CN114315976A

CN114315976A - Combined preparation for treating prostatic cancer and medical application thereof

Info

Publication number: CN114315976A
Application number: CN202210149596.6A
Authority: CN
Inventors: 尹乐; 顾雨春
Original assignee: Chengnuo Regenerative Medical Technology Zhuhai Hengqin New Area Co ltd
Current assignee: Chengnuo Regenerative Medical Technology Zhuhai Hengqin New Area Co ltd
Priority date: 2021-07-14
Filing date: 2022-02-18
Publication date: 2022-04-12
Anticipated expiration: 2042-02-18
Also published as: CN114315977B; CN114835781A; CN114315977A; CN113527434A; CN114315976B; CN114560910A; CN114835781B

Abstract

The combined preparation is a preparation formed by combining a tumor antigen binding peptide-engineered immune cell, a PD-L1 inhibitor and a co-cultured CIK cell, and is applied to the treatment of the prostate cancer for the first time, the combined application of the three can obviously enhance the inhibition effect on the tumor cell, the treatment effect on the prostate cancer is good, and the biological treatment prospect is good.

Description

Combined preparation for treating prostatic cancer and medical application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a combined preparation for treating prostatic cancer and medical application thereof, more particularly, the combined preparation comprises tumor antigen binding peptide-engineered immune cells, a PD-L1 inhibitor and co-cultured CIK cells.

Background

The prostate cancer is a genitourinary system malignant tumor which is frequently generated in middle-aged and old men, the incidence of the prostate cancer is a result of combined action of multiple factors such as heredity and environment, and the cancer statistical data in 2019 show that the incidence of the prostate cancer is the first in the male tumor, the second in the mortality, and the second in the lung cancer. In recent years, the incidence of prostate cancer has been increasing and the prostate cancer has been becoming younger in the world with rapid development of economic society, changes in lifestyle of people, prolongation of life expectancy of people, and improvement in diagnostic techniques. Because the tumor is far away from the urethra in the early stage, the early clinical symptoms of the prostate cancer patient are lack, so that the prostate cancer patient is not easy to find, when the patient is in symptom treatment, prostate cancer cells infiltrate into the urethra, and even distant metastasis of bones, pelvic cavities, spinal cords and other parts occurs, so that the death rate and disability rate are high.

The current common treatment modes of the prostate cancer comprise radical operation of the prostate cancer, castration treatment, chemotherapy, radiotherapy and the like, and the castration treatment is the basis and standard of all treatment modes. Patients relapse about 30% after receiving surgical treatment, and about 80% progress to Castration Resistant Prostate Cancer (CRPC) around 18 months after receiving Castration treatment, and eventually to Metastatic Castration resistant prostate cancer (mCRPC) with a poor prognosis. For mCRPC immunotherapy, passive immunization including specific monoclonal antibodies (mabs) of Immune Checkpoint Blockade (ICB) or Tumor-associated antigen (TAA) and active immunization of Tumor-specific antigen vaccination are mainly included.

One of the main directions of prostate cancer immunotherapy is to block the binding of inhibitory immunoreceptors and their ligands, and PD-1/PD-L1 constitutes a major immune tolerance mechanism. The PD-1 receptor is expressed in activated effector T cells and regulatory T cells. PD-1 binds to PD-L1 and PD-L2 on cancer cells, checks for peripheral autoimmunity and promotes escape of autoimmunity. Expression of PD-L1 promotes immune evasion of Tumor cells within the Tumor Microenvironment (TME) and can be upregulated by activated Tumor-targeted interferon made by T cells. The immune checkpoint inhibitor can be combined with different receptors on the surfaces of T cells or tumor cells, has an action mechanism that the tumor cells generate a large amount of exogenous antigens in a host immune system, and is combined with Major Histocompatibility Complex (MHC) molecules on the surfaces of antigen presenting cells to activate the T cells, trigger an organism to generate immune response and achieve the aim of killing the tumor cells.

In recent years, DC-CIK cell immunotherapy tumor becomes a hot spot of domestic and foreign research, Cytokine-induced killer Cells (CIK) are a group of heterogeneous cells obtained by co-culturing human Peripheral Blood Mononuclear Cells (PBMC) in vitro by a plurality of cytokines, and the cells have strong tumor killing activity of T cells and the tumor killing characteristic of non-major histocompatibility complex restriction of NK cells and have strong killing power on tumor cells; dendritic Cells (DCs) are currently the most powerful antigen-presenting cells known to humans and have a strong ability to activate CD4+ helper T cells, CD8+ cytotoxic T cells. DC cells and CIK cells are two important cells for tumor immunotherapy. The DC-CIK is characterized in that the DC-CIK and the CIK are cultured together in vitro, so that the maturation of DC cells is promoted, and the tumor killing activity of CIK cells is enhanced.

One of the most promising approaches to tumor immunotherapy currently under development is Chimeric Antigen Receptor (CAR), a genetically engineered receptor designed to target specific antigens (e.g., tumor antigens). This targeting specificity can lead to cytotoxicity to tumor cells. However, current CAR cell therapies rely on TAA-specific tumor types, and if tumor-associated antigens of the tumors are not defined or identified, single-chain antibody sequences capable of specific recognition cannot be prepared based on the tumor-associated antigens, and then specific CARs capable of effectively recognizing tumor cells and CAR-T or CAR-NK cells capable of specifically recognizing and killing tumor cells cannot be constructed.

In order to solve the technical problems existing in the field at present, the extracellular recognition region polypeptide WTN of TABP-EIC (Tumor Antigen Binding Peptide-engineered Immune Cell) is screened by a phage-polypeptide library, and can be used for aiming at tumors with undefined antigens, and in addition, the invention firstly combines the Tumor Antigen Binding Peptide-engineered Immune Cell (TABP-EIC-WTN), a PD-L1 inhibitor and a co-cultured CIK Cell to be applied to the treatment of prostate cancer.

Disclosure of Invention

In order to overcome the technical defects existing in the field at present, the invention aims to provide a combined preparation for treating the prostate cancer and medical application thereof, wherein the combined preparation comprises a tumor antigen binding peptide-engineered immune cell, a PD-L1 inhibitor and a co-cultured CIK cell, the combined effect of the tumor antigen binding peptide-engineered immune cell, the PD-L1 inhibitor and the co-cultured CIK cell obviously enhances the anti-tumor effect on the prostate cancer, and the combined preparation has excellent clinical application value and prospect.

The above object of the present invention is achieved by the following technical solutions:

the first aspect of the invention provides the use of a tumor antigen binding peptide-engineered immune cell, a PD-L1 inhibitor and a co-cultured CIK cell in combination for the preparation of a combined preparation for the treatment of cancer;

preferably, the cancer comprises cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, cutaneous squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, stomach cancer, esophageal cancer, thyroid cancer, transitional epithelial carcinoma of the bladder, leukemia, brain tumor, stomach cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, cancer of the female reproductive tract, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma; more preferably, the cancer is prostate cancer.

Further, the tumor antigen binding peptide-the composition of the tumor antigen binding peptide in the engineered immune cell is WTN polypeptide region-hinge region-transmembrane domain-costimulatory domain-primary signaling domain or WTN polypeptide region-hinge region-transmembrane domain-primary signaling domain;

preferably, the tumor antigen binding peptide is composed of a WTN polypeptide region-hinge region-transmembrane domain-costimulatory domain-primary signaling domain; preferably, the WTN polypeptide region comprises a linker between the plurality of repeats of the WTN polypeptide and the plurality of repeats of the WTN polypeptide; more preferably, the WTN polypeptide is a polypeptide that specifically binds PSMA; most preferably, the WTN polypeptide is selected from any one of the following group:

(1) 1, as shown in SEQ ID NO;

(2) 1, or a product formed by substitution, deletion or addition at one or more of

positions

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 of the polypeptide shown in SEQ ID NO;

(3) 1, amino, carboxyl, sulfydryl, phenolic hydroxyl, imidazolyl, guanidino, indolyl and methylthio at one or more sites of the tail end of the main chain or side chain of the polypeptide as shown in SEQ ID NO;

most preferably, the WTN polypeptide is a polypeptide as shown in SEQ ID NO. 1; most preferably, the nucleotide sequence of the WTN polypeptide is shown as SEQ ID NO. 2.

Further, the hinge region of the tumor antigen binding peptide is a CD8 a hinge region; preferably, the amino acid sequence of the CD8 alpha hinge region is shown as SEQ ID NO. 3; more preferably, the nucleotide sequence of the CD8 a hinge region is shown in SEQ ID NO 4; preferably, the transmembrane domain is a 2B4 transmembrane domain; more preferably, the amino acid sequence of the transmembrane domain of 2B4 is shown in SEQ ID NO 5; most preferably, the nucleotide sequence of the transmembrane domain of 2B4 is shown in SEQ ID NO 6; preferably, the co-stimulatory domain is a 2B4 co-stimulatory domain; more preferably, the amino acid sequence of the 2B4 co-stimulatory domain is shown in SEQ ID NO 7; most preferably, the nucleotide sequence of the 2B4 co-stimulatory domain is shown in SEQ ID NO 8; preferably, the primary signaling domain is the NKG2D primary signaling domain; more preferably, the amino acid sequence of the NKG2D primary signaling domain is as set forth in SEQ ID NO 9; most preferably, the NKG2D primary signaling domain has the nucleotide sequence shown in SEQ ID NO 10;

most preferably, the tumor antigen binding peptide consists of the WTN polypeptide region-CD 8 α hinge region-2B 4 transmembrane domain-2B 4 costimulatory domain-NKG 2D primary signaling domain; most preferably, the amino acid sequence of the tumor antigen binding peptide is shown in SEQ ID NO. 11; most preferably, the nucleotide sequence of the tumor antigen binding peptide is shown in SEQ ID NO 12.

Further, the tumor antigen binding peptide-engineered immune cells include tumor antigen binding peptide-engineered NK cells, tumor antigen binding peptide-engineered T cells, tumor antigen binding peptide-engineered B cells, tumor antigen binding peptide-engineered macrophages; preferably, the tumor antigen binding peptide-engineered immune cells are tumor antigen binding peptide-engineered NK cells.

Further, the PD-L1 inhibitors include atezolizumab, avelumab, durvalumab; preferably, the PD-L1 inhibitor is atezolizumab.

Further, the co-cultured CIK cells are DC-CIK cells co-cultured with prostate cancer cells; preferably, the co-cultured CIK cells comprise CD8+ T cells.

In a specific embodiment of the present invention, the present invention proves that the co-cultured CIK cells of the present invention mainly play a role in CD8+ T cells, and therefore, the use of the combination of CD8+ T cells and tumor antigen binding peptide-engineered immune cells and PD-L1 inhibitor in the preparation of combined preparations for treating prostate cancer is also included in the protection scope of the present invention, so that the combination of tumor antigen binding peptide-engineered immune cells, PD-L1 inhibitor and co-cultured CIK cells of the present invention includes the following combinations:

(1) tumor antigen binding peptide-engineered immune cells, PD-L1 inhibitor and co-cultured CIK cells;

(2) tumor antigen binding peptide-engineered immune cells, PD-L1 inhibitors, and CD8+ T cells.

Further, the invention also provides the use of the combination of tumor antigen binding peptide-engineered immune cells, a PD-1 inhibitor and co-cultured CIK cells for the preparation of a combined preparation for the treatment of prostate cancer, said PD-1 inhibitor comprising nivolumab, pembrolizumab, cemipimab, preferably said PD-1 inhibitor is nivolumab.

Further, the co-cultured CIK cell is prepared by the following method:

(1) culturing the DC cells;

(2) culturing the CIK cells;

(3) and (3) mixing the DC cells obtained by the culture in the step (1) and the CIK cells obtained by the culture in the step (2) in a culture medium, adding prostate cancer cells for co-culture, and collecting the suspended DC-CIK mixture to obtain the co-culture CIK cells.

Further, the step (1) comprises the steps of:

(a) on day 0, PBMC cells were cultured in DC cell growth medium;

(b) on day 3, the DC cell growth medium was changed;

(c) on day 6, DC cell maturation factors were added to the DC cell growth medium;

(d) collecting cells on 8 th day to obtain DC cells;

preferably, the substances added to the DC cell growth medium in step (a) comprise FBS, DC cell culture factors; more preferably, the substance added to the DC cell growth medium in step (a) comprises 5% FBS, 2U/mL DC cell culture factor; preferably, the final concentration of DC cell maturation factor in step (c) is 2U/mL.

Further, the step (2) comprises the steps of:

(a) on day 0, PBL cells were cultured in CIK cell activation medium;

(b) supplementing the cell culture with CIK cell proliferation medium on

days

3, 4, and 6;

(c) collecting cells on 8 days to obtain CIK cells;

preferably, the substances added to the CIK cell activation medium in step (a) include KBM551, FBS, anti-CD 3 antibody, IFN-gamma, IL-1 alpha, IL-2; more preferably, the substances added to the CIK cell activation medium in step (a) include KBM551, 3% FBS, 50ng/mL anti-CD 3 antibody, 1000U/mL IFN-. gamma.100U/mL IL-1. alpha., 500U/mL IL-2; preferably, the PBL cells of step (a) have a cell density of 1.5X 10⁶Per mL; preferably, the substances added to the CIK cell proliferation medium in step (b) include KBM551, FBS, IL-2; more preferably, the substance added to the CIK cell proliferation medium in step (b) comprises KBM551, 3% FBS and 500U/mL IL-2.

Further, the culture medium in the step (3) is a CIK cell proliferation culture medium; preferably, the DC cells have a cell density of 4X 10⁷Per mL; preferably, the cell density of the CIK cells is 1 × 10⁸Per mL; preferably, the prostate cancer cells have a cell density of 1 × 10⁷Per mL; preferably, the CO-cultivation conditions are 5% CO₂At 37 ℃ for 24 h; more preferably, the prostate cancer cells comprise C4-2, LNCaP, PC-3, DU 145; more preferably, the prostate cancer cell is C4-2.

A second aspect of the invention provides a combined preparation for use in the treatment of cancer;

preferably, the cancer is prostate cancer.

The combined preparation comprises tumor antigen binding peptide-engineered immune cells, a PD-L1 inhibitor and co-cultured CIK cells.

Further, the tumor antigen-binding peptide-engineered immune cell is the tumor antigen-binding peptide-engineered immune cell according to the first aspect of the present invention.

Further, the PD-L1 inhibitor is a PD-L1 inhibitor according to the first aspect of the invention.

Further, the co-cultured CIK cells are the co-cultured CIK cells according to the first aspect of the invention.

In addition, the present invention provides a method of treating a prostate cancer patient, the method comprising: administering to a subject in need thereof an effective amount of a tumor antigen-binding peptide-engineered immune cell (TABP-EIC-WTN cell), a PD-L1 inhibitor, co-cultured CIK cells, and/or a combined preparation according to the second aspect of the invention.

The invention has the advantages and beneficial effects that:

the tumor antigen binding peptide-engineered immune cells (TABP-EIC-WTN cells), the PD-L1 inhibitor and the co-cultured CIK cells are jointly applied to the treatment of the prostate cancer for the first time, and experiments prove that the combination of the three remarkably enhances the anti-tumor effect on the prostate cancer, so that the tumor antigen binding peptide-engineered immune cells, the PD-L1 inhibitor and the co-cultured CIK cells has extremely good clinical application value and prospect.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a graph showing the results of immunofluorescence of Lncap cell lines with higher expression levels of polypeptides WTN and PSMA;

FIG. 2 is a graph showing the immunofluorescence results of PC3 cell line with low expression of polypeptide WTN and PSMA;

FIG. 3 is a graph showing the results of flow assays of C4-2 GFP cells co-cultured with TABP-EIC-WTN cells for 2, 6, 12, and 24 hours;

FIG. 4 is a graph showing the results of measuring IFN-. gamma.secretion levels by ELISA;

FIG. 5 is a graph showing the results of measuring the expression level of PD-L1 on C4-2 cells at different concentrations of IFN-. gamma.;

FIG. 6 is a graph showing the results of measuring the expression level of PD-L1 in C4-2 cells co-cultured with TABP-EIC-WTN cells for 24 hours;

FIG. 7 is a graph showing the results of measuring the expression level of PD-L1 in TABP-EIC-WTN cells co-cultured with C4-2 cells;

FIG. 8 is a graph showing the results of measuring the expression level of PD-1 in TABP-EIC-WTN cells co-cultured with C4-2 cells;

FIG. 9 is a graph showing the results of measuring the expression level of PD-L1 in TABP-EIC-WTN cells under different concentrations of IFN-. gamma.stimulation;

FIG. 10 is a graph showing the results of a flow cytometry plot and summary data, in which the expression amount of PD-L1 on TABP-EIC-WTN cells cultured using a common medium and the supernatant obtained after co-culturing TABP-EIC-WTN + C4-2 cells for 12 hours, the percentage of PD-L1 expression did not differ significantly between TABP-EIC-WTN cells cultured in both media;

FIG. 11 is a schematic view of a cell co-incubation apparatus in which C4-2 cells are seeded at the bottom and TABP-EIC-WTN cells are seeded at the upper region, the inner and outer spaces being separated by a filter membrane which allows cytokines to pass through but prevents TABP-EIC-WTN cells from coming into direct contact with C4-2 cells;

FIG. 12 is a graph of flow cytometry plots and results of summary data;

FIG. 13 is a graph showing the results of transcriptional differential analysis of co-cultured/cultured TABP-EIC-WTN/C4-2 cells alone, in which: volcano plot of deregulated genes between co-cultured TABP-EIC-WTN and separately cultured TABP-EIC-WTN cells, panel B: volcano plots of deregulated genes between co-cultured C4-2 and C4-2 cells cultured alone;

FIG. 14 is a graph of the results of a KEGG pathway enrichment analysis, Panel A: up-regulated genes in co-cultured TABP-EIC-WTN, panel B: down-regulated genes in co-cultured TABP-EIC-WTN, panel C: up-regulated genes in co-cultured C4-2 cells;

FIG. 15 is a graph showing the results of flow assay, Panel A: expression level of NKG2D on TABP-EIC-WTN cells, FIG. B: the expression level of MICA/B on C4-2 cells;

FIG. 16 is a graph showing the results of Western blot analysis of proteins involved in the signal pathway expressed by PD-L1, Panel A: graph of assay results without NKG2D blocker, B: graph of assay results with addition of NKG2D blocker;

FIG. 17 is a graph showing the statistics of the expression levels of proteins involved in the signal pathway of PD-L1 expression, Panel A: PD-L1, Panel B: p-PI3K, Panel C: p-AKT, Panel D: p-mTOR, E Panel: p-JAK1, panel F: p-JAK2, G panel: p-STAT 1;

FIG. 18 is a graph showing the results of measurement of bioluminescence intensity (BLI) of C4-2 GFP cells co-cultured with TABP-EIC-WTN, in which: test result chart, B chart: a statistical result graph;

FIG. 19 is a graph showing the results of increasing the tumor cell inhibition rate of TABP-EIC-WTN in terms of atezolizumab or nivolumab, panel A: e: t is 1:1, B diagram: e: t is 5: 1;

FIG. 20 is a graph showing the results of measurement of IFN-. gamma.secretion from TABP-EIC-WTN cells when Atezolizumab or nivolumab was added;

fig. 21 is a graph of flow cytometry plots and summary data results, panel a: flow cytometry plot, panel B: FIG. 1 is a graph showing the expression of CD107a induced by C4-2 cells on TABP-EIC-WTN cells (when atezolizumab or nivolumab is added), the expression of TABP-EIC-WTN cells and C4-2 cells being expressed at a ratio of 1:1 ratio at 37 ℃ for 20 hours, collecting and treating TABP-EIC-WTN cells with atezolizumab (20. mu.g/mL) or nivolumab (20. mu.g/mL), and then performing detection;

FIG. 22 is a graph showing the results of tumor size measurements in groups of mice on

days

7, 14, and 21;

FIG. 23 is a graph showing the results of tumor suppression with and without co-cultured CIK, Panel A: tumor suppression without co-cultured CIK, panel B: tumor suppression when co-cultured CIK was added;

FIG. 24 is a photograph of immunohistochemical staining of tumor cells, Panel A: staining result graph, panel B: a statistical result graph;

fig. 25 is a graph showing statistical results of statistics of tumor sizes of mice treated with and without co-cultured CIK, respectively, a graph: CIK with co-culture, Panel B: CIK without co-culture;

FIG. 26 is a graph showing a real image of a tumor after resection and a statistical result of tumor weight, in which FIG. A: material object diagram, B diagram: counting results;

FIG. 27 shows the measurement of expression levels of CD3, CD8, and CD56 in co-cultured CIK, Panel A: control, panel B: CD3, panel C: CD8, panel D: CD56, panel E: CD3 CD56, panel F: a statistical result graph;

FIG. 28 is a graph showing the results of tumor growth in mice from the control group, CIK-treated group and co-cultured CIK-treated group, Panel A: mouse tumor growth, panel B: statistical analysis of BLI measurements;

FIG. 29 is a graph showing the statistical results of tumor volumes of mice in the control group, CIK-treated group and co-cultured CIK-treated group at

days

5, 10 and 20;

figure 30 is a graph of serum testosterone and PSA levels after ADT in a prostate cancer patient, panel a: serum testosterone, panel B: PSA;

FIG. 31 is a graph of HE staining of tumor tissues of prostate cancer patients (100-fold and 200-fold), panel A: 100 times, panel B: 200 times of the total weight of the powder;

fig. 32 is a flow cytometry plot and summary data histogram, panel a: flow cytometry plot, panel B: statistical result graph, graph C: statistical results graph, wherein the graph shows the expression of PD-L1 on primary prostate cancer cells incubated with TABP-EIC-WTN cells for 24 hours, (right) CCK-8 assay results showing that atezolizumab (20 μ g/mL) rather than nivolumab (20 μ g/mL) significantly enhances the inhibitory effect on TABP-EIC-WTN, E: t is 1: 1.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Example 1 peptide library screening

1. Purpose of experiment

The invention adopts a Ph.D. -12 phage display peptide library kit to screen out the polypeptide WTN specifically combined with PSMA.

2. Ph.D. -12 phage display peptide library kit composition

Random dodecapeptide phage display library: 100 μ L, 1.5X 10¹³pfu/mL, stored in TBS solution with 50% Glycerol, complexity 2.7X 10⁹Transforming the cells; -28gIII sequencing primer: 5 '-HOGTATGGGATTTTTGCTAAAACAA C-3', 100pmol, 1 pmol/. mu.L; -96gIII sequencing primers: 5 '-HOCCCTCATAGTTAGCGTAACG-3', 100 pmol/. mu.L, 1 pmol/. mu.L; coli ER2738 host strain F' lacIq Δ (lacZ) M15 proA + B + zzf Tn10 (TetR)/fhaA 2 supE thi Δ (lac-proAB) Δ (hsdMS-mcrB)5 (rk-mk-McrBC-): the strain is provided in the form of a thallus culture containing 50% of glycerol, and non-competent cells are stored at-70 ℃; streptavidin, 1.5mg of freeze-dried powder; biotin: 10mM 100. mu.L.

3. Experimental methods

Day one

The panning experiments were performed on single sterile polystyrene petri dishes, 12 or 24 well plates, 96 well microplates, with at least one plate (or well) coated with each target molecule, depending on the number and type of target molecules on which the panning of the library was performed simultaneously, and the amounts given in the following methods are the amount of 60 x 15mm petri dishes, in brackets the amount of microplates, and the other medium-sized wells adjusted accordingly, but in each case the same number of phage was added: 1.5X 10¹¹A virus seed;

(1) a100. mu.g/mL solution of the target molecule (NaHCO dissolved in 0.1M, pH 8.6) was prepared₃) If it is desired to stabilize the target molecule, other buffers of similar ionic strength (containing metal ions, etc.) may also be used;

(2) adding 1.5mL (150 μ L per well of microporous plate) of the above solution into each plate (well), and repeatedly rotating until the surface is completely wet;

(3) shaking slightly at 4 deg.C in a humidifying container (such as sealable plastic box arranged with wet paper towel), incubating overnight, and storing the plate in the container at 4 deg.C;

the next day

(4) Selecting ER2738 monoclonal (plate paved when measuring bacteriophage titer) in 10mL LB liquid culture medium, if amplifying eluted bacteriophage on the same day, also inoculating ER2738 in 20mL LB liquid culture medium, using 250mL triangular flask, shaking culture at 37 deg.C;

(5) pouring out the coating liquid in each plate, inverting the plate, patting and throwing the plate on a clean paper towel forcibly to remove residual solution, filling sealing liquid in each plate (or hole), and acting at 4 ℃ for at least 1 h;

(6) spin wash plate 6 times, spin each time to wash the bottom and edge of the plate or well, pour off buffer, shake-off upside down on clean paper towel to remove residual solution (or use an automatic plate washer);

(7) 4X 10 dilutions were made in 1mL (100. mu.L for microwell plates) of TBST buffer¹⁰The phage (i.e., 10. mu.L of the original library) were then added to the coated plate and gently shaken at room temperature for 10-60 min;

(8) pouring to remove the unbound phage, inverting the plate, and patting on a clean paper towel to remove the residual solution;

(9) wash the plate 10 times with TBST buffer as described in 6, change clean paper towel each time to avoid cross contamination;

(10) according to the intermolecular interactions studied, the bound phage were eluted with 1mL (100. mu.L for microwell plates) of the appropriate elution buffer, the known ligand for the target molecule was dissolved in TBS solution at a concentration of 0.1-1mM or the bound phage were competitively eluted from the immobilized target with free target solution (-100. mu.g/mL in TBS), gently shaken at room temperature for 10-60min, and the eluate was aspirated into another clean microfuge tube; non-specific buffers such as 0.2M Glycine-HCl (pH 2.2), 1mg/mL BSA can also be used to separate the bound molecules: gently shake for >10min, the eluate is aspirated into another clean microcentrifuge tube, and the eluate is neutralized with 150. mu.L (15. mu.L for microwells) 1M Tris-HCl (pH 9.1);

(11) the titers of the small amounts (. about.1. mu.L) of the eluates were determined as described above in the conventional M13 procedure, and plaques from the first or second round of eluate titer determination were sequenced as needed as follows: if necessary, the remaining eluate may be stored at 4 ℃ overnight and expanded the next day, in which case ER2738 may be cultured overnight in LB-Tet medium, the next day the culture 1:100 is diluted in 20mL LB (contained in a 250mL Erlenmeyer flask), the unexpanded eluate is added, and the culture is vigorously shaken at 37 ℃ for 4.5h, and step 13 is continued;

(12) amplification of the remaining eluate: adding the eluate into 20mL of ER2738 culture (the thallus is in the early stage of logarithm), and culturing at 37 ℃ for 4.5h by shaking vigorously;

(13) the culture was transferred into a centrifuge tube and then centrifuged at 10,000rpm at 4 ℃ for 10 min. Transferring the supernatant into another centrifugal tube, and centrifuging;

(14) transferring the upper 80% of the supernatant to a fresh tube, adding 1/6 volume of PEG/NaCl, and allowing the phage to precipitate at 4 ℃ for at least 60min overnight;

the third day

(15) Centrifuging PEG at 4 deg.C and 10,000rpm for 15min, discarding supernatant, centrifuging for a short time, and removing residual supernatant;

(16) the precipitate was resuspended in 1mL TBS, the suspension was transferred to a microcentrifuge tube and centrifuged at 4 ℃ for 5min to pellet the residual cells;

(17) transferring the supernatant into another fresh microfuge tube, reprecipitating with 1/6 volume of PEG/NaCl, incubating on ice for 15-60min, centrifuging at 4 deg.C for 10min, discarding supernatant, centrifuging for a short time, and removing residual supernatant with micropipette;

(18) the pellet was resuspended in 200. mu.L TBS, 0.02% NaN₃Centrifuging for 1min, precipitating any residual insoluble substances, and transferring the supernatant into a fresh tube, wherein the supernatant is the eluate after amplification;

(19) titrating the amplified eluate with LB/IPTG/Xgal plates according to the conventional M13 method, and storing at 4 ℃;

(20) coating a plate or hole for the second round of elutriation;

the fourth and fifth days

(21) The titer was determined by counting the number of blue spots on the plate and this value was used to calculate a titer corresponding to 1-2X 10¹¹The amount of pfu added; if the titer is too low, the next rounds of panning may be performed down to 10⁹Testing the phage addition amount of pfu;

(22) and (3) carrying out a second round of panning: the eluate obtained by the first panning and amplification is 1-2X 10¹¹Repeating steps 4-18 for the amount of phage in pfu, increasing the concentration of Tween to 0.5% (v/v) in the washing step;

(23) the titer of the eluate obtained from the second round of panning after amplification was determined on LB/IPTG/Xgal plates;

(24) coating a plate or a hole for a third round of elutriation;

day six

(25) Performing a third panning: 2X 10 of the eluate amplified by the second panning¹¹The phage amount of pfu repeats steps 4-11, with the washing step again using 0.5% (v/v) Tween;

(26) the titers of the eluates from the third round of panning were determined on LB/IPTG/Xgal plates without amplification, and the eluates from the third round were not necessarily amplified unless a fourth round of panning was performed, and plaques obtained from the titer determination were used for sequencing: as long as the plate culture time is not longer than 18h, the culture time is too long, the loss is easy to occur, and the rest eluates are stored at 4 ℃;

(27) one ER2738 monoclonal was selected and cultured overnight in LB-Tet medium.

4. Results of the experiment

The experimental result shows that the amino acid sequence of the polypeptide WTN specifically combined with PSMA obtained by screening is WTNHHQHSKVRE (SEQ ID NO: 1).

Example 2 validation of WTN polypeptide specificity

1. Experimental methods

The WTN polypeptide is respectively subjected to immunofluorescence detection with an Lncap cell line with higher PSMA expression level and a PC3 cell line with low PSMA expression level, and the used fluorescence marker is FITC (namely FITC-labeled WTN polypeptide).

2. Results of the experiment

Fluorescence detection of WTN polypeptide and Lncap cell line is shown in FIG. 1, and fluorescence detection of WTN polypeptide and PC3 cell line is shown in FIG. 2; in FIG. 1, the center of the dot is the nucleus and the light color around the dot is the fluorescence exhibited by the binding of WTN to the cell surface antigen PSMA; only the cell nucleus is stained in fig. 2, and it can be seen that the cell surface fluorescence labeled by WTN appears only in fig. 1, demonstrating that the binding of WTN to the cell surface antigen PSMA has high specificity.

EXAMPLE 3 cell line according to the present invention, and culture method and construction method thereof

1. Construction method of TABP-EIC-WTN

(1) Preparation of TABP-EIC-WTN cells

The NK cells used in the experiment are all obtained by amplifying Peripheral Blood Mononuclear Cells (PBMC).

(2) Construction of tumor antigen-binding peptide expression vector

The tumor antigen binding peptide structure (complete structure: WTN polypeptide region-CD 8 alpha hinge region-2B 4 transmembrane domain-2B 4 co-stimulation domain-NKG 2D primary signal conduction domain, nucleic acid sequence is shown in SEQ ID NO: 12) is obtained by gene synthesis (general purpose organism), and the expression vector is pLenti-EF1a-Backbone (NN) (adddge # 27961). The restriction sites of the structure of the tumor antigen-binding peptide are BsiWI and EcoRI (i.e., the sequence shown in SEQ ID NO:12 replaces the sequence between the restriction sites BsiWI-EcoRI). The carrier is called TABP-EIC-WTN skeleton carrier after inserting the tumor antigen binding peptide structure, and the carrier can express the tumor antigen binding peptide with the amino acid sequence of SEQ ID NO. 11.

(3) Lentiviral packaging

TABP-EIC-WTN backbone vector and auxiliary vector pMD2.G (addgen #12259), pMDLg/pRRE (addgen #12251), pRSV-Rev (addgen #12253) were mixed at a ratio of 10:7:5:3, and 293T cells were transfected with 20. mu.g of plasmid per 10mL of transfection system. Supernatants were collected 48 hours and 72 hours after transfection, purified and concentrated to obtain lentiviruses.

(4) Lentiviral transduction

Mixing the concentrated lentivirus with NK cells, purifying the lentivirus at 200 μ L per 100 ten thousand cells, and placing the mixture in an incubator at 37 ℃ with 5% CO₂Culturing under the condition, and completely replacing the culture solution after 24 hours.

(5) Amplification of TABP-EIC-WTN cells: and (3) normally culturing and amplifying TABP-EIC cells obtained after lentivirus infection.

(6) Detection of tumor antigen binding peptide expression efficiency of TABP-EIC-WTN cells: (100% positive for a single clone).

2. Methods of culturing other cell lines

(1) C4-2 cells: human CRPC cell line expressing PSMA (prostate cancer cell line), C4-2 cells were cultured in RPMI-1640 medium (Sericebio) containing 10% fetal bovine serum FBS (biological industries) and 1% penicillin/streptomycin (Hyclone);

in order to conveniently observe the tumor inhibition condition, a C4-2 cell line which stably expresses GFP and is hereinafter referred to as C4-2 GFP is established by a single-cell cloning method.

(2) NK92 cells: human malignant non-Hodgkin lymphoma Natural killer cell lines, NK92 and TABP-EIC-WTN cells were cultured in medium supplemented with 20% FBS, 0.2mM myo-inositol (Sigma), 0.1mM beta-mercaptoethanol (PAN-Biotech), 0.02mM folic acid (Sigma), 200U/mL recombinant human IL-2(SL Pharm) and 1% penicillin-streptomycin alpha MEM (Gibco).

(3)293T cells: human embryonic kidney cell lines, from American type culture Collection ATCC, 293T cells in DMEM medium (Hyclone) or opti-MEM (Gibco);

(4) PCa cells (prostate cancer cells): prostate tissue from one CRPC patient undergoing radical prostatectomy.

The cell culture of the invention is carried out at 37 ℃ and 5% CO₂Is cultured in a humid environment.

Example 4 expression of PD-L1 on C4-2 cells co-cultured with TABP-EIC-WTN cells was up-regulated depending on IFN-. gamma.

C4-2 GFP cells were co-cultured with TABP-EIC-WTN cells, and flow-assayed at 2, 6, 12, and 24 hours, with the results shown in FIG. 3: and the detection of PD-L1 expressed by C4-2 cells is shown in FIG. 3: the Mean Fluorescence Intensity (MFI) of PD-L1 and the percentage of C4-2 cells expressing PD-L1 of C4-2 cells were significantly upregulated with increasing time.

TABP-EIC-WTN cells were cultured and IFN-. gamma.secretion levels were measured at 2, 6, 12, and 24 hour time points in the culture supernatants by ELISA in the presence or absence of C4-2 GFP co-culture, as shown in FIG. 4.

IFN-gamma was added to C4-2 cells and the expression level of PD-L1 on C4-2 cells at different concentrations of IFN-gamma was shown in FIG. 5: both the Mean Fluorescence Intensity (MFI) of PD-L1 and the percentage of C4-2 cells expressing PD-L1 increased in a concentration-dependent manner.

The results of measuring the expression level of PD-L1 on C4-2 cells co-cultured with TABP-EIC-WTN cells for 24 hours in the case of using an IFN γ blocker (IFN γ mab) are shown in FIG. 6: IFN gamma blockers completely reversed the up-regulation of PD-L1 of C4-2 co-cultured with TABP-EIC-WTN cells.

The above experimental results show that the up-regulation of PD-L1 on C4-2 cells co-cultured with TABP-EIC-WTN cells is dependent on IFN-. gamma.cells.

Example 5 upregulation of PD-L1 expression on TABP-EIC-WTN cells co-cultured with C4-2 cells dependent on direct cell contact

TABP-EIC-WTN cells and C4-2 cells were co-cultured, and the expression levels of PD-L1 and PD-1 of TABP-EIC-WTN were measured at 2, 6, 12 and 24 hours, as shown in FIG. 7 and FIG. 8, respectively. The results show that: the percentage of TABP-EIC-WTN that was PD-L1 or PD-1 positive was significantly upregulated over time.

The flow-through assay of the expression level of PD-L1 on TABP-EIC-WTN cells stimulated with different concentrations of IFN-. gamma.is shown in FIG. 9. The expression level of PD-L1 in TABP-EIC-WTN cells was not significantly changed after IFN-gamma stimulation.

Example 6 analysis of transcriptional Difference between Co-culture/independent culture of TABP-EIC-WTN/C4-2 cells

Total RNA was extracted from the samples using trizol (invitrogen). DNA digestion was performed after RNA extraction by dnase i. RNA quality was determined by examining A260/A280 using a NanodropTM OnEC spectrophotometer (Thermo Fisher Scientific Inc). RNA integrity was confirmed by 1.5% agarose gel electrophoresis. Finally, qualified RNA was quantified by Qubit3.0 and QubitTM RNA Broad Range Assay kit (Life Technologies).

Using KC-Digital TM-Stranded mRNA Library Prep Kit for

(Seqhealth Technology Co., Ltd.) 2. mu.g of total RNA was used for RNA sequencing library preparation. Library products corresponding to 200-500bps were enriched, quantified and finally sequenced on Illumina Novaseq 6000.

Raw sequencing data was first filtered by trimmatic (version 0.36) and clear Reads were further processed using internal scripts to eliminate the repetitive bias introduced in library preparation and sequencing. Briefly, clean reads are first clustered according to UMI sequences, where reads with the same UMI sequence are grouped into the same cluster, resulting in 65, 536 clusters. Reads in the same cluster are compared to each other by pairwise alignment, and reads with more than 95% sequence identity are then extracted into new sub-clusters. After the sub-clusters are generated, multiple sequence alignments are performed to obtain one consensus sequence for each sub-cluster. After these steps, any errors and deviations introduced by PCR amplification or sequencing are eliminated.

They were mapped to Homo sapiens reference genome data from Homo sapiens grch38 (ftp:// ftp. ensembl. org/pub/release-87/fasta/Homo sapiens/dnas /) using STAR software (version 2.5.3 a). Reads mapped to exon regions of each gene were calculated by feature counting (Suclean-1.5.1; Bioconductor) and then RPKM was calculated. The edgeR package (version 3.12.1) was used to identify genes differentially expressed between groups. FDR corrected p-value cutoff was 0.05 and fold change cutoff was 2 for statistical significance of gene expression differences.

Gene Ontology (GO) analysis and KEGG enrichment analysis of differentially expressed genes were performed by KOBAS software (version: 2.1.1) with a corrected P cut-off of 0.05 to determine statistically significant enrichment. The alternative splicing event was detected by using rMATS (version 3.2.5) with an FDR value cut off of 0.05 and an absolute Δ ψ value of 0.05.

The volcano of deregulated genes between co-cultured TABP-EIC-WTN and separately cultured TABP-EIC-WTN cells is shown in FIG. 13A, and the volcano of deregulated genes between co-cultured C4-2 and separately cultured C4-2 cells is shown in FIG. 13B. In fig. 13, differentially expressed genes with fold change greater than 2.0 and P <0.05 were color labeled. P values were calculated using a two-sided unpaired student t-test.

The KEGG pathway enrichment analysis for up-regulated genes in the co-culture of fig. 13A is shown in fig. 14A, and down-regulated genes in fig. 14B. The KEGG pathway enrichment analysis of the up-regulated genes in the co-culture in FIG. 13B is shown in FIG. 14C. In fig. 14, the color of the dots represents the enrichment factor and the size represents the number of inputs per KEGG entry. The horizontal axis represents the importance of enrichment. The vertical axis represents the enriched KEGG pathway.

FIG. 15A shows the measurement of the expression level of NKG2D on TABP-EIC-WTN cells. The MICA/B expression level on C4-2 cells was measured as shown in FIG. 15B. FIG. 15 shows the detection of marker on the cell surface (determination of the expression level of the corresponding marker).

Example 7 signalling pathways involved in PD-L1 expression in the co-culture of TABP-EIC-WTN and C4-2 cells

TABP-EIC-WTN was cultured alone or in combination with C4-2 cells (E: T ═ 1:1) for 24 hours, and then protein was extracted for detection.

FIGS. 16-17 are assays for protein level detection, and Western blot analysis of PD-L1 (FIGS. 16A, 17A), p-PI3K (FIGS. 16A, 17B), p-AKT (FIGS. 16A, 17C), and p-mTOR (FIGS. 16A, 17D) in co-cultured TABP-EIC-WTN, respectively, are shown as the labels in the figures. TABP-EIC-WTN, blocking TABP-EIC-WTN, NK92, co-culturing NK92, blocking NK92 cells.

NKG2D blocker (NKG2D mab) was added to TABP-EIC-WTN or NK92 cells and incubated at 37 ℃ for 1 hour prior to co-culture with C4-2 cells. NKG2D blockers inhibit the activation of the PI3K/AKT/mTOR pathway in NK92 cells but not TABP-EIC-WTN cells.

Western blot analysis of IFN γ -mediated JAK/STAT activator members included p-JAK1 (fig. 16B, 17E), p-JAK2 (fig. 16B, 17H), and p-STAT1 (fig. 16B, 17G) in C4-2 cells co-cultured with TABP-EIC-WTN cells compared to C4-2 cells without any treatment.

The above experimental results indicate that TABP-EIC-WTN and C4-2 cells are involved in the signaling pathway of PD-L1 expression when co-cultured.

Example 8 detection of cytotoxic Activity of TABP-EIC-WTN cells treated with atezolizumab or nivolumab

The results of the measurement of the luminescence intensity of TABP-EIC-WTN co-cultured with C4-2 GFP cells are shown in FIG. 18: bioluminescence intensity (BLI) of C4-2 GFP cells co-cultured with TABP-EIC-WTN when treated with atezolizumab or nivolumab at concentrations of 10, 20, 40. mu.g/mL.

The CCK-8 test result shows that atezolizumab (20 mug/mL) remarkably enhances the inhibition rate of TABP-EIC-WTN on the prostate cancer cells, while nivolumab (20 mug/mL) slightly enhances the inhibition rate of TABP-EIC-WTN on the prostate cancer cells. E: t is 1: 1. 5: the results at1 are shown in fig. 19A and 19B, respectively. The combination of TABP-EIC-WTN and PD-L1 inhibitors is shown to be effective in treating prostate cancer.

The effect of Atezolizumab or nivolumab on IFN-. gamma.secretion from TABP-EIC-WTN cells was examined at

hours

2 and 6 of the addition of Atezolizumab or nivolumab using ELISA, and the results are shown in FIG. 20: atezolizumab (10, 20, 40. mu.g/mL) increased IFN-. gamma.secretion from TABP-EIC-WTN cells incubated with C4-2 cells.

TABP-EIC-WTN cells were cultured at 37 ℃ in a 1:1 for 20 hours after co-incubation with C4-2 cells, TABP-EIC-WTN cells were then harvested and treated with atezolizumab (20 μ g/mL) or nivolumab (20 μ g/mL), and then CD107a expression in TABP-EIC-WTN cells induced by C4-2 cells in the presence of atezolizumab or nivolumab was examined using a flow cytometer, the results being shown in fig. 21, indicating that activated NK cells produce cytotoxic effects mainly through two cell signaling pathways, one involving perforin and granzyme B and the other involving target cell death ligands, including TRAIL and FASL. In the presence of atezolizumab, the expression of CD107a in TABP-EIC-WTN cells was significantly increased in an ADCC-dependent manner. In addition, the results of FIGS. 19-21 indicate that atezolizumab is significantly more effective than nivolumab.

Example 9 anti-tumor Effect of TABP-EIC-WTN and atezolizumab or nivolumab on C4-2 cells in the Presence or absence of CD8+ T cells in vivo

1. Preparation of DC-CIK Co-cultured with C4-2 cells

Peripheral Blood Mononuclear Cells (PBMC) or lymphocytes (PBL) were isolated from a donated blood sample from a normal healthy subject by Ficoll-Paque (TBD science), followed by centrifugation on a 460g density gradient for 40 minutes, washed twice with saline and cultured in DC adherent medium consisting of X-VIVO (Lonza) and 5% FBS (Gibco). After 1 hour incubation, adherent PBMCs (monocytes) were collected for DC culture and suspended PBLs were collected for Cytokine Induced Killer (CIK) cell culture (day 0).

Adherent monocytes were cultured in DC growth medium consisting of X-VIVO (Lonza) and also containing 5% FBS and 2U/HmL DC culture factor (Novoprotein), CO at 37 ℃₂Culture in the incubator, then half of the DC growth medium was changed on day 3, DC maturation factor (Novoprotein) was added on day 6 (final concentration of 2U/mL), and final harvest was performed on day 8.

For CIK cultures, suspended PBLs were adjusted to a density of 1.5X 10⁶And cultured in CIK activation medium containing KBM551 (corning), 3% FBS, 50ng/mL anti-CD 3 antibody (Beijing Tianlian Biotech), 1000U/mL IFN-. gamma. (Beijing Tianlian Biotech), 100U/mL IL-1. alpha. (Beijing Tianlong Biotech), and 500U/mL IL-2(SL Pharm). Cell cultures were supplemented with CIK proliferation medium containing KBM551, 3% FBS and 500U/mL IL-2 on

days

3, 4 and 6, during which cell density was maintained at 1.5X 10⁶More than one cell/mL.

On day 8, harvest was about 4X 10⁷DC and 1X 10⁸CIK, centrifuged, and then mixed in 150mL CIK proliferation medium. The DC-CIK mixture was then added to a seed containing 1X 10⁷C4-2 adherent cells in a T75 flask at 5% CO₂Co-culturing at 37 ℃ in a humid environment.

After 24 hours of co-cultivation, the suspended DC-CIK mixture was harvested and designated "co-cultivated CIK (labeled as cocultured CIK" on the figure).

2. Construction and culture of model mouse

Male NOD/SCID mice at 5 weeks of age were purchased from Tokyo Vittal river laboratory animal technology, Inc., and housed in a facility for animals without specific pathogens in key laboratories in the national molecular oncology center for cancer. All experimental procedures were approved by the ethical committee of our hospital and were performed according to the principles of experimental animal care (NIH publication volume 25, revision 28 th 1996). Mice were inoculated subcutaneously in the upper abdomen with 2X 10 cells suspended in 200. mu.L PBS⁶C4-2 cells. Tumor volume reached 100-³Treatment was started and C4-2 vaccinated mice were randomized into seven groups:

(i) in the Control (Control) group,

(ii) the TABP-EIC-WTN group,

(iii) TABP-EIC-WTN + nivolumab group,

(iv) TABP-EIC-WTN + atezolizumab group,

(v) TABP-EIC-WTN + co-cultured CIK (cultured CIK) group,

(vi) TABP-EIC-WTN + nivolumab + CIK group co-cultured,

(vii) TABP-EIC-WTN + atezolizumab + group of co-cultured CIK.

The PD-L1 antibody atezolizumab (GlpBio, GC32704) (20mg/kg) or PD-1 antibody nivolumab (GlpBio, GC34218) (10mg/kg) or control PBS tail vein was administered on

days

7, 9, 11, 13, 15 post tumor vaccination for 5 doses, while TABP-EIC-WTN treatment was 5X 10 injections on

days

8, 10, 12, 14, 16 post PD-L1/PD-1 antibody treatment⁶TABP-EIC-WTN, for co-intravenous injection of 5 doses.

On day 17, CIK was co-cultured by i.v. injection based on TABP-EIC-WTN treatment, with or without the addition of PD-L1/PD-1 antibody.

Tumor volume calculation formula: LxW²L and W represent the longest and shortest diameters measured by calipers on

days

7, 10, 14, 16, 18, 20. Also by using

Spectral CT detection of bioluminescent intensity (BLI) tumor size before and after treatment was assessed, and BLI was measured and expressed as radiance (p/sec/cm2/sr) as described at

days

7, 14, 21 after tumor cell implantation.

All mice were sacrificed on day 21, tumors were collected, photographed, weighed and collected for histological examination.

3. Results of the experiment

The tumor sizes of the C4-2 GFP cell implanted mice were measured on

days

7, 14, and 21 (n-3-4), and the results are shown in fig. 22. Tumor size was counted separately for mice treated with and without co-cultured CIK, tumor volume was counted for each group without co-cultured CIK as in fig. 25A, and tumor volume was counted for each group with and without co-cultured CIK as in fig. 25B.

A physical map of the excised tumor is shown in fig. 26A, a statistical analysis of tumor weight is shown in fig. 26B, values on the map are expressed as mean ± SD, representing P < 0.05; NS represents no significant difference.

To analyze the tumor-inhibiting effect of co-cultured CIK, the tumor-inhibiting effect was measured with or without co-cultured CIK, and quantitative BLI test was performed on the control group, TABP-EIC-WTN treated group, TABP-EIC-WTN + nivolumab treated group, TABP-EIC-WTN + atezolizumab treated group on

days

7, 14, and 21, and the results are shown in FIG. 23A. Quantitative BLI detection was performed on the control group, TABP-EIC-WTN treated group, TABP-EIC-WTN + co-cultured CIK treated group, TABP-EIC-WTN + nivolumab + co-cultured CIK treated group, TABP-EIC-WTN + atezolizumab + co-cultured CIK treated group, and the results are shown in FIG. 23B.

Immunohistochemical staining of tumor cells, fixing of necrotic tissue with 10% neutral buffered formalin, paraffin embedding, cutting into 4 μm thick sections, staining with Hematoxylin and Eosin (HE) for light microscopy. The necrotic area was stained red under an optical microscope with no intact cellular structures. View2 viewer software (Hamamatsu Photonics, Shizuoka, Japan) was used to quantify the percentage of red necrotic regions relative to the entire region, with large vessels excluded from the image analysis above. As a result, as shown in FIG. 24A, significant tumor necrosis was observed in the TABP-EIC-WTN-treated group, and the addition of atezolizumab significantly increased the severity of tumor necrosis in the TABP-EIC-WTN-treated group, and the statistical result is shown in FIG. 24B.

The above experiment results show that TABP-EIC-WTN and/or atezolizumab and/or co-cultured CIK combination can effectively treat tumors, namely TABP-EIC-WTN and atezolizumab combination can effectively treat tumors; the TABP-EIC-WTN can effectively treat tumors by combining with the co-cultured CIK; the combination of TABP-EIC-WTN, atezolizumab and co-cultured CIK can effectively treat tumors.

Example 10 composition and functional characterization of Co-cultured CIK

Flow cytometry was used to detect CD3, CD8, and CD56 expression in co-cultured CIK, in which cells were stained with the following antibodies: CD3-APC, CD8-FITC, CD56-PerCP from BD Bioscience; the results of the statistical analysis of the detection results are shown in fig. 27.

Fluorescence imaging assays were performed on day 5 and day 10 in control, CIK-treated and co-cultured CIK-treated mice, and BLI measurements and statistical analysis (n-3) of tumor activity of C4-2 GFP engraftment are shown in figure 28. Measurements were taken on

days

5, 10, and 20 and tumor volumes were calculated, and the results are shown in fig. 29.

The experimental results show that the CIK and the co-cultured CIK have certain treatment effects on tumors, and the co-cultured CIK prepared by the invention has better treatment effect and is obviously superior to a control group and a CIK group.

Example 11Atezolizumab enhances the cytotoxicity of TABP-EIC-WTN in vitro on CRPC cells from a prostate cancer patient

One prostate cancer patient was randomly selected and their serum testosterone and PSA levels after ADT were recorded, with the PSA level rising continuously above nadir from 5 months 2021 at the post ADT testosterone castration level as shown in fig. 30A and fig. 30B, respectively.

On 26/5/2021, the patient successfully received robot-assisted radical prostatectomy (RARP) and bilateral pelvic lymphadenectomy under general anesthesia. A portion of the fresh specimens was sent to the laboratory for primary culture of PCa cells (prostate cancer cells) within 2 hours after removal from the body. The procedure was as follows: CRPC tissue was rinsed several times with sterile saline and cut into 3mm thick pieces. The pieces were then digested with 0.1% collagenase I (Sigma) in alpha-MEM (Corning) for 2 hours at 37 ℃ in a shaking incubator. The pieces were washed 3 times with PBS and centrifuged at 300g for 5 minutes at low speed to remove collagenase I. Cell culture dishes of 10cm were precoated with FBS (Gibco).

The tissue was seeded on FBS-free culture dishes at 37 ℃ in 5% CO₂Was cultured for 24 hours in a humidified incubator, and then 10mL of α -MEM was added and 10% FBS was added to the culture dish for further cell culture.

The tumor sections were HE stained (x 100) and the staining results are shown in fig. 31A, and fig. 31B is an optical micrograph of primary prostate cancer cells (x 200) at day 14.

After incubating PCa (prostate cancer cells) with TABP-EIC-WTN cells for 24 hours, the expression of PD-L1 on PCa was detected by flow measurement, and the results are shown in FIG. 32A and FIG. 32B, which demonstrates that Atezolizumab enhances the cytotoxicity of TABP-EIC-WTN on CRPC cells.

CCK-8 assay analyzes the tumor inhibition effect of TABP-EIC-WTN cells when atezolizumab/nivolumab is added (E: T ═ 1:1), statistics are shown in FIG. 32C, and the results show that the inhibition rate of TABP-EIC-WTN is remarkably enhanced by atezolizumab (20 μ g/mL) (E: T ═ 1:1), and further show that the combination of PD-L1 inhibitor and TABP-EIC-WTN can effectively treat prostate cancer.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

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Asn His Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr

180 185 190

Ser Met Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu Pro Ala

195 200 205

Tyr Thr Leu Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly Ser Arg

210 215 220

Lys Arg Asn His Ser Pro Ser Phe Asn Ser Thr Ile Tyr Glu Val Ile

225 230 235 240

Gly Lys Ser Gln Pro Lys Ala Gln Asn Pro Ala Arg Leu Ser Arg Lys

245 250 255

Glu Leu Glu Asn Phe Asp Val Tyr Ser Met Gly Trp Ile Arg Gly Arg

260 265 270

Arg Ser Arg His Ser Trp Glu Met Ser Glu Phe His Asn Tyr Asn Leu

275 280 285

Asp Leu Lys Lys Ser Asp Phe Ser Thr Arg Trp Gln Lys Gln Arg Cys

290 295 300

Pro Val Val Lys Ser Lys Cys Arg Glu Asn Ala Ser

305 310 315

<210> 12

<211> 951

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgtggacca accaccacca gcacagcaag gtgagagagg gcggcggcag ctggaccaac 120

caccaccagc acagcaaggt gagagagggc ggcggcagct ggaccaacca ccaccagcac 180

agcaaggtga gagagaccac taccccagca ccgaggccac ccaccccggc tcctaccatc 240

gcctcccagc ctctgtccct gcgtccggag gcatgtagac ccgcagctgg tggggccgtg 300

catacccggg gtcttgactt cgcctgcgat caggactgtc agaatgccca tcaggaattc 360

agattttggc cgtttttggt gatcatcgtg attctaagcg cactgttcct tggcaccctt 420

gcctgcttct gtgtgtggag gagaaagagg aaggagaagc agtcagagac cagtcccaag 480

gaatttttga caatttacga agatgtcaag gatctgaaaa ccaggagaaa tcacgagcag 540

gagcagactt ttcctggagg ggggagcacc atctactcta tgatccagtc ccagtcttct 600

gctcccacgt cacaagaacc agcatataca ttatattcat taattcagcc ttccaggaag 660

tctggttcca ggaagaggaa ccacagccct tccttcaata gcactatcta tgaagtgatt 720

ggaaagagtc aacctaaagc ccagaaccct gctcgattga gccgcaaaga gctggagaac 780

tttgatgttt attccatggg gtggattcgt ggtcggaggt ctcgacacag ctgggagatg 840

agtgaatttc ataattataa cttggatctg aagaagagtg atttttcaac acgatggcaa 900

aagcaaagat gtccagtagt caaaagcaaa tgtagagaaa atgcatctta a 951

Claims

1. Use of a tumor antigen binding peptide-engineered immune cell, a PD-L1 inhibitor and a co-cultured CIK cell in combination for the preparation of a combined preparation for the treatment of cancer;

preferably, the cancer is prostate cancer.

2. The use according to claim 1, wherein the tumor antigen binding peptide-the composition of the tumor antigen binding peptide in the engineered immune cell is WTN polypeptide region-hinge region-transmembrane domain-costimulatory domain-primary signaling domain or WTN polypeptide region-hinge region-transmembrane domain-primary signaling domain;

preferably, the tumor antigen binding peptide is composed of a WTN polypeptide region-hinge region-transmembrane domain-costimulatory domain-primary signaling domain;

preferably, the WTN polypeptide region comprises a linker between the plurality of repeats of the WTN polypeptide and the plurality of repeats of the WTN polypeptide;

more preferably, the WTN polypeptide is a polypeptide that specifically binds PSMA;

most preferably, the WTN polypeptide is selected from any one of the following group:

(1) 1, as shown in SEQ ID NO;

(2) 1, or a product formed by substitution, deletion or addition at one or more of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 of the polypeptide shown in SEQ ID NO;

most preferably, the WTN polypeptide is a polypeptide as shown in SEQ ID NO. 1;

most preferably, the nucleotide sequence of the WTN polypeptide is shown as SEQ ID NO. 2.

3. The use of claim 2, wherein the hinge region of the tumor antigen binding peptide is a CD8 a hinge region;

preferably, the amino acid sequence of the CD8 alpha hinge region is shown as SEQ ID NO. 3;

more preferably, the nucleotide sequence of the CD8 a hinge region is shown in SEQ ID NO 4;

preferably, the transmembrane domain is a 2B4 transmembrane domain;

more preferably, the amino acid sequence of the transmembrane domain of 2B4 is shown in SEQ ID NO 5;

most preferably, the nucleotide sequence of the transmembrane domain of 2B4 is shown in SEQ ID NO 6;

preferably, the co-stimulatory domain is a 2B4 co-stimulatory domain;

more preferably, the amino acid sequence of the 2B4 co-stimulatory domain is shown in SEQ ID NO 7;

most preferably, the nucleotide sequence of the 2B4 co-stimulatory domain is shown in SEQ ID NO 8;

preferably, the primary signaling domain is the NKG2D primary signaling domain;

more preferably, the amino acid sequence of the NKG2D primary signaling domain is as set forth in SEQ ID NO 9;

most preferably, the NKG2D primary signaling domain has the nucleotide sequence shown in SEQ ID NO 10;

most preferably, the tumor antigen binding peptide consists of the WTN polypeptide region-CD 8 α hinge region-2B 4 transmembrane domain-2B 4 costimulatory domain-NKG 2D primary signaling domain;

most preferably, the amino acid sequence of the tumor antigen binding peptide is shown in SEQ ID NO. 11;

most preferably, the nucleotide sequence of the tumor antigen binding peptide is shown in SEQ ID NO 12.

4. The use of claim 1, wherein the tumor antigen binding peptide-engineered immune cells comprise tumor antigen binding peptide-engineered NK cells, tumor antigen binding peptide-engineered T cells, tumor antigen binding peptide-engineered B cells, tumor antigen binding peptide-engineered macrophages;

preferably, the tumor antigen binding peptide-engineered immune cells are tumor antigen binding peptide-engineered NK cells.

5. The use of claim 1, wherein the PD-L1 inhibitor comprises atezolizumab, avelumab, durvalumab;

preferably, the PD-L1 inhibitor is atezolizumab.

6. The use of claim 1, wherein the co-cultured CIK cells are DC-CIK cells co-cultured with prostate cancer cells;

preferably, the co-cultured CIK cells comprise CD8+ T cells;

more preferably, the preparation method of the co-cultured CIK cells comprises the following steps:

(1) culturing the DC cells;

(2) culturing the CIK cells;

(3) mixing the DC cells obtained by culturing in the step (1) and the CIK cells obtained by culturing in the step (2) in a culture medium, adding prostate cancer cells for co-culture, and collecting the suspended DC-CIK mixture to obtain co-culture CIK cells;

most preferably, the step (1) comprises the steps of:

(a) on day 0, PBMC cells were cultured in DC cell growth medium;

(b) on day 3, the DC cell growth medium was changed;

(d) collecting cells on 8 th day to obtain DC cells;

most preferably, the substances added to the DC cell growth medium in step (a) comprise FBS, DC cell culture factors;

most preferably, the substance added to the DC cell growth medium in step (a) comprises 5% FBS, 2U/mL DC cell culture factor;

most preferably, the final concentration of the DC cell maturation factor in step (c) is 2U/mL;

most preferably, the step (2) comprises the steps of:

(a) on day 0, PBL cells were cultured in CIK cell activation medium;

(b) supplementing the cell culture with CIK cell proliferation medium on days 3, 4, and 6;

(c) collecting cells on 8 days to obtain CIK cells;

most preferably, the substances added to the CIK cell activation medium in step (a) include KBM551, FBS, anti-CD 3 antibody, IFN-gamma, IL-1 alpha, IL-2;

most preferably, the substance added to the CIK cell activation medium in step (a) comprises KBM551, 3% FBS, 50ng/mL anti-CD 3 antibody, 1000U/mLIFN-gamma, 100U/mLIL-1 alpha, 500U/mL IL-2;

most preferably, the PBL cells of step (a) have a cell density of 1.5X 10⁶/mL；

Most preferably, the substances added to the CIK cell proliferation medium in step (b) include KBM551, FBS, IL-2;

most preferably, the substances added to the CIK cell proliferation medium in step (b) include KBM551, 3% FBS and 500U/mL IL-2;

most preferably, the medium in step (3) is a CIK cell proliferation medium;

most preferably, the DC cells have a cell density of 4X 10⁷/mL；

Most preferably, the cell density of the CIK cells is 1 × 10⁸/mL；

Most preferably, the prostate cancer cells have a cell density of 1 × 10⁷/mL；

Most preferably, the CO-cultivation conditions are 5% CO₂、37℃、24h；

Most preferably, the prostate cancer cells comprise C4-2, LNCaP, PC-3, DU 145;

most preferably, the prostate cancer cell is C4-2.

7. A combined preparation for use in the treatment of cancer, comprising a tumor antigen binding peptide-engineered immune cell, a PD-L1 inhibitor, co-cultured CIK cells;

preferably, the cancer is prostate cancer.

8. The combination preparation of claim 7, wherein the tumor antigen-binding peptide-engineered immune cell is the tumor antigen-binding peptide-engineered immune cell of any one of claims 1 to 4.

9. The combination formulation of claim 7, wherein the PD-L1 inhibitor is the PD-L1 inhibitor of claim 5.

10. Combined preparation according to claim 7, wherein the co-cultured CIK cells are the co-cultured CIK cells according to claim 6.