CN114560910A

CN114560910A - Use of a PD-L1 inhibitor in combination with a tumor antigen binding peptide-engineered immune cell in the treatment of prostate cancer

Info

Publication number: CN114560910A
Application number: CN202210149771.1A
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: 2022-05-31
Also published as: CN114315976A; CN113527434A; CN114315977A; CN114835781A; CN114315976B; CN114835781B

Abstract

The invention discloses an application of a PD-L1 inhibitor and a tumor antigen binding peptide-engineered immune cell in prostate cancer treatment, wherein the PD-L1 inhibitor and the tumor antigen binding peptide-engineered immune cell are combined and applied to the treatment of the prostate cancer for the first time, and the invention shows better treatment effect.

Description

Use of a PD-L1 inhibitor in combination with a tumor antigen binding peptide-engineered immune cell in the treatment of prostate cancer

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of a PD-L1 inhibitor and a tumor antigen binding peptide-engineered immune cell in prostate cancer treatment.

Background

The current tumor treatment methods mainly comprise surgical treatment, radiotherapy, chemotherapy, targeted drug therapy and the like, wherein the surgical treatment is conventional surgical resection of tumors, and the problems that tumor cells are difficult to be completely resected, postoperative recurrence is easy to occur and the like exist; radiotherapy and chemotherapy can kill normal cells while killing tumor cells, thus causing great harm to the organism and the psychology of a patient; although the targeted drug therapy can reduce the adverse reaction of the drug, the drug resistance of the tumor is easy to generate, thereby causing the tumor recurrence. With the development of molecular biology and tumor biology, tumor immunotherapy (Cancer immunotherapy) changes the traditional tumor treatment mode and becomes a new tumor treatment means, and is different from the traditional tumor treatment means in that the target of tumor immunotherapy is mainly immune cells, and the immune system of an organism is activated by inhibiting immune negative regulatory factors, enhancing the recognition capability of the immune cells on the surface antigen of tumor cells and the like, so that the tumor cells are eliminated, and the tumor immunotherapy has the advantages of good effect, small adverse reaction, relapse prevention and the like.

In recent years, with further understanding of the immune escape mechanism of tumors, various novel tumor immunotherapies have been developed. Tumor immunotherapy falls into two categories: one is an immune checkpoint inhibitor and the other is cellular immunotherapy, among which the most widely studied immune checkpoints are CTLA-4 and PD-L1/PD-1. CTLA-4 is a T cell surface receptor, as an immunosuppressive molecule, capable of participating in the transmission of immunosuppressive signals, and in 2011, the FDA approved the first antibody drug ipilimumab (ipilimumab) directed against immune checkpoint CTLA-4; PD-1 is another common immunosuppressive molecule on the surface of a T cell, the ligand PD-L1 of the PD-L1 is proved to be expressed on the surface of various tumor cells, and the tumor cells expressing the PD-L1 inhibit the activation of the T cells by binding with the PD-1 on the surface of the T cells, so that the tumor immune escape is realized. PD-L1/PD-1 inhibitors block these immune checkpoints and enhance the killing activity of T cells against tumor cells. Currently, PD-1 monoclonal antibodies such as nivolumab (Opdivo), pembrolizumab (Keytruda), Cemiplimab (Libtayo), and PD-L1 monoclonal antibodies such as atezolizumab (Tecntriq), avelumab (Bavencio), durvalumab (Imfinzi) are approved by the FDA.

Among cellular immunotherapies, Chimeric antigen receptor modified T cells (CAR-T) and Chimeric antigen receptor modified NK cells (CAR-NK) are two types of tumor immunotherapies that are currently the most rapidly developed and have good application prospects. The T cell or NK cell modified by the CAR can specifically recognize tumor-associated antigens on the surface of tumor cells, so that the targeting property, killing activity and durability of the effector T cell or NK cell are higher than those of immune cells applied conventionally. However, current CAR cell therapy depends on a Tumor type with a specific Tumor-associated antigen (TAA), if the Tumor-associated antigen of the Tumor is not clear or identified, a single-chain antibody sequence with a related specificity cannot be prepared based on the Tumor-associated antigen, and thus a specific CAR capable of effectively recognizing the Tumor cell and a CAR-T or CAR-NK cell capable of specifically recognizing and killing the Tumor cell cannot be constructed.

In order to solve the technical problems in the art, the present invention aims to provide an application of a PD-L1 inhibitor and TABP-EIC-WTN (Tumor Antigen Binding Peptide-engineered Immune Cell) in combination for treating prostate cancer, wherein the combination of the PD-L1 inhibitor and the TABP-EIC-WTN has a good therapeutic effect on prostate cancer. In addition, the WTN is a polypeptide which is obtained by screening a phage-polypeptide library according to the present invention, can specifically bind to tumor cells, and has high binding efficiency. At present, no research reports that the PD-L1 inhibitor and TABP-EIC-WTN are combined to be applied to the treatment of tumors.

Disclosure of Invention

In view of the above, in order to overcome the above existing defects in the field, the present invention aims to provide an application of a combination of a PD-L1 inhibitor and a tumor antigen binding peptide-engineered immune cell in prostate cancer treatment, and experimental verification proves that the combination of the PD-L1 inhibitor and the tumor antigen binding peptide-engineered immune cell has a better treatment effect on prostate cancer, significantly enhances an anti-tumor effect, and is significantly better than the treatment effect of the single tumor antigen binding peptide-engineered immune cell.

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

the invention provides the application of the combination of the PD-L1 inhibitor and the tumor antigen binding peptide-engineered immune cell in the preparation of the cancer targeted therapy medicine;

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, gastric cancer, esophageal cancer, thyroid cancer, transitional epithelial carcinoma of the wing, leukemia, brain tumor, gastric 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 to 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.

In a specific embodiment of the invention, the WTN polypeptide is a novel polypeptide that is obtained by screening a phage-polypeptide library and has high binding efficiency and can specifically bind to tumor cells (prostate cancer cells).

In a specific embodiment of the invention, the PD-L1 inhibitor is atezolizumab, but the PD-L1 inhibitor of the invention is not limited to atezolizumab, and both avelumab, durvalumab and the PD-L1 inhibitor under study, which are now approved by the FDA, or PD-L1 inhibitor which is approved by the FDA in the future and PD-L1 inhibitor which will be studied in the future are within the scope of the invention.

A second aspect of the invention provides a pharmaceutical composition for the treatment of cancer;

preferably, the cancer is prostate cancer.

Further, the pharmaceutical composition comprises a PD-L1 inhibitor and a tumor antigen binding peptide-engineered immune cell.

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

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

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;

preferably, the hinge region is a CD8 a hinge region;

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

most preferably, the nucleotide sequence of the CD8 a hinge region is set forth in SEQ ID NO 4;

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

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

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

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

most 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.

A third aspect of the present invention provides a method of screening for a potential substance for treating cancer;

preferably, the cancer is prostate cancer.

Further, the method comprises the steps of:

(1) providing a candidate substance and a positive control substance, wherein the positive control substance is a combination of a PD-L1 inhibitor and a tumor antigen binding peptide-engineered immune cell;

(2) detecting the effect of the candidate substance on the proliferation, migration, and/or invasion of the cancer cells in the test group, and comparing the result with the experiment results of the positive control group and the negative control group;

(3) if the candidate substance has a significantly higher inhibition degree on the proliferation, migration and/or invasion of the cancer cells than the negative control group and is more than 75% of the positive control group, the candidate substance is suggested to be a potential substance for treating the cancer cells;

preferably, the cancer is prostate cancer.

In a fourth aspect, the invention provides the use of a PD-L1 inhibitor in combination with a tumor antigen-binding peptide-engineered immune cell for screening for potential agents for the treatment of cancer;

preferably, the cancer is prostate cancer.

Furthermore, the present invention provides a method of treating a patient with prostate cancer, the method comprising administering to a subject in need thereof an effective amount of a PD-L1 inhibitor and TABP-EIC-WTN, and/or a pharmaceutical composition according to the second aspect of the invention.

The invention has the advantages and beneficial effects that:

the invention firstly applies the combination of the PD-L1 inhibitor and TABP-EIC-WTN to the treatment of the prostatic cancer, and experimental verification proves that the combination of the PD-L1 inhibitor and the tumor antigen binding peptide-engineered immune cell has better treatment effect on the prostatic cancer and is obviously superior to the treatment effect of a single PD-L1 inhibitor or a single tumor antigen binding peptide-engineered immune cell.

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 immunofluorescence results of Lncap cell line with higher expression of polypeptide WTN and PSMA;

FIG. 2 is a graph showing the immunofluorescence results of the 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 on C4-2 cells cocultured 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 level of PD-L1 on TABP-EIC-WTN cells cultured using a common medium and supernatants obtained after 12 hours of co-culture from TABP-EIC-WTN + C4-2 cells were shown, and the percentage of PD-L1 expression did not significantly differ in the 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, plot 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 was measured 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 the 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 the 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 primers: 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 bacterial culture containing 50% glycerol, non-competent cells, 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 mu L per well of the microporous plate) of the 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 flat 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 are eluted with 1mL (100. mu.L in microwell plates) of the appropriate elution buffer, the known ligand of the target molecule is dissolved in TBS solution at a concentration of 0.1-1mM or the bound phage are competitively eluted from the immobilized target molecule with free target molecule solution (-100. mu.g/mL in TBS), the elution is taken up in another clean microfuge tube with gentle shaking at room temperature for 10-60 min; 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 shaking for >10min, and absorbing the eluate into another clean microcentrifuge tube, and then neutralizing the eluate with 150. mu.L (15. mu.L for microwell) of 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, 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 precipitate 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 as low as 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 titer of the eluate from the third round of panning, which was not amplified, was determined on LB/IPTG/Xgal plates and, unless a further round of panning was performed, plaques obtained at the time of 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 costimulation 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 tumor antigen-binding peptide structure 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) carrying out normal culture and amplification on the 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 result of detecting PD-L1 expressed by C4-2 cells is shown in figure 3: the Mean Fluorescence Intensity (MFI) of PD-L1 of C4-2 cells and the percentage of C4-2 cells expressing PD-L1 were significantly upregulated over time.

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

The expression level of PD-L1 on C4-2 cells at various concentrations of IFN- γ, and the addition of IFN- γ to C4-2 cells, is 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.

Both Gene Ontology (GO) analysis and KEGG enrichment analysis of differentially expressed genes were performed with KOBAS software (version: 2.1.1) to correct the P-cut to 0.05 to determine statistically significant enrichment. By detecting alternative splicing events using rMATS (version 3.2.5), the FDR value cutoff was 0.05 and the absolute value of. DELTA.. psi was 0.05.

The volcano pattern of deregulated genes between co-cultured TABP-EIC-WTN and separately cultured TABP-EIC-WTN cells is shown in FIG. 13A, and the volcano pattern 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 non-paired student t-test.

The KEGG pathway enrichment analysis for up-regulated genes in co-culture in fig. 13A is shown in fig. 14A, and down-regulated genes are shown 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 enriched KEGG pathways.

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 blocking agent (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. The NKG2D blocker inhibited 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, 17F), 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 the signal pathway involved in PD-L1 expression is involved in the co-culture of TABP-EIC-WTN and C4-2 cells.

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 prostate cancer cells, while nivolumab (20 mug/mL) slightly enhances the inhibition rate of TABP-EIC-WTN on prostate cancer cells. E: t is 1: 1. 5: the results at1 are shown in fig. 19A and 19B, respectively. Shows that the combination of TABP-EIC-WTN and PD-L1 inhibitor can effectively treat prostatic 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 the expression of CD107a in TABP-EIC-WTN cells induced by C4-2 cells in the presence of atezolizumab or nivolumab was examined using flow cytometry, with the results shown in figure 21, indicating that activated NK cells produce cytotoxic effects primarily 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 with 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) containing 5% FBS and 2U/mL DC culture factor (Novoprotein) at 37 deg.C, CO₂Culture 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 proceduresApproved by the ethical committee of our hospital and conducted according to the principles of laboratory animal care (NIH publication volume 25, revision 28 of 1996). Mice were inoculated subcutaneously in the upper abdomen 2X 10 suspended in 200. mu.L PBS⁶C4-2 cells. Tumor volume of 100-200mm at day 7³Treatment 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 the PD-1 antibody nivolumab (GlpBio, GC34218) (10mg/kg) or the control PBS tail vein were administered on

days

7, 9, 11, 13, 15 post-tumor vaccination for a total of 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

Bioluminescence intensity (BLI) by spectral CT measurements tumor size before and after treatment was assessed, and BLI was measured and expressed as radiometric (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 graph 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. Necrotic areas were 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 11 Atezolizumab 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).

Tissue inoculated in a culture mediumFBS in Petri 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 result shows that the inhibition rate of TABP-EIC-WTN is remarkably enhanced by atezolizumab (20 μ g/mL) (E: T ═ 1:1), and further shows 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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<141> 2022-02-17

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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

The use of a combination of a PD-L1 inhibitor and a tumor antigen binding peptide-engineered immune cell in the preparation of a medicament for the targeted 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;

(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 in 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 as shown in SEQ ID NO. 7;

most preferably, the nucleotide sequence of the 2B4 co-stimulatory domain is shown as 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 any one of claims 1-3, wherein said 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. A pharmaceutical composition for treating cancer, comprising a PD-L1 inhibitor and a tumor antigen-binding peptide-engineered immune cell;

preferably, the cancer is prostate cancer.
7. The pharmaceutical composition of claim 6, wherein the tumor antigen binding peptide-the tumor antigen binding peptide in the engineered immune cell is comprised of a WTN polypeptide region-a hinge region-a transmembrane domain-a costimulatory domain-a primary signaling domain;

preferably, the WTN polypeptide region includes linkers between the plurality of WTN polypeptide repeats and the plurality of WTN polypeptide repeats;

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

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;

preferably, the hinge region is a CD8 a hinge region;

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

most preferably, the nucleotide sequence of the CD8 a hinge region is set forth 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 as 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 as SEQ ID NO 12;

most preferably, the tumor antigen-binding peptide-engineered immune cells are tumor antigen-binding peptide-engineered NK cells.
8. The pharmaceutical composition of claim 6, wherein the PD-L1 inhibitor comprises atezolizumab, avelumab, durvalumab;

preferably, the PD-L1 inhibitor is atezolizumab.
9. A method of screening for potential agents for the treatment of cancer, the method comprising the steps of:

(1) providing a candidate substance and a positive control substance, wherein the positive control substance is a combination of a PD-L1 inhibitor and a tumor antigen binding peptide-engineered immune cell;

(2) detecting the effect of the candidate substance on the proliferation, migration and/or invasion of the cancer cells in the test group, and comparing the result with the experimental results of the positive control group and the negative control group;

(3) if the candidate substance has a significantly higher inhibition degree on the proliferation, migration and/or invasion of the cancer cells than the negative control group and is more than 75% of the positive control group, indicating that the candidate substance is a potential substance for treating the cancer;

preferably, the cancer is prostate cancer.
Use of a PD-L1 inhibitor in combination with a tumor antigen binding peptide-engineered immune cell for screening for potential agents for the treatment of cancer;

preferably, the cancer is prostate cancer.