A biomarker for predicting or judging the effect of tumor immunotherapy on a subject and its application

Abstract

The present invention belongs to the field of biotechnology, and discloses a biomarker for predicting or judging the effect of tumor immunotherapy on a subject and its application, wherein the biomarker is an important effector protein of the human innate immune system, namely, MxA protein. The biomarker can be detected in samples from tumor patients to predict the clinical effect of immunotherapy, thereby helping to determine whether the tumor patient can benefit from immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a biomarker for predicting or judging the tumor immunotherapy effect of a subject and its application. Background Art

[0002] Glioblastoma multiforme (GBM) is one of the most lethal brain cancers, accounting for 48.6% of all primary brain malignancies, and poses a huge threat to human health because current treatments are minimally effective. Initial surgical resection, adjuvant radiotherapy, and temozolomide chemotherapy are the standard of care for GBM. However, patient survival has only slightly improved, with a reported median overall survival (OS) of 14.6 months and a 5-year survival rate of less than 10%. Therefore, new treatments are urgently needed to improve the prognosis of patients with this deadly brain cancer.

[0003] Immunotherapies such as immune checkpoint blockade have brought substantial improvements in survival for a variety of solid malignancies, including melanoma, non-small cell lung cancer, breast cancer, and non-Hodgkin lymphoma. Several new immunotherapies are under development for the treatment of GBMs and have shown encouraging results in preclinical studies and early-stage trials. Glycoprotein 96-kDa (Gp96), a heat shock protein, belongs to the heat shock protein family and is primarily located in the endoplasmic reticulum (ER) as a master chaperone. Gp96 has a natural ability to bind tumor-associated antigens (peptides), thereby forming Gp96-peptide complexes, which can be taken up by antigen-presenting cells (e.g., dendritic cells) and then elicit innate and adaptive antitumor immune responses. Therefore, after purifying the complexes from patient tumors, Gp96-peptide complexes can be developed as personalized multivalent cancer therapeutic vaccines, commonly referred to as heat shock protein peptide complex 96 (HSPPC-96).

[0004] HSPPC-96 is a personalized multivalent anti-tumor vaccine that has demonstrated low toxicity and positive clinical results in preclinical models and early trials, showing safety and clinical efficacy of more than 60% in the treatment of various malignancies, including recurrent and newly diagnosed GBMs. However, the high variability of immunotherapy efficacy remains one of the biggest challenges facing this immunotherapy. In our HSPPC-96 vaccination cohort, OS time was highly variable, ranging from 7.5 to 68.2 months. Therefore, identifying patients who are more likely to respond to or benefit from immunotherapy is critical for this new treatment modality. Although the lack of correlation between clinical activity and immune response has been widely noted, we still used ELISPOT analysis and TCR sequencing to discover biomarkers associated with immune response to predict the treatment outcome of HSPPC-96 vaccine. We observed that TSIR and a panel of TCR clones after vaccination are potential biomarkers for predicting better clinical outcomes. However, the timing of TSIR detection after vaccination, the cost of TCR sequencing, and the difficulty of performing it in routine clinical settings limit the widespread application of these biomarkers. As protein detection methods such as immunohistochemistry (IHC) staining and enzyme-linked immunosorbent assay (ELISA) are routinely used in clinical settings, protein markers have more opportunities to be tested in routine laboratories.

[0005] MxA protein belongs to a large GTPase family and has been widely studied for its broad antiviral activity. It is induced by interferon-α\β and represents a classic interferon-stimulated gene product. Therefore, a large number of studies have used MxA protein expression as an indicator to measure the activity of the interferon-α\β signaling pathway. At the same time, recent findings have shown that MxA is an oncoprotein in breast cancer that promotes the invasion and proliferation of tumor cells. High expression of MxA protein reflects strong autocrine activation of the interferon-α\β signaling pathway, which has been shown to inhibit antitumor immune responses by impairing the immunogenicity of glioma cells. In addition, the tumor-promoting effect of MxA protein itself will also mediate its negative impact on the effect of vaccination, suggesting a possible mechanism for tumor cells to evade vaccine immunity, thereby providing a molecular target for further improving the therapeutic effect. Summary of the invention

[0006] The purpose of the present invention is to provide a biomarker for predicting or judging the effect of tumor immunotherapy, aiming to predict or judge the effect of immunotherapy on tumor patients.

[0007] In order to achieve the above invention purpose, the technical solution adopted is:

[0008] A biomarker for predicting or judging the effect of tumor immunotherapy on a subject, comprising an MxA protein, wherein the MxA protein is selected from the macromolecular GTPase protein superfamily.

[0009] The present invention is further configured as follows: the MxA protein includes a first region, a second region and a third region.

[0010] The present invention is further configured as follows: the first region is the active region of the GTPase, the second region is the transition region of the GTPase, and the third region is the effector region of the GTPase.

[0011] The present invention is further configured as follows: the MxA protein is selected from any one of tumor pathological examination tissue, surgically removed tumor tissue, serum, plasma, interstitial fluid, cerebrospinal fluid and urine of the subject before receiving tumor immunotherapy;

[0012] The immunotherapy is any one of tumor therapeutic vaccine preparation therapy and immune checkpoint inhibitor therapy;

[0013] The present invention is further configured as follows: the tumor is any one of glioblastoma, renal cancer, liver cancer, gastric cancer, melanoma, colorectal cancer and lung cancer.

[0014] The present invention is further configured as follows: the immunotherapy is treatment with the tumor therapeutic vaccine preparation alone or a combination of the immune checkpoint inhibitor treatment and chemotherapy, radiotherapy or targeted therapy;

[0015] Wherein, the tumor therapeutic vaccine preparation is selected from any one of HSPPC-96, HSPPC-70, and BCG vaccine, and the immune checkpoint inhibitor is selected from any one of PD-1, PD-L1, CTLA-4, BTLA, TIM-3, LAG-3, TIGIT, and LAIR1;

[0016] Furthermore, the present invention provides a reagent for detecting the above-mentioned biomarkers, which is used for immunohistochemistry staining, immunofluorescence, ELISA, ELISA, protein peptide chip detection, immunoblotting, microbead immunoassay or microfluidic immunoassay.

[0017] Furthermore, the present invention provides the use of the above-mentioned biomarkers or the above-mentioned reagents in the preparation of products for predicting or judging the effect of tumor immunotherapy on a subject.

[0018] The present invention is further configured such that: the subject is a mammal.

[0019] Furthermore, the present invention provides a kit comprising the above reagents, wherein the kit is a kit for immunohistochemistry staining, immunofluorescence, enzyme-linked immunosorbent assay, protein peptide chip detection, immunoblotting, microbead immunoassay or microfluidic immunoassay.

[0020] In summary, compared with the prior art, the present invention discloses a biomarker for predicting or judging the effect of tumor immunotherapy on a subject. The biomarker provided by the present invention can be used to predict or judge the effect of HSPPC-96 vaccination on tumors, especially glioblastoma multiforme (GBM). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Representative renderings of CD4+, CD8+, PD-1+ cells, gp96, and MxA proteins in GBM tissue provided in this example;

[0022] Figure 2 The correlation diagram between low expression of MxA protein and good prognosis provided in this example;

[0023] Figure 3 A correlation diagram between low expression of MxA protein and long-term survival provided in this example;

[0024] Figure 4 This is a relationship diagram of the MxA protein expression TCR clone and TSIR provided in this example. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the protection scope of the present invention.

[0026] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0027] In a first aspect of this embodiment, a biomarker for predicting or judging the effect of tumor immunotherapy on a subject is provided, including MxA protein. The MxA protein is selected from the macromolecular GTPase protein superfamily. It should be noted that the biomarker of this embodiment is an important effector protein of the human innate immune system, namely, the MxA protein.

[0028] Furthermore, the MxA protein used includes a first region, a second region and a third region. It should be noted that:

[0029] The first region is the active region of the GTPase, the second region is the transition region of the GTPase, and the third region is the effector region of the GTPase.

[0030] Furthermore, the MxA protein is selected from any one of tumor pathological examination tissue, surgically removed tumor tissue, serum, plasma, interstitial fluid, cerebrospinal fluid and urine of the subject before receiving tumor immunotherapy. In the specific implementation process, the tumor pathological examination tissue is preferred in this embodiment.

[0031] Furthermore, the tumor is any one of glioblastoma, renal cancer, liver cancer, gastric cancer, melanoma, colorectal cancer and lung cancer. In the specific implementation process, glioblastoma is the preferred embodiment of this embodiment.

[0032] Furthermore, immunotherapy is any one of tumor therapeutic vaccine preparation treatment and immune checkpoint inhibitor treatment. In the present embodiment, immunotherapy is tumor therapeutic vaccine preparation treatment alone or immune checkpoint inhibitor treatment combined with chemotherapy, radiotherapy or targeted therapy. The tumor therapeutic vaccine preparation is selected from any one of HSPPC-96, HSPPC-70, and BCG vaccine. The immune checkpoint inhibitor is selected from any one of PD-1, PD-L1, CTLA-4, BTLA, TIM-3, LAG-3, TIGIT, and LAIR1. In the specific implementation process, HSPPC-96 tumor vaccine is preferred in this embodiment.

[0033] In the second aspect of this embodiment, a reagent for detecting the above-mentioned biomarkers is provided, and the reagent is used for immunohistochemistry (IHC) staining, immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), protein peptide chip detection, immunoblotting, microbead immunoassay and microfluidic immunoassay. In the specific implementation process, as a preference, the reagent is used to detect the biomarkers through antigen-antibody reaction, for example, through IHC, IF and ELISA.

[0034] The third aspect of this embodiment provides the use of the above-mentioned biomarkers or reagents in the preparation of products for predicting or judging the effect of tumor immunotherapy on a subject.

[0035] Furthermore, the subject is a mammal. In the specific implementation process, primate mammals are preferred, and humans are more preferred.

[0036] In a fourth aspect of the present embodiment, a kit is provided, comprising the above-mentioned reagents. The kit is a kit for immunohistochemistry (IHC) staining, immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), protein peptide chip detection, immunoblotting, microbead immunoassay, and microfluidic immunoassay. In the specific implementation process, the kit is used to perform in situ detection of pathological tissue of biomarkers through antigen-antibody reaction. Preferably, the kit is an MxA protein immunofluorescence (IF) detection kit.

[0037] In a fifth aspect of this embodiment, the following test is provided:

[0038] Patient samples in this trial were retrospectively examined for expression of the study protein (i.e., MxA protein) using formalin-fixed and paraffin-embedded (FFPE) tumor tissues. These tumor tissues were collected during neurosurgical resection (before vaccination) from a group of GBM patients who received HSPPC-96 vaccination in an open 95-label, single-arm, phase I clinical trial. The trial was designed to determine the safety and preliminary efficacy of the HSPPC-96 vaccine in the treatment of newly diagnosed GBMs with standard of care therapy. After postoperative concurrent chemoradiotherapy, all patients received a total of 6 doses of the vaccine, 25 μg per week. The vaccine was generated by extracting gp96 and its binding peptides from fresh tumor tissue according to the above steps. The production of the vaccine followed the GMP manufacturing standard guidelines. In this trial, a total of 20 patients received 102 vaccinations, and 19 patients with complete follow-up information were included for survival analysis, resulting in a median progression-free survival (PFS) of 11.0 months and a median OS of 31.4 months for these patients. Approximately 1 year after the end of the trial, this trial continued to follow up all survivors and updated survival data, with 3-year PFS and OS of 20% and 40% for all vaccinated patients, respectively. This clinical trial was approved by the Ethics Committee of Beijing Tiantan Hospital (JS2012-001-03) and registered in ClinicalTrials, and written informed consent was obtained from all participants. The study report included the baseline characteristics of the patients, such as sex, age at diagnosis, Karnofsky performance status (KPS) scale (range 0-100%), MGMT promoter methylation status, IDH1 / 2 mutation, and TERT promoter mutation.

[0039] Furthermore, this study used multidimensional immunofluorescence staining (MIF) to detect immune features, including the density of immune cells (CD4+T cells, CD8+T cells, and PD1+ cells) and the protein expression of gp96 (a component of the HSPPC-96 vaccine) and MxA (an interferon-stimulated gene product, whose expression reflects the activity of the interferon pathway), such as Figure 1 As shown. Among the FFPE tumor samples collected from all 19 patients before vaccination, patients with complete follow-up (24) were included. Consistent with the results of previous studies (35), the infiltration levels of immune cells in this sample varied greatly, with the median levels of CD4+T cells, CD8+T cells, and PD1+ cells being 78 / mm2 (range 2-356 / mm2), 20 / mm2 (range 1-185 / mm2), and 14 / mm2 (range 1-101 mm2), respectively. This trial also observed that the infiltration rate of CD4+T cells was higher than that of CD8+T cells. As Figure 1As shown, gp96 and MxA proteins were detected by cytoplasmic staining and were ubiquitously expressed in all samples. However, the extent of staining in each sample also varied from sample to sample, ranging from 2.2% to 22% for gp96 and 1.5% to 49% for MxA proteins, respectively. These results suggest that there are significant differences in the immune profiles of tumor samples before vaccination, which provides a basis for further analysis of their impact on clinical outcomes.

[0040] After the latest median follow-up of 58.9 months, the median PFS and OS were 11.0 months and 31.4 months, respectively. After 3 years, the PFS of all vaccinated patients was 20% (4 / 20) and the OS was 40% (8 / 20). Based on the average levels of these immune characteristics, this trial divided patients into high and low groups. This trial did not observe a significant effect of the density of immune cells (including CD4+T cells, CD8+T cells, and PD1+ cells) on the survival rate of these patients, such as Figure 2 -A. Figure 2 -B and Figure 2 -C. However, high gp96 expression is often negatively correlated with patient OS, as shown in Figure 2 -D. The expression of MxA protein had a negative impact on the OS of these patients. The median OS of the MxA low expression group was 48 months, while the median OS of the MxA high expression group was 20 months (p = 0.038). Figure 2 -E. This indicates the potential of MxA protein as a predictive biomarker for the efficacy of HSPPC-96 vaccination.

[0041] Furthermore, this trial divided the vaccinated patients into long-term survivors (LTS) and short-term survivors (STS) according to whether the patients survived for more than three years, which is a common survival cutoff value for defining LTS in GBM clinical studies. Among the 19 patients, 8 were LTS and 11 were STS. As expected in this trial, the expression of MxA protein in the LTS group was significantly lower than that in the STS group (P = 0.0328). Figure 3 In addition, the receiver operating characteristic (ROC) curve analysis showed that MxA protein expression was a good indicator for distinguishing LTS from STS (AUC = 0.7955, P = 0.0318). Figure 3 Since LTS are the subset of patients most likely to respond to immunotherapy, these results suggest that the MxA protein could be a potential biomarker for early identification of these responders prior to vaccination.

[0042] Furthermore, this trial found that MxA protein expression was associated with prognosis and pre-existing TCR clones, but not TSIR. Next, this trial investigated the potential mechanism by which low expression is associated with better clinical outcomes. This trial previously used ELISPOT analysis to evaluate TSIR levels in PBMCs collected before and after HSPPC-96 vaccination. This trial observed that HSPPC-96 vaccination increased TSIR levels by 2.3-fold, and TSIR levels after vaccination were closely correlated with patient survival. Based on these observations, low MxA protein expression is associated with increased TSIR levels after vaccination, leading to favorable outcomes. However, this trial did not observe any significant correlation between MxA protein expression and the three TSIR index levels, including TSIR before vaccination (TSIR\u-pre\u-vac, such as Figure 4 -A), TSIR post-vaccination (TSIR__-post__-vac, as shown in Figure 4 -B) and the fold change of TSIR from pre- to post-vaccination (TSIR__-FC__-VAC, as shown in Figure 4 -C). In addition, the fitted Cox regression model including age, TSIR\Upost\Uvac, and MxA protein expression also showed that MxA protein expression was an independent predictor of prognosis. Therefore, MxA protein expression and TSIR\upost\uvac independently affected the outcome of this vaccination cohort.

[0043] Furthermore, this trial used TCR sequencing to reveal the TCR sequence characteristics of tumor-infiltrating lymphocytes in LTS and found that four TCR clones were significantly enriched in the LTS group and the presence of these clones was associated with a favorable outcome. Therefore, this trial next explored whether the association between MxA protein expression and outcome was associated with an unbalanced distribution of these TCR clones. This trial compared the frequency of these clones, represented by the complementarity determining region 3 (CDR3), which determines the specificity of a given TCR clone between high and low MxA protein expression groups. Therefore, this trial found that CDR3-2 was absent in the high MxA protein expression group (p=0.021), as Figure 4 Since the presence of CDR3-2 was associated with long-term survival in this cohort and in another peptide vaccine trial for glioma, this study inferred that the negative impact of MxA protein expression on the presence of CDR3-2 might be inversely correlated with the survival time of patients.

[0044] In summary, the MxA protein provided in this example can be used as a biomarker for predicting or judging the therapeutic effect of HSPPC-96 vaccination on tumors, especially glioblastoma multiforme (GBM).

[0045] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of a reagent for detecting MxA protein in tumor tissue in the preparation of a product for predicting the effect of HSPPC-96 immunotherapy on glioblastoma patients, characterized in that: Included is an MxA protein selected from the macromolecular GTPase protein superfamily.

2. Use of the reagent for detecting MxA protein in tumor tissue according to claim 1 in the preparation of a product for predicting the effect of HSPPC-96 immunotherapy on glioblastoma patients, characterized in that: The MxA protein includes a first region, a second region and a third region.

3. Use of the reagent for detecting MxA protein in tumor tissue according to claim 2 in the preparation of a product for predicting the effect of HSPPC-96 immunotherapy on glioblastoma patients, characterized in that: The first region is the active region of the GTPase, the second region is the transition region of the GTPase, and the third region is the effector region of the GTPase.

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