CN115078727A - A marker, kit and application for predicting the effect of cancer immunotherapy - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to a marker, a kit and an application for predicting the effect of cancer immunotherapy. A marker for predicting the effect of cancer immunotherapy, the marker is CypB. Described CypB and PD-L1, tumor mutation burden TMB, EGFR mutation, tumor infiltrating lymphocyte TIL, DDR gene (DNA damage response gene), circulating tumor cell CTC, circulating tumor DNA (ctDNA), neutrophil/lymphocyte Any one or more combinations of cell ratio NLRs are used as markers for predicting the effect of cancer immunotherapy. The advantages of the present invention are: (1) high sensitivity and specificity. (2) The curative effect prediction accuracy rate is high. (3) The operation is simple and time-consuming. (4) Strong applicability and easy to promote.

Description

A marker, kit and application for predicting the effect of cancer immunotherapy

technical field

The invention relates to the field of biotechnology, in particular to a marker, a kit and an application for predicting the effect of cancer immunotherapy.

Background technique

Malignant tumors are one of the top three causes of death in the world, posing a great threat to human survival, and the incidence of malignant tumors is increasing year by year. In 2020, there will be 19.29 million new cancer cases worldwide and 1.8 million lung cancer deaths among the 9.96 million cancer deaths worldwide, far exceeding other cancer types and ranking first in cancer deaths.

With the continuous research on cancer, interventional therapy, targeted therapy and immunotherapy have emerged in the traditional treatment methods of surgery, radiotherapy and chemotherapy, among which immunotherapy has achieved good results in advanced cancer patients. Tumor immunotherapy is based on the principle of blocking the PD-1/PD-L1 immune checkpoint pathway. The specific antibodies of PD-1 or PD-L1 are injected into tumor patients, so that the tumor no longer has the ability to evade the immune system. Thereby promoting the body to remove tumor cells. The application of immune checkpoint inhibitors (ICIs) in the treatment of NSCLC is developing rapidly. However, there is still a lack of comprehensive and effective ICIs efficacy predictive markers to accurately screen the benefit population of ICIs.

Immunotherapy for NSCLC is mainly divided into: immune checkpoint inhibitors, cellular immunotherapy with adoptive infusion of immune cells, monoclonal antibodies, immune vaccines, and viral fusion therapy. Since 2015, a variety of immunosuppressive drugs have been approved for second-line or even first-line treatment of patients with advanced NSCLC, such as nivolumab, pembrolizumab, and atezolizumab. (Atezolizumab) and durvalumab (Durvalumab). Both nivolumab and pembrolizumab are anti-PD-1 (programmed death receptor 1) monoclonal antibodies that act on PD-1/PD-L1 (programmed death receptor ligand 1). , programmed death ligand 1) pathway. PD-1 is one of the most well-studied pathways to date. PD-1 inhibits the proliferation and differentiation of T cells by restricting the interaction between MHC molecular complexes and T cells by binding to its ligands. PD-1 inhibitors can achieve anti-tumor effects by inhibiting the blocking signal of T cell activation signals, so that T cells continue to activate.

The mechanism of immune checkpoint inhibitors represented by PD-1 and PD-L1 inhibitors is that the activation of immune cells increases the expression of immune checkpoints (such as PD-1/PD-L1) in the co-inhibitory signaling pathway, while the immune Inhibitors restore or even enhance the ability of immune cells to kill tumor cells by blocking the immune checkpoint of the co-inhibitory signaling pathway.

At present, the effective rate of PD-1 monoclonal antibody can reach about 20%, and there are still a large number of patients who cannot benefit from it.

Immunotherapy is expensive and has a low efficacy rate, which brings certain difficulties to the treatment of patients. Therefore, it is particularly important to accurately screen out the beneficiaries of ICIs and reduce the burden on patients. The expression of PD-L1 in tumor cells is considered to be an important predictive marker of efficacy, but there is no unified international standard for the detection of PD-L1 expression. Responses to ICIs can also be observed in about 10% of patients with negative PD-L1 expression, while a considerable proportion of patients with high PD-L1 expression do not respond to ICIs. With the expansion of application scope and the accumulation of data, more and more clinical observations show that PD-L1 as a predictor of immunotherapy is not perfect and has certain limitations.

CypB (Cyclophilins B, cyclophilin B, cycloprotein B), also known as Peptidyl-prolyl cis-trans isomerase B (PPIB), is the smallest 3-hydroxylated collagen complex. a member of . It is a protein encoded by the CypB gene, mainly present in the endoplasmic reticulum (ER), nucleus and extracellular space, and has peptidylprolyl cis-trans isomerase activity. CypB protein is expressed in almost all tissues, but the expression level is lower than that of CypA, and it has 65% sequence homology with CypA. CypB is involved in various processes such as protein folding, redox regulation, endoplasmic reticulum stress, ribosome synthesis and prolactin signaling. There have been a lot of reports on the up-regulated expression of CypA in human tumors, but there are not many reports on the expression of CypB in human tumors. , liver cancer and colorectal cancer occurrence and development are closely related.

In the era of lung cancer immunotherapy, the indications of immunotherapy are gradually expanding, but there are still problems such as low efficacy rate and large individual differences in practical application. How to effectively predict individualized efficacy of tumor patients and improve the accuracy of clinical treatment decisions based on specific markers is a key issue in current immunotherapy.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a marker for predicting the effect of cancer immunotherapy with high sensitivity and specificity, high curative effect prediction accuracy, strong adaptability, and easy promotion. To achieve the above purpose, the present invention adopts the following technical scheme :

In a first aspect, the present invention provides a marker for predicting the effect of cancer immunotherapy, and the marker is CypB.

In a specific embodiment, the CypB and PD-L1, tumor mutation burden TMB, EGFR mutation, tumor infiltrating lymphocyte TIL, DDR gene (DNA damage response gene), circulating tumor cell CTC, circulating tumor DNA (ctDNA) ), neutrophil/lymphocyte ratio (NLR), any one or more combinations are used as markers for predicting the effect of cancer immunotherapy.

In a second aspect, the present invention provides a kit for predicting the effect of cancer immunotherapy, the kit comprising at least an antibody of CypB.

In a third aspect, the present invention provides an application of CypB in the preparation of a kit for predicting the effect of cancer immunotherapy.

In a specific embodiment, the cancer patient is suffering from a cancer selected from the group consisting of renal cell carcinoma (RCC), colorectal cancer (CRC), gastric cancer (GC), melanoma, non-small cell lung cancer (NSCLC), glioblastoma tumor and any type of adenocarcinoma.

In a specific embodiment, an application of CypB in the preparation of a kit for predicting the effect of cancer immunotherapy, wherein the effect of immunotherapy is predicted by the following method, and the method comprises the following steps:

S1 Before immunotherapy, the CypB content in the blood of the subjects was detected;

S2 compares the detected value of CypB content in step S1 with a preset cutoff value, and if the detected value is less than the preset cutoff value, it is predicted that the immunotherapy is effective.

In a specific embodiment,

The method for determining the preset cutoff value is as follows: the subjects of retrospective or prospective studies are defined into two groups of treatment benefit and non-benefit according to their immunotherapy efficacy response, and the detection value of CypB content in the patient's blood is measured. ROC curve analysis was performed, and the detection value of CypB content at the Youden index was used as the cutoff value.

In a specific embodiment, the detection method of CypB content in the blood of the subject is any one of ELISA method, immunoblotting method, flow cytometry and liquid phase chip.

In a specific embodiment, the subject blood includes, but is not limited to, peripheral blood plasma, serum and exosomes in blood.

In another specific embodiment, the immunotherapy includes but is not limited to monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy, small molecule inhibitors and immune system modulators in immunotherapy any one or more combinations.

Compared with the prior art, the advantages of the marker, kit and application for predicting the effect of cancer immunotherapy provided by the present invention are:

(1) High sensitivity and specificity.

(2) The curative effect prediction accuracy rate is high.

(3) The operation is simple and time-consuming.

(4) Strong applicability and easy to promote.

Description of drawings

Figure 1 shows the relationship between the baseline CypB expression level of patients and the prognosis of immunotherapy.

In the figure, PD is disease progression, and immunotherapy is not beneficial; PR is disease remission, and immunotherapy is beneficial.

Figure 2 shows the ROC curve of baseline CypB levels predicting efficacy.

Detailed ways

In order to make those skilled in the art better understand the technical solution of the present invention, the following examples will further describe the present invention in detail, and the following examples are only used to illustrate the invention, but are not used to limit the scope of the invention.

Example 1

Using CypB (alias PPIB) as a marker, by detecting the plasma CypB protein level of patients before immunotherapy, according to the cutoff value, predict the efficacy of immunotherapy, evaluate the prognosis of immunotherapy, and whether they can benefit from immunotherapy.

1. Before the patient undergoes immunotherapy, the peripheral blood of the patient is collected and the plasma is separated.

2. ELISA method to detect the expression level of CypB in plasma

The standard was diluted according to the concentration gradient, and the serum to be tested was diluted 1:10.

Add 100 μl of the standard and the serum to be tested in turn to the ELISA microplate coated with the primary antibody. Incubate at 37°C for 2h.

Aspirate the standard and serum, add 300 μl of washing solution to each well, shake the microplate slightly, and then pour off the washing solution. Repeat the above washing process three times.

Add 100 μl of the diluted detection secondary antibody to each well and incubate at 37°C for 2 h.

Aspirate the standard and serum, add 300 μl of washing solution to each well, shake the microplate slightly, and then pour off the washing solution. Repeat the above washing process three times.

Add diluted HRP (horseradish peroxidase)-labeled secondary antibody, and incubate at 37°C for 45min.

Add 100 μl of the diluted detection secondary antibody to each well and incubate at 37°C for 2 h.

Aspirate the standard and serum, add 300 μl of washing solution to each well, shake the microplate slightly, and then pour off the washing solution. Repeat the above washing process three times.

100 μl of color developing solution was added, and the color was developed at room temperature for 20 min.

Add 50 μl of stop solution, and read by microplate reader at OD480nm wavelength.

The standard curve was drawn according to the OD value, and the serum CypB concentration was calculated.

3. Use the patient's plasma before treatment to predict the patient's immunotherapy efficacy (responsiveness)

Select the cutoff value (retrospective or prospective study subjects are defined as treatment benefit and non-benefit groups according to their response to immunotherapy, ROC curve analysis is performed on the detection value of CypB content in the blood of patients, Youden index The detection value of CypB content at the place is taken as the cutoff value), if the detection value is greater than the cutoff, the patient's curative effect response is poor, and immunotherapy may not be effective. If the detection value is less than cutoff, the patient's curative effect response is good, and the immunotherapy has long-term effect.

Example 2

Example 1 result analysis

Among the 21 patients tested, 13 patients received immunosuppressive + chemotherapy combined therapy (No. 8-18, 20, 21), and 8 patients received immunosuppressive single-agent (No. 1-7, No. 19) treatment. Patient information See Table 1 for information on immunotherapy.

Table 1 Patient information and immunotherapy information

In the evaluation of efficacy results, 14 patients had disease remission (PR), and the treatment benefited; 7 patients had disease progression (PD), and the treatment did not benefit. The relationship between CypB plasma level and disease efficacy is shown in Figure 1, and the ROC curve predicting the efficacy of immunotherapy is shown in Figure 2.

Figure 1 shows that the median value of plasma CypB level in PD group was significantly higher than that in PR group, and the difference was statistically significant (P<001).

The area under the ROC curve was AUC=0.816, p=0.021. According to the Youden index, the cutoff value was 3.092. At this cutoff value, the sensitivity and specificity of predicting the benefit of immunotherapy were 85.71% and 78.57%.

Among the 21 patients, 19 patients had PD-L1 immunohistochemical test results at the same time. The results are shown in Table 2.

The sensitivity, specificity, diagnostic accuracy, positive predictive value and negative predictive value of PD-L1 and CypB in predicting the benefit (PR) of immunotherapy were analyzed by using the four-table diagnostic test. The results are shown in Tables 3 and 4.

Table 2 Immunohistochemical test results

patient number baseline PD-L1 immunohistochemical results tumor type TNM Staging 1 1.641 80% squamous cell carcinoma T2aN2M1b 4 2 2.142 95% adenocarcinoma T4N2M1c 4 3 2.509 90% squamous cell carcinoma T4N3M0 3 4 4.756 55% squamous cell carcinoma T4N0M1a 4 5 56.425 95% adenocarcinoma T2aN3M1a 4 6 1.825 0% adenocarcinoma T3N2M1c 4 7 65.117 0% squamous cell carcinoma T4N3M1b 4 8 1.271 75% adenocarcinoma T4N2Mx 4 9 2.783 95% squamous cell carcinoma T4N1M1a 4 10 3.021 60% squamous cell carcinoma T4N2M1c 4 11 3.163 90% adenocarcinoma T4N3M1a 4 12 0.758 cps score=5 small cells T4N2M1b 13 1.251 40% adenocarcinoma T2N3M0 3 14 1.546 40% adenocarcinoma T4N2M1a 4 15 2.509 10% adenocarcinoma T2N3M0 3 16 2.574 0% squamous cell carcinoma T3N3M0 4 17 7.145 5% squamous cell carcinoma T3N3M1c 4 18 38.830 1% adenocarcinoma T2bN2M1a 4 19 4.227 NA squamous cell carcinoma T1bN2M1c 4 20 4.391 NA adenocarcinoma T4N3M1c 4 twenty one 3.981 NA squamous cell carcinoma T3N2M1a 4

Table 2PD-L1 predicts patient immunotherapy benefit (PR) results

Table 4CypB predicts the results of immunotherapy benefit (PR) in patients

From the results in Tables 3 and 4, it can be seen that the diagnostic ability of CypB is better than that of PD-L1.

In addition, among patients with high PD-L1 expression, 3 patients did not benefit from treatment, and their CypB expression levels were all higher than the cutoff value.

Among the patients with low PD-L1 expression, there were 5 cases with very low expression, less than or equal to 5%, which is generally considered to indicate that immunotherapy is not beneficial. The actual treatment outcome of these 5 patients was 3 responses (PR) and 2 responses (PD). The accuracy of CypB for the prediction of immunotherapy efficacy in these 5 patients was 60%.

The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above-mentioned embodiments. Within the scope of the technical concept of the present invention, various changes can be made to the technical solutions of the present invention, and these simple changes are all It belongs to the protection scope of the present invention.

In addition, it should be noted that the specific technical features and steps described in the above-mentioned specific embodiments can be combined in any suitable manner unless they are inconsistent. The possible combinations are not specified otherwise.

In addition, the various embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the spirit of the present invention, they should also be regarded as the contents disclosed in the present invention.

Claims (10)

1. A marker for predicting the effect of cancer immunotherapy, comprising: the marker is CypB.

2. The marker of claim 1, wherein: any one or more of CypB, PD-L1, tumor mutation load TMB, EGFR mutation, tumor infiltrating lymphocyte TIL, DDR gene (DNA damage response gene), circulating tumor cell CTC, circulating tumor DNA (ctDNA) and neutrophil/lymphocyte ratio NLR is/are combined to be used as a marker for predicting the cancer immunotherapy effect.

3. A kit for predicting the effect of cancer immunotherapy, characterized by: the kit comprises at least antibodies to CypB.

4. An application of CypB in preparing the reagent kit for predicting the immunotherapy effect of cancer is disclosed.

5. Use according to claim 4, characterized in that: the cancer patient is experiencing an adenocarcinoma selected from the group consisting of Renal Cell Carcinoma (RCC), colorectal cancer (CRC), Gastric Cancer (GC), melanoma, non-small cell lung cancer (NSCLC), glioblastoma, and any type of adenocarcinoma.

6. Use of CypB in the manufacture of a kit for predicting the effect of an immunotherapy for cancer, characterized in that it comprises the following steps:

s1 detecting the content of CypB in the blood of the subject;

s2, the detection value of step S1 is compared with the preset cutoff value, and if the detection value is smaller than the preset cutoff value, the immunotherapy is predicted to be effective.

7. The application according to claim 6, wherein the predetermined cutoff value is determined by: the retrospective or prospective study subjects were defined as two groups of treatment benefit and non-benefit according to the response of their immunotherapy, and ROC curve analysis was performed on the measured CypB content in the patient's blood, and the measured CypB content at the John's index was taken as the cutoff value.

8. Use according to claim 6, characterized in that: the detection method of CypB content in blood of the subject is any one of ELISA method, immunoblotting method, flow cytometry and liquid phase chip.

9. Use according to claim 6, characterized in that: the subject's blood includes, but is not limited to, peripheral blood plasma, serum, and exosomes in the blood.

10. Use according to claim 6, characterized in that: the immunotherapy includes but is not limited to the combination of any one or more of monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy, small molecule inhibitors and immune system modulator immunotherapy.

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