid tumors is still slow, mainly due to lack of tumor antigen expression, and complex tumor microenvironment.
PD-1 (programmed cell death, programmed cell death factor) is mainly expressed on the surface of immune cells such as T cells, while a ligand PDL-1 of PD-1 is mainly and highly expressed on tumor cells, and targeting PD-1/PDL-1 to block the binding of the ligand can lead the immune cells to be activated again to attack the tumor cells. And monoclonal antibody drugs against PD-1/PDL-1 have been used in a variety of neoplastic diseases.
But less than 50% of tumor patients have an effect on the treatment of monoclonal antibodies to PD-1. And the monoclonal antibody using PD-1 has large systemic side effect and quick drug resistance. Thus, targeting additional inhibitory molecules in the tumor microenvironment simultaneously acts synergistically to enhance anti-tumor effects.
TREM2 is a receptor that interacts with a number of ligands, many of which are markers of tissue damage. Coding variations in TREM2 increase the risk of alzheimer's disease and other neurodegenerative diseases, TREM2 is also expressed by tumor-infiltrated macrophages, and there is increasing evidence that TREM2 plays a role in tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), and that anti-tumor effects are enhanced when TREM2 is completely knocked out in combination with monoclonal antibodies to PD1, suggesting that anti-TREM 2 and PD1 co-therapy is of great significance for anti-tumor.
At present, a chimeric antigen receptor and related modified immune cell immunotherapy which take a tumor antigen as a target and integrate a bispecific antibody PD-1-TREM2 with self secretion and antagonism at the same time have not been researched and reported.
Disclosure of Invention
The technical problem to be solved by the present invention is to overcome the above-mentioned drawbacks described in the prior art by first providing a chimeric antigen receptor for an autocrine bispecific antibody.
It is a second object of the present invention to provide genes expressing the chimeric antigen receptor of the above-described autocrine bispecific antibody.
It is a third object of the present invention to provide a lentiviral expression vector.
It is a fourth object of the present invention to provide a method for modifying immune cells using chimeric antigen receptors that can auto-secrete PD1-TREM2 bispecific antibodies and target tumor antigens.
A fifth object of the present invention is to provide the use of the immune cells obtained by the above method for preparing an antitumor drug.
The invention aims at realizing the following technical scheme:
a chimeric antigen receptor of an autocrine bispecific antibody is formed by concatenating a CD8 alpha signal peptide, CEAscFV, a CD8 transmembrane region, a CD28 costimulatory molecule, a CD3 zeta intracellular signal region, T2A, a secretory peptide, PD-1scFV, linker, TREM2scFV.
Preferably, the chimeric antigen receptor sequence is as set forth in SEQ ID NO: 1.
Preferably, the amino acid sequence of the CD8 alpha signal peptide is shown in SEQ ID NO:2, the amino acid sequence of the CEAscFV is shown in SEQ ID NO:3, the amino acid sequence of the CD8 transmembrane region is shown as SEQ ID NO:4, the amino acid sequence of the CD28 co-stimulatory molecule is shown in SEQ ID NO:5, the amino acid sequence of the intracellular signal region of the CD3 zeta is shown as SEQ ID NO:6, the amino acid sequence of the T2A is shown as SEQ ID NO:7, the amino acid sequence of the secretory peptide is shown as SEQ ID NO:8, the amino acid sequence of the PD-1scFV is shown in SEQ ID NO:9, the amino acid sequence of the Linker is shown as SEQ ID NO:10, the amino acid sequence of the TREM2scFV is shown in SEQ ID NO: 11.
The invention also provides a lentiviral expression vector comprising a gene sequence expressed by a chimeric antigen receptor.
Preferably, the recombinant plasmid used in the expression vector is pCDH-CMV-MCS-EF 1-copGGFP.
The invention also provides a method for modifying immune cells by using the chimeric antigen receptor which can automatically secrete PD1-TREM2 bispecific antibody and target tumor antigens, which comprises the following steps:
(1) Obtaining a nucleotide sequence of a specific PD1-TREM2 bispecific antibody;
(2) Cloning a nucleotide sequence molecule of a chimeric antigen receptor which can automatically secrete PD1-TREM2 bispecific antibodies and target tumor antigens into a lentiviral expression vector to obtain a TA sPD-1-TREM 2CAR expression plasmid;
(3) Transfecting the expression plasmid into 293T cells, packaging to obtain virus particles, and centrifugally concentrating to obtain virus concentrated solution;
(4) Infecting the immune cells with the lentiviral concentrate to obtain chimeric antigen receptor modified immune cells capable of autocrine PD1-TREM2 and targeting tumor antigens;
(5) The secreted PD1-TREM2 bispecific antibody was purified.
The invention also provides an application of the immune cells obtained by the method in preparing antitumor drugs.
Preferably, in the above application, the tumor is a cancer whose cancer cell surface abnormally expresses CEA.
More preferably, the tumor is a CEA-expressing positive tumor.
More preferably, the cancer is selected from: lung cancer, liver cancer, colon cancer, colorectal cancer, and ovarian cancer.
Compared with the prior art, the invention has the following beneficial effects:
the chimeric antigen receptor modified immune cell (sBsAb CAR for short) of the autocrine bispecific antibody can specifically kill tumor cells with CEA antigen expressed in high degree, and simultaneously autocrine PD1-TREM2 bispecific antibody, thereby improving the inhibition signal of tumor microenvironment, reactivating immune cells and promoting the killing effect on tumor cells. Meanwhile, the half-life period of the bispecific antibody is improved, the concentration of the bispecific antibody at local parts of tumors is high, and the systemic side effect of the monoclonal antibody is avoided.
Drawings
FIG. 1 is a schematic structural diagram of the CAR obtained in example 1;
FIG. 2 is an expression diagram of a CAR in an example;
FIG. 3 is a purification chart of the bispecific antibody of PD1-TREM2 in the example;
FIG. 4 is a CAR of an embodiment CEA T cell, secreted CAR CEA/PD1 T cells and CARs CEA/PD1-TREM2 T cell pairTumor volume effect profile of tumor-bearing mice.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
EXAMPLE 1 Synthesis of CAR-expressed genes
The chimeric antigen receptor of the self-secreting bispecific antibody provided by the invention is formed by connecting a CD8 alpha signal peptide, CEAscFV, a CD8 transmembrane region, a CD28 costimulatory molecule, a CD3 zeta intracellular signal region, T2A, a secretory peptide, PD-1scFV, linker and TREM2scFV in series, and the structure is shown in figure 1; the anti-CEA and PD1, TREM2 single-chain antibody sequences are derived from monoclonal antibody sequences constructed in the laboratory, and are subjected to codon optimization so as to ensure that the expression of T cells is more suitable under the condition that the heterocoded amino acid sequences are unchanged.
EXAMPLE 2 construction of CAR plasmids
Splice of each structure of the CAR in example 1 was accomplished separately using overlap PCR and other molecular cloning techniques to obtain the CAR CEA Secretory CAR CEA/PD1 CAR (CAR) CEA/PD1-TREM2 。
Converting the spliced products into STB3 competent, picking monoclonal culture, extracting plasmid and sequencing to obtain CAR CEA Secretory CAR CEA/PD1 And CAR CEA/PD1-TREM2 。
EXAMPLE 3 packaging of CAR lentiviruses
The steps are as follows:
(1) Expanding and culturing 293T cells with good growth state into a 15cm culture dish, and growing the 293T cells to about 70% when the confluency of the cells is high;
(2) The CAR lentiviral plasmid is transfected to 293T cells by PEI, and then placed into an incubator for culture;
(3) After 48 hours of culture, cell supernatants were collected by centrifugation;
(4) The collected cell supernatant was concentrated with PEG to collect CAR lentiviruses.
Example 4T cell isolation and CAR lentiviral transduction
The T cells are subjected to Ficoll density gradient centrifugation to obtain mouse T cells, IL-2 is added to continue culturing for 48 hours, then collected lentivirus is used for removing infected T cells, and then flow sorting is used for sorting out the T cells with high expression CAR. See fig. 2.
EXAMPLE 5 concentration purification of secreted PD1-TREM2
CAR-T cells from which PD1-TREM2 was secreted were grown in an expanded manner, and the supernatant was collected, and then the bispecific antibody of PD1-TREM2 in the supernatant was purified by a nickel column, and the expression of the bispecific antibody was detected by SDS-PAGE (FIG. 3)
Example 6 killing of tumor-bearing mice by CAR-T cells that autocrine PD1-TREM2
6-week-old C57 healthy mice reached a tumor volume of 200-300mm after 1 week of inguinal subcutaneous injection of MC38 cells 3 Animal experiments were performed: randomly dividing the mice into 3 groups, and injecting PBS and secreted CAR via tail vein at day 0,3,6,9, 12, 15 after tumor formation CEA/PD1 T cells and CARs CEA/PD1-TREM2 T cells, observed changes in tumor volume size during treatment, found: t cells expressing chimeric antigen receptor can inhibit tumor growth, compared to secreted CAR CEA/PD1 T cells, expressing CAR CEA/PD1-TREM2 T cells have greater anti-tumor capacity (fig. 4).
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Sequence listing
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Ala Ser Gly Phe Thr Phe Ser Asn Tyr Tyr Met Ala Trp Val Arg Gln
820 825 830
Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Leu Thr Asn Ser Gly
835 840 845
Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
850 855 860
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
865 870 875 880
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Glu Trp Ala Gly Ser
885 890 895
Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
900 905 910
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
915 920 925
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
930 935 940
Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn Leu
945 950 955 960
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
965 970 975
Tyr Thr Ser Asn Arg Phe Thr Gly Val Pro Ser Arg Phe Ser Gly Ser
980 985 990
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
995 1000 1005
Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Ile Tyr Asn Ser Pro Trp Thr
1010 1015 1020
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys His His His His His
1025 1030 1035
<210> 2
<211> 45
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 3
<211> 233
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
Ala Ile Gly Met Thr Gly Ser Pro Ser Leu Leu Ser Ala Ser Thr Gly
1 5 10 15
Ala Ala Val Thr Ile Ser Cys Ala Ala Ser Gly Ser Ile Ser Ser Thr
20 25 30
Leu Ala Thr Thr Gly Gly Leu Pro Gly Leu Ala Pro Leu Leu Leu Ile
35 40 45
Thr Ala Ala Ser Ser Leu Gly Ser Gly Val Pro Ser Ala Pro Ser Gly
50 55 60
Ser Gly Ser Gly Thr Ala Pro Thr Leu Thr Ile Ser Ser Leu Gly Pro
65 70 75 80
Gly Ala Pro Ala Thr Thr Thr Cys Gly Gly Ser Thr Ser Thr Pro His
85 90 95
Thr Pro Gly Arg Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Glu
100 105 110
Gly Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Phe Ser Asp Tyr
130 135 140
Phe Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
145 150 155 160
Ala His Ile Tyr Thr Lys Ser Tyr Asn Tyr Ala Thr Tyr Tyr Ser Gly
165 170 175
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Arg Ser Met
180 185 190
Val Tyr Leu Gln Met Asn Asn Leu Arg Thr Glu Asp Thr Ala Thr Tyr
195 200 205
Tyr Cys Thr Arg Asp Gly Ser Gly Tyr Pro Ser Leu Asp Phe Trp Gly
210 215 220
Gln Gly Thr Gln Val Thr Val Ser Ser
225 230
<210> 4
<211> 72
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 4
Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro Thr
1 5 10 15
Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser
20 25 30
Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
35 40 45
Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile Thr
50 55 60
Leu Ile Cys Tyr His Arg Ser Arg
65 70
<210> 5
<211> 41
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 5
Asn Ser Arg Arg Asn Arg Leu Leu Gln Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala Pro
20 25 30
Ala Arg Asp Phe Ala Ala Tyr Arg Pro
35 40
<210> 6
<211> 104
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 6
Gln Gln Glu Cys Arg Asp Cys Cys Gln Pro Ala Gly Pro Gln Pro Ala
1 5 10 15
Leu Gln Ala Gln Ser Arg Ala Lys Arg Gly Ile Arg Leu Gly Glu Glu
20 25 30
Ala Gly Ser Gly Ser Arg Asp Gly Arg Gln Thr Ala Glu Glu Glu Glu
35 40 45
Pro Pro Gly Arg Arg Ile Gln Cys Thr Ala Glu Arg Gln Asp Gly Arg
50 55 60
Ser Leu Gln Asp Arg His Lys Arg Arg Glu Ala Glu Arg Gln Gly Ala
65 70 75 80
Arg Trp Pro Leu Pro Gly Ser Gln His Cys His Gln Gly His Leu Cys
85 90 95
Pro Ala Tyr Ala Asp Pro Gly Pro
100
<210> 7
<211> 18
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 7
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
1 5 10 15
Gly Pro
<210> 8
<211> 20
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 8
Met Val Ser Thr Pro Gln Phe Leu Val Phe Leu Leu Phe Trp Ile Pro
1 5 10 15
Ala Ser Arg Leu
20
<210> 9
<211> 244
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 9
Arg Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Glu Gly Ser Leu
1 5 10 15
Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr Phe Met
20 25 30
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala His
35 40 45
Ile Tyr Thr Lys Ser Tyr Asn Tyr Ala Thr Tyr Tyr Ser Gly Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Arg Ser Met Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Arg Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Thr Arg Asp Gly Ser Gly Tyr Pro Ser Leu Asp Phe Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser
130 135 140
Val Asn Val Gly Glu Thr Val Lys Ile Thr Cys Ser Gly Asp Gln Leu
145 150 155 160
Pro Lys Tyr Phe Ala Asp Trp Phe His Gln Arg Ser Asp Gln Thr Ile
165 170 175
Leu Gln Val Ile Tyr Asp Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu
180 185 190
Arg Ile Ser Gly Ser Ser Ser Gly Thr Thr Ala Thr Leu Thr Ile Arg
195 200 205
Asp Val Arg Ala Glu Asp Glu Gly Asp Tyr Tyr Cys Phe Ser Gly Tyr
210 215 220
Val Asp Ser Asp Ser Lys Leu Tyr Val Phe Gly Ser Gly Thr Gln Leu
225 230 235 240
Thr Val Leu Gly
<210> 10
<211> 16
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 10
Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 11
<211> 241
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 11
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Leu Thr Asn Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Glu Trp Ala Gly Ser Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
130 135 140
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
145 150 155 160
Gln Asn Val Gly Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
165 170 175
Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Asn Arg Phe Thr Gly Val
180 185 190
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
195 200 205
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg
210 215 220
Ile Tyr Asn Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
225 230 235 240
Lys

Claims (6)

1. A chimeric antigen receptor for an autocrine bispecific antibody, which is formed by a cascade of a CD8 a signal peptide, a CEAscFV, a CD8 transmembrane region, a CD28 costimulatory molecule, a CD3 zeta intracellular signal region, T2A, a secretory peptide, a PD-1scfv, a linker, a tr 2 scfv; the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 1.
2. A lentiviral expression vector comprising the amino acid sequence of claim 1.
3. The expression vector of claim 2, wherein the recombinant plasmid used is pCDH-CMV-MCS-EF1-copGFP.
4. A method of modifying immune cells using the chimeric antigen receptor that can auto-secrete PD1-TREM2 bispecific antibody of claim 1 and target a tumor antigen, comprising the steps of:
(1) Obtaining a nucleotide sequence of a specific PD1-TREM2 bispecific antibody;
(2) Cloning a nucleotide sequence molecule of a chimeric antigen receptor which can automatically secrete PD1-TREM2 bispecific antibodies and target tumor antigens into a lentiviral expression vector to obtain a TA sPD-1-TREM 2CAR expression plasmid;
(3) Transfecting the expression plasmid into 293T cells, packaging to obtain virus particles, and centrifugally concentrating to obtain virus concentrated solution;
(4) Infecting the immune cells with the lentiviral concentrate to obtain chimeric antigen receptor modified immune cells capable of autocrine PD1-TREM2 and targeting tumor antigens;
(5) The secreted PD1-TREM2 bispecific antibody was purified.
5. The use of the immune cells obtained by the method of claim 4 for preparing an antitumor drug.
6. The use according to claim 5, wherein the tumor is selected from lung cancer, liver cancer, colon cancer, colorectal cancer, ovarian cancer.
CN202111063869.7A 2021-09-10 2021-09-10 Chimeric antigen receptor immune cell capable of automatically secreting PD1-TREM2 bispecific antibody and application thereof Active CN113929785B (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111032085A (en) * 2017-06-05 2020-04-17 昆士兰医学研究所理事会 Combination of an immune checkpoint antagonist and a RANK-L (NF-KB ligand) antagonist or bispecific binding molecules thereof for use in cancer treatment or prevention and uses thereof
CN112119097A (en) * 2018-04-13 2020-12-22 艾菲默德有限责任公司 Natural killer cell-conjugated antibody fusion constructs
CN112424357A (en) * 2018-06-26 2021-02-26 协和麒麟株式会社 Antibodies that bind to chondroitin sulfate proteoglycan 5
CN113194994A (en) * 2018-12-11 2021-07-30 开拓免疫医疗公司 Methods of using anti-TREM 2 antibodies

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111032085A (en) * 2017-06-05 2020-04-17 昆士兰医学研究所理事会 Combination of an immune checkpoint antagonist and a RANK-L (NF-KB ligand) antagonist or bispecific binding molecules thereof for use in cancer treatment or prevention and uses thereof
CN112119097A (en) * 2018-04-13 2020-12-22 艾菲默德有限责任公司 Natural killer cell-conjugated antibody fusion constructs
CN112424357A (en) * 2018-06-26 2021-02-26 协和麒麟株式会社 Antibodies that bind to chondroitin sulfate proteoglycan 5
CN113194994A (en) * 2018-12-11 2021-07-30 开拓免疫医疗公司 Methods of using anti-TREM 2 antibodies

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