CN113528436A

CN113528436A - Lymphocyte-based homologous targeting artificial antigen presenting cell and construction and application thereof

Info

Publication number: CN113528436A
Application number: CN202110891900.XA
Authority: CN
Inventors: 刘庄; 孙乐乐
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-08-04
Filing date: 2021-08-04
Publication date: 2021-10-22
Anticipated expiration: 2041-08-04
Also published as: CN113528436B; WO2023010777A1

Abstract

The invention relates to a lymphocyte-based homologous targeting artificial antigen presenting cell, which takes lymphocytes as a carrier and also comprises a compound and a co-stimulatory molecule which are coupled on the surfaces of the lymphocytes and are formed by tumor antigen peptides and a major histocompatibility complex I. The lymphocyte-based homologous targeting artificial antigen presenting cell can specifically migrate to peripheral lymphoid organs in vivo, so that the antigen-specific CD8 positive T cells of an organism are activated, and the lymphocyte-based homologous targeting artificial antigen presenting cell has a potential application prospect in the aspect of preparing tumor immunotherapy medicaments.

Description

Lymphocyte-based homologous targeting artificial antigen presenting cell and construction and application thereof

Technical Field

The invention relates to the technical field of immunotherapy, in particular to a lymphocyte-based homologous targeting artificial antigen presenting cell and construction and application thereof.

Background

Tumors are a malignant disease that threatens human survival. With the aging of the world population and the change of living environment and living habits, the number of people who die due to tumors is rapidly increasing every year. At present, chemotherapy and radiotherapy are the main methods for treating tumors clinically except for surgical resection, but have strong toxic and side effects. Cancer immunotherapy is a research hotspot in recent years. Antigen-specific CD8 positive T cell activation plays an important role in tumor immunotherapy. In this process, the presentation of tumor-specific antigens by Antigen Presenting Cells (APCs) to T cells via the class I major histocompatibility complex (MHC-I) is a critical step. Previous studies have shown that tumor antigens taken up by professional APCs such as Dendritic Cells (DCs) require cross-presentation before they can be presented to the cell surface as antigenic peptide-class I major histocompatibility complex (pMHC-I). However, the efficiency of spontaneous antigen cross-presentation is limited, and therefore DC cell vaccines have been developed to activate tumor antigen-specific CD 8-positive T cells in patients. The DC cell vaccine is prepared by firstly obtaining DC cells by inducing the differentiation of autologous stem cells and then enhancing the cross presentation of tumor specific antigens in vitro through manual intervention. This is an expensive and labor intensive process, and such DC vaccines are cross-presented by manual induction, making it difficult to ensure product batch stability.

Artificial antigen presenting cells (aapcs) are mimics of natural APCs, used for T cell expansion in vitro, and play an important role in adoptive immunotherapy. aAPCs are constructed by chemically modifying T cell activating ligands, including pMHC (T cell receptor activating ligand) and costimulatory molecules, onto the surface of biocompatible materials. However, most aAPCs reported previously were constructed using synthetic nano-or micro-sized particles as matrix material, and the ligand distribution on these aAPCs was very different from that on natural APCs. Zhuang Liu et al (DNA-amplified Ligand Positioning on Red Blood Cells to Enable Optimized T Cell Activation for additive immunological analysis. Angew. chem. int. Ed.2020,59, 14842-14853.) an aAPC was constructed using Red Blood Cells (RBC) using lipid-functionalized DNA-induced self-assembly techniques. However, none of these aAPCs specifically migrate to T cell-rich regions in peripheral lymphoid tissues that favor T cell proliferation, and thus optimal autoantigen-specific T cell activation in vivo is not achieved.

Disclosure of Invention

In order to solve the technical problems, the invention provides a lymphocyte-based homologous targeting artificial antigen presenting cell which can specifically migrate to peripheral lymphoid organs in vivo, so that the antigen-specific CD8 positive T cells of an organism are activated, tumor cells are further killed, tumor growth is inhibited, and the life cycle is prolonged.

The invention relates to a lymphocyte-based homologous targeting artificial antigen presenting cell, which takes lymphocytes as a carrier and also comprises a compound and a co-stimulatory molecule which are coupled on the surfaces of the lymphocytes, wherein the compound is formed by tumor antigen peptide and a major histocompatibility complex I.

Neither traditional synthetic-based aapcs nor RBC-based aapcs are able to specifically migrate to T cell-rich regions in peripheral lymphoid tissue, which are favorable sites for T cell expansion. The artificial antigen presenting cell constructed by taking the lymphocyte as a carrier can migrate to peripheral lymph organs including spleen and lymph nodes in a targeted manner through a homing receptor on the surface of the lymphocyte, so that the artificial antigen presenting cell is in close contact with T cells in the peripheral lymph organs, and further effectively activates the antigen-specific CD8 positive T cells of an organism.

Further, in the lymphocyte-based homologous targeting artificial antigen presenting cell, the lymphocyte and the compound are connected through double-stranded DNA, wherein the lymphocyte and the double-stranded DNA are connected through lipid, and the compound and the double-stranded DNA are combined through non-covalent interaction;

lymphocytes are linked to a co-stimulatory molecule by a fully complementary double stranded DNA, wherein the lymphocytes are linked to one strand of the double stranded DNA by a lipid and the co-stimulatory molecule is linked to the other strand of the double stranded DNA.

Further, the complex is linked to the double-stranded DNA via biotin and avidin. The DNA and the major histocompatibility complex I can also be covalently linked, for example, a thiol group is modified at one end of the DNA, and the DNA is coupled with the amino group on the surface of the protein by a common small molecule bifunctional cross-linking agent (SMCC, sulfo-SMCC, SPDP and the like). However, in order to achieve a distribution that mimics the major histocompatibility complex of the natural APC surface, a biotin-avidin linkage was chosen for assembly of major histocompatibility complex I. If the major histocompatibility complex I is covalently modified by DNA and then assembled into cells, it is impossible to mimic the distribution of the major histocompatibility complex on the surface of natural APC.

Further, the co-stimulatory molecule may be covalently or non-covalently bound to another strand of DNA, preferably the co-stimulatory molecule is linked to the other strand of DNA by avidin and biotin.

Furthermore, the surface of the lymphocyte is modified with single-stranded DNA1 and single-stranded DNA2 respectively, wherein the single-stranded DNA1 is double-modified by lipid and biotin and is connected with a single-stranded cDNA1 which is completely complementary with the single-stranded DNA, the single-stranded DNA2 is modified by lipid and is connected with a single-stranded cDNA2 which is completely complementary with the single-stranded DNA, the single-stranded cDNA2 is modified by biotin, the compound is modified by biotin, and the co-stimulatory molecule is modified by biotin; the single-stranded DNA1 is specifically bound to the complex through avidin; the single stranded cDNA2 is specifically bound to a costimulatory molecule by avidin.

In the prior art, modification of T cell activating ligands on lymphocyte surfaces by general chemical coupling methods affects the function of lymphocyte surface receptor proteins, particularly the function of homing receptors. In addition, coupling of T cell activating ligands on the surface of living cells by general chemical methods also fails to regulate the distribution of T cell activating ligands, and thus fails to mimic the distribution profile of T cell activating ligands on the surface of natural antigen presenting cells. In the invention, a compound formed by tumor antigen peptide and major histocompatibility complex I and co-stimulation molecules are assembled on the surface of lymphocyte through DNA, lipid, avidin and biotin, the distribution appearance of the surface T cell activation ligand is closer to that of natural antigen presenting cells, and the spatial distribution of the activation ligand on aAPCs can be accurately controlled by simulating natural APCs, thereby realizing the optimization of T cell activation.

Further, lipids include sterols and phospholipids, such as cholesterol, sphingomyelin, gangliosides, or long-chain alkanes, and the like.

Further, biotin is a biotin reactive with amino groups, such as N-hydroxysuccinimide biotin (the amino group on the major histocompatibility complex I and costimulatory molecule forms a covalent bond with N-hydroxysuccinimide on N-hydroxysuccinimide biotin) or carboxy-functionalized biotin (EDC mediated coupling of the amino and carboxy groups on the major histocompatibility complex I and costimulatory molecule).

Further, in the single-stranded DNA1, lipid modification is at the 5 'end of DNA1, and biotin modification is at the 3' end of DNA 1; in the single-stranded cDNA2, biotin was modified at the 5' end of cDNA 2; in single-stranded DNA2, the lipid modification is at the 5' end of DNA 2.

Further, lymphocytes are derived from autologous peripheral blood. Peripheral blood lymphocytes mainly include T cells and B cells, and peripheral blood taken from the body is intended to alleviate rejection.

Further, the co-stimulatory molecule is selected from one or more of CD28 antibody, CD40 antibody, OX4O antibody, CD27 antibody, CD137 antibody and GITR antibody, preferably CD28 antibody.

Further, the tumor antigen peptide is selected from one or more of peptides of tumor-associated antigens, autoantigens, alloantigens and infectious agent antigens, such as OVA_257-264Mc38 tumor antigen peptide.

Further, the sequence length of the single-stranded DNA2 and the single-stranded DNA1 is 15-60 bases, preferably 20-30 bases, so that the stability of DNA hybridization is ensured.

The invention claims the application of the lymphocyte-based homologous targeting artificial antigen presenting cell in the preparation of tumor immunotherapy drugs.

Furthermore, when the lymphocyte-based homologous targeting artificial antigen presenting cell is applied to preparing a tumor immunotherapy medicament, the tumor antigen peptide is an antigen peptide screened from a tumor or an antigen peptide commonly existing in the same type of tumor.

The tumor treatment preparation comprises the lymphocyte-based homologous targeting artificial antigen presenting cell.

Further, the tumor treatment preparation further comprises a PD1 antibody.

Furthermore, tumor treatment is performed in multiple times, and multiple immunizations can stimulate stronger immune response.

Further, the administration mode of the tumor therapeutic agent is intravenous injection.

Further, the tumor therapeutic agent can be used for treating colon cancer, melanoma, breast cancer, lung cancer, head and neck tumor, gastric cancer, bladder cancer, bone cancer, laryngeal cancer, liver cancer, pancreatic cancer or nasopharyngeal cancer.

The method for constructing the artificial antigen presenting cell of the present invention comprises the following steps:

(1) reacting the major histocompatibility complex I with biotin in a buffer solution to obtain a biotin-modified major histocompatibility complex I, and incubating and combining the biotin-modified major histocompatibility complex I with tumor antigen peptide to obtain a complex formed by the tumor antigen peptide and the major histocompatibility complex I;

reacting the costimulatory molecule with biotin in a buffer solution to obtain biotin-modified costimulatory molecule, incubating and combining the biotin-modified costimulatory molecule with avidin to obtain avidin-modified costimulatory molecule, and incubating and combining the avidin-modified costimulatory molecule with biotin-modified single-stranded cDNA2 to obtain cDNA 2-modified costimulatory molecule;

incubating and combining lymphocytes with lipid and biotin double-modified single-stranded DNA1 and cDNA1 in a buffer solution to obtain DNA modified lymphocytes, and incubating and combining the DNA modified lymphocytes with avidin in the buffer solution to obtain avidin modified lymphocytes;

(2) incubating and combining the avidin modified lymphocyte, a compound formed by a tumor antigen peptide and a major histocompatibility complex I and lipid modified single-stranded DNA2 in a buffer solution to obtain a compound and a DNA2 double-modified lymphocyte;

(3) and (3) incubating and combining the compound and the lymphocyte doubly modified by the DNA2 and the co-stimulatory molecule modified by the cDNA2 in a buffer solution to obtain the lymphocyte-based homologous targeting artificial antigen presenting cell. The schematic construction flow is shown in FIG. 1.

Further, in step (1), the molar ratio of major histocompatibility complex I to biotin is 1: 20-40, preferably 1: 20.

further, in step (1), the molar ratio of co-stimulatory molecule to biotin is 1: 10-20. Preferably 1: 10.

further, in step (1), the molar ratio of biotin-modified co-stimulatory molecule to avidin is 1: 3-5, preferably 1: 3.

further, in step (1), the molar ratio of the avidin-modified co-stimulatory molecule to the biotin-modified single-stranded cDNA2 was 1: 6-10, preferably 1: 6.

further, in step (1), the molar ratio of lymphocytes to lipid and biotin double-modified single-stranded DNA1 was 1: 1X 10⁷-3×10⁷Preferably 1: 1X 10⁷。

Further, in step (1), the molar ratio of lymphocytes to cDNA1 was 1: 2X 10⁷-6×10⁷Preferably 1: 2X 10⁷。

Further, in step (1), the molar ratio of DNA-modified lymphocytes to avidin is 1: 5X 10⁷-1.5×10⁸Preferably 1: 5X 10⁷。

Further, in step (2), the molar ratio of the avidin-modified lymphocytes, the complex formed by the tumor antigen peptide and the major histocompatibility complex I, and the lipid-modified single-stranded DNA2 was 1: 1X 10⁴-5×10⁶：5×10⁶-1×10⁷. Preferably 1: 1X 10⁴：5×10⁶。

Further, in step (3), the molar ratio of the complex to the DNA2 double modified lymphocyte, cDNA2 modified co-stimulatory molecule is 1: 1X 10⁴-1×10⁶。

In the preparation process of the DNA engineering lymphocyte-based homologous targeting artificial antigen presenting cell, the major histocompatibility complex I and the costimulatory molecule are subjected to biotin modification by forming covalent bonds between amino groups on the major histocompatibility complex I and the costimulatory molecule and biotin. Lymphocyte cells are DNA modified by spontaneous binding of lipids to the cell membrane. The co-stimulatory molecule is DNA modified by avidin-biotin specific binding. The co-stimulatory molecules are coupled to the lymphocyte cell surface by DNA-specific hybridization. The complex formed by the tumor antigen peptide and the major histocompatibility complex I is coupled to the surface of the lymphocyte through the avidin-biotin specific binding, and aggregation occurs due to the multiple avidin-biotin specific binding, thereby simulating the distribution morphology of the major histocompatibility complex I on the surface of the natural antigen presenting cell.

By the scheme, the invention at least has the following advantages:

(1) the invention provides a lymphocyte-based homologous targeting artificial antigen presenting cell, which can migrate to a peripheral lymph organ in a targeted manner in vivo and is in close contact with T cells in the peripheral lymph organ, thereby being more beneficial to the activation of antigen-specific T cells.

(2) Compared with a vaccine technology based on natural antigen presenting cells, the lymphocyte-based homologous targeting artificial antigen presenting cells are easier to control the number of surface T cell activating ligands, so that products with stable quality in batches are prepared. In addition, compared with the vaccine technology based on natural antigen presenting cells, the invention does not need processes such as cell culture and antigen cross induction, so the cost is lower, and the treatment can be applied to patients in shorter time.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.

FIG. 1 is a schematic diagram of the construction process of lymphocyte-based homologous targeting artificial antigen presenting cells of the present invention;

FIG. 2 is the imaging of laser confocal scanning microscope for assembling pMHC-I and CD28 antibodies on the surface of lymphocytes in example 1;

FIG. 3 is a graph showing the results of lymphocyte-based targeting of the artificial antigen-presenting cells for in vitro antigen-specific T cell expansion in example 2;

FIG. 4 is a graph showing the results of using the lymphocyte-based homologously targeting artificial antigen-presenting cells in example 3 for peripheral lymphoid organ targeting in vivo;

FIG. 5 is a graph showing the results of lymphocyte-based targeting of an artificial antigen-presenting cell for stimulating the activation of exogenous antigen-specific T cells in vivo in example 4;

FIG. 6 is a graph showing the results of lymphocyte-based targeting of the artificial antigen-presenting cells for stimulating the activation of endogenous antigen-specific T cells in vivo in example 5;

FIG. 7 is a graph of the results of personalized immunotherapy based on lymphocyte-based, cognate targeting artificial antigen presenting cells in combination with immune checkpoint inhibitors for the mouse B16-OVA melanoma model in example 6;

FIG. 8 is a graph of the results of the immune memory function evaluation of the mice cured in the chemotherapy sensitization of the mice Mc38 colon carcinoma subcutaneous tumor model by the lymphocyte-based targeting artificial antigen presenting cells combined with immune checkpoint inhibitors in example 7.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

Preparation and characterization of lymphocyte-based homologous targeting artificial antigen presenting cells for mouse B16-OVA tumor model treatment

(1) A20-fold excess of N-hydroxysuccinimide atto488 and N-hydroxysuccinimide biotin was added sequentially to 50 μ L of 0.5mg/mL major histocompatibility complex I (MHC-I). After 1 hour incubation at 4 ℃, excess atto488 and biotin were removed by two ultrafiltration runs using a 100KDa ultrafiltration tube. The concentration of modified MHC-I was then detected by BCA quantification kit. The average number of atto488 on one MHC-I was determined by uv-visible absorption.The average number of biotin on one MHC-I was estimated to be the same as atto 488. To prepare a complex of antigenic peptide and MHC-I (pMHC-I), biotin and atto 488-labeled MHC-I were combined with OVA_257-264The peptides were incubated at 4 ℃ for 8 hours. Excess peptide was then removed by two ultrafiltration runs using a 100kDa ultrafiltration tube. The concentration of pMHC-I was measured by BCA protein quantification kit.

(2) A10-fold excess of N-hydroxysuccinimide alexa647 and N-hydroxysuccinimide biotin was added to 50 μ L of 1mg/mL CD28 antibody (aCD 28). Purification of the modified aCD28 was performed in the same manner as described above for the modified pMHC-I. The concentration of modified aCD28 was determined by BCA. The average number of alexa647 on one aCD28 was measured by uv-visible absorption. The average amount of biotin on one CD28 antibody was estimated to be the same as alexa 647. To ligate the DNA to aCD28, aCD28-alexa 647-biotin was first incubated with a 4-fold excess of avidin at 4 ℃ for 30 minutes. Thereafter, biotin-labeled DNA2(5' biotin-cDNA2) was added to an antibody solution in an equimolar amount to avidin, followed by incubation for 30 minutes.

(3) Peripheral blood was collected from the mouse orbit using a capillary tube, and density gradient centrifugation was performed using a mouse peripheral blood leukocyte separation kit according to the manufacturer's instructions to obtain lymphocytes. After counting with a cytometer, lymphocytes (1X 10)⁷) First incubated with 5 'cholesterol and 3' biotin modified DNA1(5'chol-DNA1-3' biotin) (2. mu.L, 100. mu.M) and cDNA1 (molar ratio of DNA1 to cDNA1 1:2) at 4 ℃ for 15 min. After removing excess DNA by centrifugation, lymphocytes were resuspended in PBS and counted.

(4) Cells were incubated with a 5-fold excess of avidin for 30 minutes at 4 ℃. After removing excess avidin by two centrifugations (1300rpm 3min), lymphocytes were resuspended in PBS and counted by a cell counter.

(5) Avidin-labeled lymphocytes were mixed with atto488 and biotin-labeled pMHC-I and 5' chol-DNA2 at 1: 5X 10, respectively⁵And 1: 5X 10⁶After incubation at 4 ℃ for 30 minutes, excess biotin was added to block unbound sites on avidin. Subsequently, by centrifugation (130)0rpm3min) to remove excess pMHC-I and 5' chol-DNA 2.

(6) The lymphocytes were resuspended in PBS buffer and mixed with 5' biotin-cDNA2 modified aCD28 at a molar ratio of 1: 5X 10⁵Mixing was performed and incubation was performed at 4 ℃ for 1 hour. Excess aCD28 was removed by two centrifugations (1300rpm 3 min).

(7) The confocal laser scanning microscope imaging is utilized to prove whether the lymphocyte surface is successfully coupled with pMHC-I and aCD28, and the distribution of pMHC-I is observed.

Among them, the sequence information of DNA1, cDNA1, DNA2 and cDNA2 is shown in Table 1.

TABLE 1 sequence information

As shown in FIG. 2, the imaging results of the confocal laser scanning microscope on the lymphocyte-based homologous targeting artificial antigen presenting cells showed that pMHC-I and aCD28 were successfully coupled to the lymphocyte surface, and the distribution of pMHC-I was in an aggregated state, simulating the distribution of pMHC-I on the surface of natural antigen presenting cells.

Example 2

Lymphocyte-based homologous targeting artificial antigen presenting cell for in vitro antigen specific T cell amplification

(1) One transgenic OT-I mouse was sacrificed and the spleen was then removed and placed in a petri dish and pre-cooled PBS was added. The spleen was ground with a syringe handle, and the tissue disruption was collected in a centrifuge tube, centrifuged at 1300rpm, the pellet collected, and 3mL of erythrocyte lysate was added. After 3min, 3mL PBS was added to stop lysis. After centrifugation, the suspension was resuspended in PBS and filtered through 400 mesh sterile gauze. Splenocytes obtained after centrifugation (1300rpm 3) were resuspended in 2mL PBS and counted in a cell counter. 2 x 10 to⁷Individual splenocytes were suspended in 10mL PBS containing 1 μ L8 mM CFSE. The cell suspension was then incubated for 15 minutes at 37 ℃ in a cell incubator. The CFSE stained cells were then collected by centrifugation (1300rpm for 5min) and resuspended 1640 medium.

(2) 2 x 10 to⁵CFSE-stained OT-I mouse splenocytes were suspended in 250. mu.L 1640 medium and incubated with lymphocyte-based, cognate, artificial antigen-presenting cells constructed according to the procedure described in example 1 in a round-bottom 96-well plate at a cell ratio of 1: 1. As a control, 2X 10 was used⁵CFSE-stained OT-I mouse splenocytes were suspended in 250. mu.L 1640 medium and incubated with free pMHC-I and aCD28, pMHC-I only coupled lymphocytes, and aCD28 only coupled lymphocytes, respectively. After 3 days of incubation, cells were harvested by centrifugation (1300rpm 3min) and the supernatant was stored at-20 ℃ for cytokine detection. The ELISA kits were used to detect IFN γ and TNF α according to the manufacturer's instruction manual.

(3) CD8 positive T cell proliferation was detected by flow cytometry. Splenocytes were resuspended in buffer and CD8 positive T cells were labeled with PE-labeled CD3 antibody, Percp-labeled CD8 antibody. PE and Percp double positive cells were gated as CD8+ T cells. Cell proliferation was analyzed by CFSE fluorescence dilution degree.

The results are shown in fig. 3 (a is a flow chart, B is a flow chart, and C is a chart of the cell factor concentration test results of the culture supernatant), and it can be seen that the lymphocyte-based homologous targeting artificial antigen presenting cells can efficiently stimulate the antigen-specific CD8 positive T cells.

Example 3

Lymphocyte-based homologous targeting artificial antigen presenting cells for peripheral lymphoid organ targeting in vivo

(1) Lymphocyte-based, cognate targeting artificial antigen-presenting cells prepared according to the procedure described in example 1 were stained with the cell membrane dye DID (λ Ex/λ Em ═ 644/663 nm).

(2) In a proportion of 1X 10 each⁷Individual numbers were injected into mice via tail vein. After 3 days, the mice were sacrificed and spleen and lymph nodes were taken to prepare cell suspensions. Cells were then stained with FITC-labeled CD3 antibody (aCD3-FITC) and PE-labeled CD19 antibody (aCD19-PE) for flow cytometry analysis.

The results are shown in FIG. 4 (A-B is a flow chart showing that lymphocyte-based cognate targeting artificial antigen presenting cells are enriched in spleen, C corresponds to the statistical results of A-B, D-E is a flow chart showing that lymphocyte-based cognate targeting artificial antigen presenting cells are enriched in lymph nodes, F corresponds to the statistical results of D-E), indicating that DNA-engineered lymphocyte-based cognate targeting artificial antigen presenting cells can efficiently migrate to peripheral lymphoid organs including spleen and lymph nodes.

Example 4

Lymphocyte-based homologous targeting artificial antigen presenting cells for stimulating exogenous antigen-specific T cell activation in vivo

(1)2×10⁷Splenocytes from individual CFSE-stained OT-I mice were injected via tail vein into C57 mice, and one day later, 5X 10 cells were injected⁶DNA-engineered lymphocyte-based cognate targeting artificial antigen presenting cells prepared according to example 1 were also injected into mice via tail vein, with free pMHC-I and aCD28 as controls.

(2) Spleen and lymph nodes of C57 mice were harvested 6 days later and spleen cell and lymph node cell suspensions were prepared. The 2X 10 pairs were then labeled with PE-labeled CD3 antibody and Percp-labeled CD8 antibody⁶Individual spleen cells or lymph node cell suspensions were stained. And then analyzed by flow cytometry after removing excess antibody. PE positive cell populations were scored as T cells and both Percp and CFSE positive cells were used to detect the extent of CFSE dilution.

The results are shown in fig. 5(a is a flow chart of proliferation of exogenous antigen-specific T cells in spleen under different treatments, B corresponds to the statistical results of the results in fig. a, C is a flow chart of proliferation of exogenous antigen-specific T cells in lymph node under different treatments, D corresponds to the statistical results of the results in fig. C), compared to the control group, the lymphocyte-based homologous targeting artificial antigen presenting cells can effectively stimulate the activation of antigen-specific CD8 positive T cells derived from OT-I mice.

Example 5

Lymphocyte-based homologous targeting artificial antigen presenting cells for stimulating endogenous antigen-specific T cell activation in vivo

(1) Will be 5X 10⁶An according toThe lymphocyte-based, cognate targeting artificial antigen presenting cells prepared in example 1 were injected directly into C57 mice via tail vein.

(2) Spleen cells were prepared 3 days later from spleen of C57 mouse. After CFSE staining, 2X 10⁵Each spleen cell was resuspended in 250. mu.L 1640 medium and cultured in round bottom 96 well plates at 37 ℃. With or without subsequent addition of OVA_257-264Peptide (1. mu.g/well). After 3 days of culture, cells were harvested and CD8 positive T cells were labeled with PE-labeled CD3 antibody and Percp-labeled CD8 antibody. Then analyzed by flow cytometry after removal of excess antibody

The results are shown in FIG. 6 (A is a flow chart showing the proliferation of CD8 positive T cells after the spleen cells of mice without the artificial antigen presenting cells injected with and without the antigenic peptide stimulation, B is a flow chart showing the proliferation of CD8 positive T cells after the spleen cells of mice with the artificial antigen presenting cells injected with and without the antigenic peptide stimulation, and C corresponds to the statistical results of the graphs A and B), compared with the control group, the lymphocyte-based homologous targeting artificial antigen presenting cells can effectively activate endogenous antigen-specific CD8 positive T cells.

Example 6

Lymphocyte-based homologous targeting artificial antigen presenting cells combined with immune checkpoint inhibitors for personalized immunotherapy of mouse B16-OVA melanoma model

By subcutaneous injection of 1X 10 in the back of female C57 mice⁶A mouse B16-OVA melanoma model was inoculated with B16-OVA cells (suspended in 50. mu.L PBS). 10 days after tumor inoculation, mice were divided into 4 groups for treatment: 1. no treatment; PD1 antibody treatment; 3. lymphocyte-based, cognate targeting artificial antigen presenting cell therapy prepared according to example 1; 4. lymphocyte-based, cognate targeting artificial antigen presenting cells prepared according to example 1 were treated in combination with PD1 antibody. The first input of artificial antigen presenting cells was performed on day 10 and the second input was performed on day 16 after tumor inoculation. PD1 antibody was injected intravenously at a dose of 15 μ g per mouse on

days

12, 15, and 18. During this period, tumor volume and survival rate of different groups of mice were closely monitored.

As shown in FIG. 7(A is a treatment flow chart, B is a tumor growth curve of mice in different treatment groups, and C is a survival curve of mice in different treatment groups), the lymphocyte-based homologous targeting artificial antigen presenting cell prepared according to example 1 can effectively inhibit tumor growth and prolong the survival time of mice when being combined with PD1 antibody.

Example 7

Lymphocyte-based homologous targeting artificial antigen presenting cells combined with immune checkpoint inhibitors for immunotherapy of mouse Mc38 model

(1) Preparation of lymphocyte-based homologous targeting Artificial antigen presenting cells was performed according to the procedure described in example 1, except that OVA was used_257-264The peptide was replaced with the Mc38 neoantigenic peptide with the sequence SIIVFNLL (from nitrogen to carbon).

(2) By subcutaneous injection of 1X 10 in the back of female C57 mice⁶A mouse Mc38 colon cancer model was inoculated with Mc38 tumor cells (suspended in 50. mu.L PBS). 5 days after tumor inoculation, mice were divided into 4 groups for treatment: 1. no treatment; PD1 antibody treatment; DNA engineered lymphocyte-based cognate targeting artificial antigen presenting cell therapy; DNA engineered lymphocyte-based cognate targeting artificial antigen presenting cells in combination with PD1 antibody therapy. The first reinfusion of artificial antigen presenting cells was performed on day 5 after tumor inoculation and the second reinfusion was performed on day 11. On

days

7, 10 and 13, PD1 antibody was injected intravenously at a dose of 15 μ g per mouse. During this period, tumor volume and survival rate of different groups of mice were closely monitored.

The results are shown in fig. 8(a is a treatment flow chart, B is a tumor growth curve of mice in different treatment groups, and C is a survival curve of mice in different treatment groups), and it can be seen that the lymphocyte-based homologous targeting artificial antigen presenting cell combined with the PD1 antibody treatment can effectively inhibit tumor growth and prolong the survival period of mice.

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 of the invention may be made without departing from the spirit or scope of the invention.

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Claims

1. The lymphocyte-based homologous targeting artificial antigen presenting cell is characterized in that the lymphocyte-based homologous targeting artificial antigen presenting cell takes lymphocytes as carriers, and further comprises a compound and a costimulatory molecule which are coupled on the surfaces of the lymphocytes, wherein the compound is formed by a tumor antigen peptide and a major histocompatibility complex I.

2. The artificial antigen presenting cell according to claim 1, wherein:

in the lymphocyte-based homologous targeting artificial antigen presenting cell, the lymphocyte and the compound are connected through double-stranded DNA, wherein the lymphocyte and the double-stranded DNA are connected through lipid, and the compound and the double-stranded DNA are combined through non-covalent action;

the lymphocyte and the costimulatory molecule are connected through a double-stranded DNA which is completely complementary, wherein, the lymphocyte and one strand of the double-stranded DNA are connected through lipid, and the costimulatory molecule and the other strand of the double-stranded DNA are connected.

3. The artificial antigen presenting cell according to claim 1, wherein:

the surface of the lymphocyte is modified with single-stranded DNA1 and single-stranded DNA2 respectively, wherein the single-stranded DNA1 is double-modified by lipid and biotin and is connected with a single-stranded cDNA1 which is completely complementary with the single-stranded DNA, the single-stranded DNA2 is modified by lipid and is connected with a single-stranded cDNA2 which is completely complementary with the single-stranded DNA, the single-stranded cDNA2 is modified by biotin, the compound is modified by biotin, and the co-stimulatory molecule is modified by biotin;

the single-stranded DNA1 is specifically bound to the complex by avidin;

the single stranded cDNA2 specifically binds to the costimulatory molecule via avidin.

4. The artificial antigen presenting cell according to claim 1, wherein: the lymphocytes are derived from autologous peripheral blood.

5. The artificial antigen presenting cell according to claim 1, wherein: the co-stimulatory molecule is one or more selected from the group consisting of a CD28 antibody, a CD40 antibody, an OX40 antibody, a CD27 antibody, a CD137 antibody and a GITR antibody.

6. The artificial antigen presenting cell according to claim 1, wherein: the tumor antigen peptide is selected from one or more of tumor-associated antigen peptide, autoantigen peptide, allotype antigen peptide and infectious agent antigen peptide.

7. Use of the lymphocyte-based, homology-targeting artificial antigen-presenting cell according to any one of claims 1 to 6 for the preparation of a medicament for immunotherapy of tumors.

8. A formulation for the treatment of tumors, characterized by: the tumor therapy preparation comprises the lymphocyte-based homologous targeting artificial antigen presenting cell according to any one of claims 1 to 6.

9. The tumor therapeutic formulation of claim 8, wherein: the tumor therapy preparation further comprises a PD1 antibody.

10. The method for constructing an artificial antigen-presenting cell according to claim 3, comprising the steps of:

(2) incubating and combining the avidin-modified lymphocyte, the complex formed by the tumor antigen peptide and the major histocompatibility complex I and the lipid-modified single-stranded DNA2 in a buffer solution to obtain a complex and a DNA2 double-modified lymphocyte;

(3) and (3) incubating and combining the compound and the lymphocyte doubly modified by the DNA2 and the co-stimulatory molecule modified by the cDNA2 in a buffer solution to obtain the lymphocyte-based homologous targeting artificial antigen presenting cell.