CN118443940A - Application of SP70 in preparation of lung cancer PD-1/PD-L1 blocking treatment efficacy prediction reagent - Google Patents
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Abstract
The invention discloses application of SP70 in preparing a reagent for predicting curative effect of lung cancer PD-1/PD-L1 blocking treatment. Application of reagent for detecting SP70 in serum in preparing reagent for predicting curative effect of lung cancer PD-1/PD-L1 blocking treatment. The seropositive detection rate of the lung cancer patients is obviously higher than that of the lung benign disease patients and healthy physical examination patients, in addition, SP70 is found to be involved in the regulation of the expression of PD-1 on the surface of CD8 + T cells in the tumor microenvironment, and the research shows that the proportion of CD8 +PD-1+ T cells in peripheral blood can predict the curative effect of PD-1/PD-L1 blocking treatment. This suggests the potential value of SP70 as a novel tumor marker for prediction of the efficacy of PD-1/PD-L1 blocking therapy in lung cancer patients. Therefore, the reagent for detecting SP70 in serum can be applied to preparing a reagent for predicting the curative effect of the lung cancer PD-1/PD-L1 blocking treatment or an auxiliary reagent for predicting.
Description
Technical Field
The invention belongs to the field of biological diagnosis, and relates to application of SP70 in preparation of a lung cancer PD-1/PD-L1 blocking treatment efficacy prediction reagent.
Background
Although conventional treatment methods of lung cancer such as surgery, radiotherapy, chemotherapy, targeted therapy and the like have advanced to some extent, the survival rate of lung cancer patients for 5 years is still low, only 15%. Immune checkpoint molecule blocking therapy represented by PD-1/PD-L1 provides a novel model for lung cancer treatment. Since 2015, two PD-1 antibodies were approved for secondary treatment of non-small cell lung cancer, but only about 20% of lung cancer patients who benefited from PD-1/PD-L1 blocking treatment, there is an urgent need for biomarkers that could predict clinical efficacy. At present, the curative effect of blocking PD-1/PD-L1 mainly depends on detecting the expression of PD-L1 in tumor tissues, but the efficiency response rate of the therapeutic effect prediction is less than 50%, and the tissue is difficult to obtain for invasive detection. In addition, the research finds that the PD-1/PD-L1 blocking therapeutic effect prediction marker which is most used clinically has limited PD-L1 prediction capability, and meanwhile, the expression of PD-L1 is unstable and is influenced by radiotherapy and chemotherapy. Therefore, the search for a reliable marker for PD-1/PD-L1 blocking treatment in clinical efficacy evaluation of lung cancer patients has important theoretical and clinical application values. Because clinical blood samples are readily available, more and more researchers have sought blood markers for the prediction of the efficacy of PD-1/PD-L1 blocking therapy.
Tumor specific protein 70 (tumor specific protein, TP70, also called SP 70) is a novel tumor marker discovered in the early stage of the laboratory, and clinical studies prove that the positive detection rate of SP70 in serum and tissues of lung cancer patients is obviously higher than that of lung benign disease patients and healthy physical examination patients. The results of the previous published articles show that the SP70 level of lung cancer chemotherapy patients is obviously reduced, and the dynamic evaluation of the chemotherapy response of the patients is carried out, namely, after every 2 chemotherapy cycles, the analysis of the tumor response chemotherapy response is divided into Complete Remission (CR), partial Remission (PR), stable illness State (SD) and illness state (PD) through CT scanning by adopting a standard of evaluation of solid tumor response (RECIST 1.1) guideline, so that the result proves that the SP70 can be used as an index for the dynamic evaluation of the chemotherapy curative effect. However, no report exists on the SP70 as a target for predicting the curative effect of the PD-1/PD-L1 blocking treatment of lung cancer, no report exists on the curative effect of the SP70 and tumor immunotherapy, and the immunotherapy represented by the PD-1/PD-L1 blocking agent treatment is a completely different tumor treatment scheme from the chemotherapy, so that whether the SP70 can be used for predicting the curative effect of the PD-1/PD-L1 blocking treatment of lung cancer is not expected.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and providing application of SP70 serving as a detection target in preparing a lung cancer PD-1/PD-L1 blocking treatment efficacy prediction reagent or an auxiliary reagent.
It is still another object of the present invention to provide the use of an agent for detecting SP70 in serum for the preparation of a reagent for predicting the efficacy of a lung cancer PD-1/PD-L1 blocking treatment or an auxiliary reagent.
The aim of the invention can be achieved by the following technical scheme:
Application of SP70 as detection target in preparing lung cancer PD-1/PD-L1 blocking treatment efficacy prediction reagent or auxiliary prediction reagent.
Application of reagent for detecting SP70 in serum in preparing reagent for predicting curative effect of lung cancer PD-1/PD-L1 blocking treatment or auxiliary reagent for predicting curative effect of lung cancer PD-1/PD-L1 blocking treatment.
In a preferred embodiment of the present invention, the reagent for detecting SP70 in serum is a reagent for quantitatively detecting SP70 in serum. As a preferred aspect of the present invention, the reagent for detecting SP70 in serum comprises monoclonal antibody NJ001-1 of SP70, wherein the monoclonal antibody NJ001-1 has a collection number of CCTCC NO: the hybridoma cell line NM001-1 of C201172 was secreted. The preservation number is CCTCC NO: the hybridoma cell line NM001-1 of C201172 is disclosed in CN 102391992A.
The beneficial effects are that:
The present invention first found that the serum SP70 concentration was significantly higher than 17 non-responders (P < 0.001) in 8 of 25 patients treated with PD-1/PD-L1 blocking, the SP70 concentration was significantly decreased (P < 0.05) after treatment with PD-1/PD-L1 blocking, and the SP70 concentration was significantly increased (P < 0.05) after treatment with PD-1/PD-L1 blocking. Subsequently, another 63 lung cancer patients receiving PD-1/PD-L1 blocking treatment were enrolled, and based on their pre-treatment SP70 concentrations and the corresponding treatment response outcomes, ROC curve analysis results showed that SP70 showed an AUC area of 0.7532 for predicting the presence or absence of a PD-1/PD-L1 blocking treatment (fig. 1d, p < 0.001). The invention also finds that SP70 participates in the regulation of the expression of PD-1 on the surface of CD8 + T cells in a tumor microenvironment, the PD-1 of the CD8 + T cells after the treatment of the SP70 is obviously increased (P < 0.001), and the PD-1 of the CD8 + T cells after the addition of the monoclonal antibody NJ001 is obviously decreased (P < 0.001); studies show that the ratio of peripheral blood CD8 +PD-1+ T cells can predict the curative effect of PD-1/PD-L1 blocking treatment. This suggests the potential value of SP70 as a novel tumor marker for prediction of the efficacy of PD-1/PD-L1 blocking therapy in lung cancer patients. Ubiquitination is one of important ways of protein modification, and an immune coprecipitation result shows that PD-1 protein and ubiquitin have a binding effect, and the binding of PD-1 and ubiquitin is reduced after SP70 treatment, so that PD-1 degradation is delayed. The lung cancer patients clinically receiving PD-1/PD-L1 blocking treatment in a group track the case information, and the serum SP70 level is found to be related to the curative effect of the PD-1/PD-L1 blocking treatment, and the SP70 can be used as a detection target for the curative effect prediction or auxiliary prediction of the PD-1/PD-L1 blocking treatment of the lung cancer.
At present, about 20% of lung cancer patients who receive PD-1/PD-L1 blocking treatment benefit from the treatment, so that reliable curative effect prediction markers are needed. The serum SP70 is convenient to detect and sample, provides a non-invasive examination index for the prediction of the curative effect of the PD-1/PD-L1 blocking treatment of a lung cancer patient, screens out a part of population benefiting from the non-invasive examination index, can provide curative effect monitoring and evaluation, and can be used for the medication guidance of the PD-1/PD-L1 blocking treatment of the lung cancer patient. The SP70 is obviously related to the ratio of peripheral blood immune cells CD8 +PD-1+ T cells, in vitro cell experiments prove that the SP70 can cause the anti-tumor immune function of cells to be damaged by up-regulating the expression of CD8 + T cells PD-1, and the anti-tumor immune function of the CD8 + T cells can be obviously recovered by adding the PD-1 inhibitor. Compared with blood immune cell detection, SP70 detection is more convenient and quick.
Drawings
FIG. 1 serum SP70 concentrations before and after treatment of lung cancer patients receiving PD-1/PD-L1 blocking treatment. Serum SP70 concentration of healthy physical examination patients and lung cancer patients; pre-treatment serum SP70 concentrations in patients receiving PD-1/PD-L1 blocking treatment with and without response; serum SP70 concentrations before and after treatment of patients receiving PD-1/PD-L1 blocking treatment; ROC curve analysis of the value of serum SP70 concentration prior to receiving PD-1/PD-L1 blocking treatment for predicted efficacy; survival curve analysis of progression free survival of patients receiving PD-1/PD-L1 blocking treatment at different concentrations of serum SP70 levels
FIG. 2 peripheral blood CD8 +PD-1+ T cell fraction prior to treatment for lung cancer patients receiving PD-1/PD-L1 blocking treatment. The ratio of CD8 +PD-1+ T cells in peripheral blood of healthy physical examination patients and lung cancer patients; the ratio of CD8 +PD-1+ T cells in peripheral blood before treatment of patients who receive PD-1/PD-L1 blocking treatment with or without response; and C, performing correlation analysis on the concentration of SP70 in serum and the proportion of CD8 +PD-1+ T cells in peripheral blood of the patient subjected to PD-1/PD-L1 blocking treatment on lung cancer.
FIG. 3 SP70 promotes increased expression of PD-1 by CD8 + T cells. A, SP70 specific monoclonal antibody NJ001 can reverse the expression promotion effect of SP70 on PD-1; immunoblotting results show that SP70 promotes CD8 + T cell PD-1 expression to be increased
FIG. 4 Co-immunoprecipitation detection of PD-1 ubiquitination of CD8 + T cell surface after SP70 culture
FIG. 5 secretion of IFN-. Gamma.and Granzyme B after treatment of CD8 + T cells with monoclonal antibodies to SP70 and PD-1
Detailed Description
Example 1
Serum SP70 concentrations before and after treatment of 25 lung cancer patients receiving PD-1/PD-L1 blocking treatment were detected using an SP70 detection kit (ELISA), 30 healthy subjects served as controls, and efficacy was evaluated according to the solid tumor efficacy criteria (RECIST) and divided into complete remission (Complete response, CR), partial Remission (PR), stabilization (Stabledisease, SD), progression (Progressive disease, PD). According to literature (The PD-1expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1blockade therapies.Nat Immunol.2020,Nov;21(11):1346-1358.DOI:10.1038/s41590-020-0769-3),CR/PR/SD(>6 months) is defined as a responder to PD-1/PD-L1 blocking treatment (Responders); SD (< 6 months)/PD is defined as Non-responders to PD-1/PD-L1 blocking therapy (Non-Responders).
As shown in fig. 1A, the serum SP70 concentration was significantly higher in lung cancer patients than in healthy physical examination subjects (P < 0.01), with 8 of the patients treated with PD-1/PD-L1 block treatment having serum SP70 concentrations significantly higher than 17 non-responses (fig. 1b, P < 0.001), with responders treated with PD-1/PD-L1 block treatment having significantly lower SP70 concentrations (fig. 1c, P < 0.05), and with non-responders treated with PD-1/PD-L1 block treatment having significantly higher SP70 concentrations (fig. 1c, P < 0.05).
Subsequently, another 63 lung cancer patients receiving PD-1/PD-L1 blocking treatment were enrolled, and based on their pre-treatment SP70 concentrations and the corresponding treatment response outcomes, ROC curve analysis results showed that SP70 showed an AUC area of 0.7532 for predicting the presence or absence of a PD-1/PD-L1 blocking treatment (fig. 1d, p < 0.001). Following 12 months of follow-up of these 63 lung cancer patients receiving PD-1/PD-LI blocking treatment, their subsequent treatment outcomes were extracted from the electronic medical record system, and lung cancer patients were divided into high SP70 concentration groups (> 4.37 ng/ml) and low SP70 concentration groups (.ltoreq.4.37 ng/ml) based on a median value of 4.37ng/ml for serum SP70 concentration of the 63 lung cancer patients, resulting in a progression free survival (Progression-free survival) plotted, showing that the progression free survival time was longer for the high SP70 concentration group of patients compared to those receiving PD-1/PD-LI blocking treatment at lower SP70 levels (FIG. 1E, P < 0.001).
Example 2
Flow Cytometry (FCM) measures the proportion of CD8 +PD-1+ T cells in peripheral blood mononuclear cells (PERIPHERAL BLOOD MONONUCLEAR CELL, PBMCs) before and after treatment in 63 lung cancer patients receiving PD-1/PD-L1 blocking treatment, and 42 healthy subjects served as controls, and efficacy was evaluated as Complete Remission (CR), partial Remission (PR), stable (SD), progression (PD) according to the solid tumor efficacy criteria (RECIST). CR/PR/SD (> 6 months) was defined as responders to PD-1/PD-L1 blocking treatment (Responders); SD (< 6 months)/PD is defined as Non-responders to PD-1/PD-L1 blocking therapy (Non-Responders).
1) Diluting and mixing the collected fresh anticoagulated whole blood with PBS in equal volume;
2) Sucking fresh anticoagulated whole blood diluted by PBS by using a sterile Pasteur pipette, carefully spreading the fresh anticoagulated whole blood on the liquid surface of the separation liquid of the Ficoll lymphocytes which is added in advance, wherein the blood spreading volume is about 1-1.5 times of the separation liquid;
3) Rotating the centrifugal machine to 2000rpm, setting the rising time to 250s, the speed falling time to 300s, and centrifuging for 20min;
4) The middle cloudy and foggy white membrane layer is peripheral blood mononuclear cells, a small amount of PBS is added for washing, centrifugation is carried out at 1500rpm for 10min, and the supernatant is discarded;
5) Adding a small amount of PBS, washing again, centrifuging at 1500rpm for 10min, and discarding the supernatant;
6) Mu.l of PBS containing 0.1% BSA was added to each tube to stain buffer resuspended cells (cell concentration approximately 1X 107/ml).
5 Μl of anti-human-CD8-FITC antibody and 20 μl of anti-human CD279-APC antibody are added into a sample tube, no antibody is added into a blank tube, only one antibody is added into a single standard tube, and after uniform mixing, the mixture is incubated for 30min at 4 ℃ in a dark place;
7) Adding a proper amount of PBS into each tube, re-suspending the cells, uniformly mixing, centrifuging at 1500rpm for 8min, and discarding the supernatant;
8) Repeating the step 7, and removing the supernatant as clean as possible after washing;
9) The cells were resuspended in an appropriate amount of PBS for each tube and then examined using a flow cytometer.
The results are shown in figure 2A, where the proportion of CD8 +PD-1+ T cells in PBMCs of lung cancer patients was significantly higher than that of healthy physical examination subjects (P < 0.001), and the proportion of CD8 +PD-1+ T cells in PBMCs prior to treatment for PD-1/PD-L1 blocking treatment subjects was significantly higher than that for non-responders (figure 2b, P < 0.05). The prior literature reports that the proportion of CD8 +PD-1+ T cells in peripheral blood PBMC can predict the curative effect of PD-1/PD-L1 blocking treatment, and as shown in FIG. 2C, the proportion of CD8 +PD-1+ T cells in peripheral blood PBMC of a lung cancer patient has a significant positive correlation with the concentration of serum SP 70.
Example 3 in vitro cell experiments found that SP70 promotes increased expression of PD-1 by healthy human CD8 + T cells
1) Collecting fresh whole blood of a healthy person, and sorting CD8 + T cells;
2) SPC-A1 cells are taken to purify SP70 protein by utilizing SP70 specific monoclonal antibody NJ 001-1;
3) Culturing CD8 + T cells by SP70 protein;
a) Culturing CD8 + T cells respectively by taking RPMI-1640 culture medium as a blank control and taking purified SP70 protein which is fully dialyzed with the RPMI-1640 culture medium as an experiment;
b) Adding monoclonal antibody NJ001 to treat cells according to experimental requirements;
c) Blank and SP70 treated groups were added with anti-CD3 (3. Mu.g/ml) and anti-CD28 (2. Mu.g/ml) to promote CD8 +
Activation of T cells in vitro culture;
d) The cells were cultured in a 5% CO2 cell incubator at 37℃for 72 hours.
4) Flow-through detection of CD8 + T cell PD-1 expression the procedure is as in example 2;
5) Immunoblotting to detect CD8 + T cell PD-1 expression;
6) Co-immunoprecipitation
As shown in the flow chart of fig. 3A, CD8 + T cells PD-1 after SP70 treatment were significantly elevated (P < 0.001), and CD8 + T cells PD-1 after addition of monoclonal antibody NJ001 were significantly reduced (P < 0.001); as shown in the immunoblot results of fig. 3B, CD8 + T cells PD-1 were significantly elevated after SP70 treatment (P < 0.05). Ubiquitination is one of the important ways of protein modification, as shown by the co-immunoprecipitation results in FIG. 4, the PD-1 protein has a binding effect with ubiquitin, and the binding of PD-1 with ubiquitin is reduced after SP70 treatment, so that the degradation of PD-1 is delayed.
Example 4 detection of CD8 + T cell function changes following SP70 treatment.
The SP70 culture of CD8 + T cells was performed as in example 3, except that PD-1 mab and isotype IgG control cultures were added. As shown in FIG. 5, the ratio of production of cytokines IFN-r and Granzyme B by CD8 + T cells after SP70 treatment was down-regulated, while the ratio of production of cytokines IFN-r and Granzyme B by CD8 + T cells after addition of PD-1 mab was up-regulated, suggesting restoration of CD8 + T cell function after addition of PD-1 inhibitor.
Claims (4)
- Application of SP70 as detection target in preparing lung cancer PD-1/PD-L1 blocking treatment efficacy prediction reagent or auxiliary prediction reagent.
- 2. Application of reagent for detecting SP70 in serum in preparing reagent for predicting curative effect of lung cancer PD-1/PD-L1 blocking treatment or auxiliary reagent for predicting curative effect of lung cancer PD-1/PD-L1 blocking treatment.
- 3. The use according to claim 2, wherein the reagent for detecting SP70 in serum is a reagent for quantitatively detecting SP70 in serum.
- 4. The use according to claim 2, wherein the reagent for detecting SP70 in serum comprises monoclonal antibody NJ001-1 of SP70, said monoclonal antibody NJ001-1 having a collection number cctccc NO: the hybridoma cell line NM001-1 of C201172 was secreted.
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