CN111848770A - Application of host defense peptide or composition thereof in preparation of tumor treatment drug - Google Patents
Application of host defense peptide or composition thereof in preparation of tumor treatment drug Download PDFInfo
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- CN111848770A CN111848770A CN202010905339.1A CN202010905339A CN111848770A CN 111848770 A CN111848770 A CN 111848770A CN 202010905339 A CN202010905339 A CN 202010905339A CN 111848770 A CN111848770 A CN 111848770A
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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Abstract
The invention belongs to the technical field of biological medicines, relates to host defense peptides or a composition thereof, and also relates to application of the host defense peptides or the composition thereof in preparing medicaments for treating tumors, in particular to application of combination of a calarin 1.1 mutant, a calarin 1.1/1.9 polypeptide, an HPV16E7 polypeptide vaccine and an immune checkpoint inhibitor in preparing anti-tumor medicaments. The invention provides a host defense peptide composition which is a calein 1.1/1.9 polypeptide composition or a calein 1.1 mutant/calein 1.9 polypeptide composition, wherein the weight ratio of a calein 1.1 peptide to a calein 1.9 peptide or the weight ratio of a calein 1.1 peptide mutant to a calein 1.9 peptide is as follows: 1: 0.1-1; the hererin 1.1 mutant and the hererin 1.1/1.9 polypeptide (or the hererin 1.1 mutant/hererin 1.9) can change the tumor microenvironment after the treatment of vaccines and PD-1 inhibitors, thereby improving the anti-tumor effect.
Description
Technical Field
The invention belongs to the technical field of biological medicines, relates to host defense peptides or a composition thereof, and also relates to application of the host defense peptides or the composition thereof in preparing medicaments for treating tumors, in particular to application of combination of a calarin 1.1 mutant, a calarin 1.1/1.9 polypeptide, an HPV16E7 polypeptide vaccine and an immune checkpoint inhibitor in preparing anti-tumor medicaments.
Background
Malignant tumors are diverse in their nature, different in involved tissues and organs, different in disease stage, and different in response to various treatments, and thus most patients need to be treated in combination. The comprehensive treatment is to comprehensively adopt measures such as surgery, chemotherapy, radiotherapy, immunotherapy, traditional Chinese medicine treatment, interventional therapy, microwave treatment and the like according to the physical condition of a patient, the pathological type of tumors, the invasion range and the like so as to greatly improve the cure rate and improve the life quality of the patient.
The Tumor Microenvironment (TME) has immunosuppressive effects, and can inhibit the function of tumor infiltrating effector T cells. TME promotes the development of tumor-associated macrophages, myeloid-derived suppressor cells, B cells, Th 2-type and regulatory T cells. Tumor Associated Macrophages (TAMs) are of increasing interest because they play a key role in tumor spread and tumor response to different therapies. TAMs can greatly accelerate the progression of untreated tumors and can also affect the efficacy of anticancer drugs, including immune checkpoint blockade immunotherapy. In particular, TAMs can have opposite phenotypes and functions that are tumoricidal (e.g., M1-like cells) or promote tumor growth (e.g., M2-like cells). Arg1 metabolizes L-arginine into urea and L-ornithine by proline and polyamines produced downstream, which are essential for cell proliferation and collagen synthesis. Therefore, Arg1+ macrophages promote wound healing and tissue fibrosis through local depletion of L-arginine, and inhibit T cell activation, promoting tumor growth.
Cancer therapy vaccines are intended to elicit effector T cells, particularly tumor antigen-specific CD8+ T cells that target tumor cells, without theoretically affecting normal cells or tissues. However, therapeutic vaccines induce T cells effective against CIN3 lesion, a precancerous lesion associated with high risk human papillomavirus infection, but no effect on cervical cancer has been observed. Simultaneous blocking of the cytokine interleukin 10(IL-10) upon immunization in a prophylactic setting greatly increases the vaccine-induced antigen-specific CD8+ T cell response and inhibits tumor growth compared to the same vaccine without IL-10 signaling block. The tumor suppression was also improved in the therapeutic setting by intraperitoneal administration of anti-IL-10 receptor antibodies, indicating that this immunization strategy may further enhance the efficacy of the anti-tumor response.
Although there are various methods for treating tumors in the prior art, there is no report on the combined use of a vaccine, an immune checkpoint inhibitor and a calein 1.1/1.9 polypeptide for preparing an antitumor drug.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a novel host defense peptide mutant obtained by the mutation of a calein 1.1 peptide.
The amino acid sequence of the calein 1.1 peptide is as follows: GLLSVLGSVAKHVLPHVVPVIAEHL-NH 2.
The amino acid sequence of the calein 1.1 peptide mutant is as follows: GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2。
Further, the invention provides application of the calein 1.1 peptide mutant and the host defense peptide composition in preparation of antitumor drugs.
The host defense peptide composition is a calrin 1.1/1.9 polypeptide composition or a calrin 1.1 mutant/calrin 1.9 polypeptide composition.
In the composition, the weight ratio of the calein 1.1 peptide to the calein 1.9 peptide is as follows: 1: 0.1-1;
in the composition, the weight ratio of the calein 1.1 peptide mutant to the calein 1.9 peptide is 1: 0.1-1;
further, when the weight ratio of the calein 1.1 peptide to the calein 1.9 peptide is: 1:0.5-1, or the weight ratio of the calein 1.1 mutant to the calein 1.9 is 1:0.5-1, the tumor microenvironment after the treatment of the vaccine and the PD-1 inhibitor can be changed to the maximum extent, and the anti-tumor effect is more obvious.
Further, the invention provides application of the caesin 1.1 mutant and the caesin 1.1/1.9 polypeptide (or caesin 1.1 mutant/caesin 1.9) in combination with a vaccine and a PD-1 inhibitor (triple therapy) in preparing antitumor drugs.
Further, the hererin 1.1 mutant, the hererin 1.1/1.9 polypeptide (or the hererin 1.1 mutant/hererin 1.9) can change the tumor microenvironment after the treatment of the vaccine and the PD-1 inhibitor, thereby improving the anti-tumor effect.
The vaccine is an HPV16E7 polypeptide vaccine and consists of 4 overlapped polypeptides EX containing HPV16E7 full length, adjuvant MPLA and anti-interleukin 10 receptor antibody.
Wherein,
EX is a mixture of A, B, C, D4 amino acids, wherein the weight ratio of A, B, C, D is 1: 1: 1: the sequence of 1, A, B, C, D4 amino acids is as follows:
A:MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEE,
B:LNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKC,
C:DRAHYNIVTFCCKCDSTLRLCVQSTHVDIR,
D:CVQSTHVDIRTLEDLLMGTLGIVCPICSQKP。
in the HPV16E7 polypeptide vaccine, the concentration of the overlapping polypeptide EX containing the HPV16E7 full length in each 100 mu l is as follows: 10 μ g, adjuvant 10 μ g MPLA and 300 μ g anti-interleukin 10 receptor antibody.
The PD-1 inhibitor is an anti-PD-1 monoclonal antibody.
The polypeptide structure of the calerin 1.1 is alpha helix-hinge-alpha helix, wherein the hinge structure corresponds to HVLP, providing the polypeptide with a degree of structural freedom to allow the alpha helix to readily interact with the cell membrane. The mutant of the invention increases the hinge structure, so that the alpha helix is easier to interact with the target site of the cell membrane, thereby improving the activity. In addition, the calein 1.1 mutant, the calein 1.1/1.9 peptide and the calein 1.1 mutant/1.9 peptide have obvious antitumor activity.
The concrete method of the Caerin1.1 mutant comprises the following steps: at the seventeenth amino acid V of GLLSVLGSVAKHVLPHVVPVIAEHL-NH2, four amino acids of LPHV are added, further improving the hydrophobicity of calein 1.1 and the positive charge amount of calein 1.1. The Caerin1.1 mutant was: GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2, synthesized by conventional polypeptide synthesis method.
Drawings
FIG. 1 shows the in vitro inhibition and killing of TC-1 and B16 tumor cells by the mutant strain of calein 1.1 and calein 1.9.
FIG. 1A TC-1 cells; FIG. 1B B16 cells.
FIG. 2 is a graph showing that triple combination therapy increases the number of immunocompetent macrophages.
FIG. 2A is a correlation analysis between six macrophage populations by single cell sequencing;
FIG. 2B Bar graph comparison shows a comparison of the cell number of each macrophage in different groups, and the fold-change in cell number for the two treatments relative to the untreated group, including the untreated group, the group to which the calein 1.1/1.9 composition was added, and the group to which the negative control polypeptide P3 was added;
FIG. 2C shows the proportion of different macrophages in each group;
FIG. 2D is a comparison of marker genes in M2 macrophages with normalized expression of other macrophages;
FIG. 2E is a representation of t-SNE of aligned gene expression data for M2 macrophages and selected genes (including Rgcc, Mgarp, Egln3, Ndrg1, Hilpda, Ero11, Gla, Furin, and Gys1) from untreated and supplemented with a cairin 1.1/1.9 composition;
FIG. 2F is a comparison of the relative expression of the first 40 marker genes of M2 macrophages in three groups;
FIG. 2G is a statistical analysis of the expression (Log2 expression values) of Arg1, Mmp13, Pxdc1, Cd93, Pf4 and Hmox 1;
FIG. 2H is a biological process of enrichment in the M2 macrophage population following treatment with a vaccine containing the composition of calein 1.1/1.9.
FIG. 3 is a graph showing that triple combination therapy further reduced the number of tumor-growth promoting B cells in TC-1 tumors.
Figure 3A is an enrichment analysis of B cell biological processes in tumors after triple therapy treatment (P value < 0.05);
figure 3B is the first 20 KEGG pathways enriched in B cells in tumors following triple therapy treatment;
figure 3C is a comparison of marker gene expression (Log2 expression values) and cell number in different treatment groups (untreated, triple therapy or P3 group).
FIG. 4 is a graph of the effect of triple combination therapy or P3 panel on TC-1 tumor infiltrating T cells.
FIG. 4A is a graph showing the ratio of the number of CD4+ CD8+, CD8+, CD4+ CD25+ and CD4-CD 8-cells in T cells extracted from TC-1 tumors from different treatment groups;
FIG. 4B is a hierarchical clustering of the first 40 marker genes of immunospecific CD8+ T cells and a comparison of expression in other types of T cells;
fig. 4C is a violin diagram comparing gene expression of selected genes, showing that CD8+ T cells were statistically significantly upregulated with other T cell populations and pdcs (including CD8a, Klrc1, lang 3, CD8b1, Nkg7, and Cxcr 6);
figure 4D is the first 20 KEGG pathways enriched in CD8+ T cells;
FIG. 4E is a gene ontology enrichment analysis of biological processes in T cells in TC-1 tumors comparing the biological processes of the first 25 enrichments in four T cell subsets, based on P-value and gene number, respectively.
Figure 5 is a triple combination therapy to significantly increase the number of active NK cells.
FIG. 5A is a two-dimensional visualization of the distribution of cells identified in NK cells with selected marker genes, including Klrb1c, Klrb1f, Car2, Gzmc, Xcl1, Klrb1a, Ncr1, and Klra 8;
figure 5B compares the number of cells expressing the selected marker gene in untreated, triple-combination and P3 group treated tumors as a violin plot, with the y-axis representing Log2 normalized expression values;
FIG. 5C is the Wikipathway signaling pathway identified in the first 60 significantly up-regulated gene-supported NK cells;
FIG. 5D statistical comparison of gene expression (Log2 values) under three conditions, statistical analysis using a two-way unpaired student t test (. P < 0.05;. P < 0.01;. P < 0.001; ns represents "not significant");
FIG. 5E is an enrichment assay for the Reactome pathway in untreated tumors;
figure 5F is an enrichment analysis of the Reactome pathway in NK cell marker genes treated with triple therapy (P < 0.01).
Figure 6 is a quantitative proteomic analysis of treated TC-1 tumors and comparison with untreated and P3 groups.
FIG. 6A is a comparison of the abundance of specific proteins in the triple-combination treatment group relative to the untreated and P3 groups, as assessed for significance using a two-tailed student's t-test;
FIG. 6B is a significantly upregulated protein-protein interaction network in the triple treatment group, with the size and color of the nodule representing the extent of protein interaction;
FIG. 6C is a protein-protein interaction network with significant upregulation in group P3, and the size and color of the knob represents the extent of protein interaction;
figure 6D shows a correlation between gene expression of proteins that were significantly upregulated (in the 19 cell populations identified by single cell sequencing) and fold changes of these proteins relative to the untreated group only in the triple treatment group;
fig. 6E is a comparison of biological processes enriched in the triple treated group and P3 group compared to the untreated group;
FIG. 6F is a comparison of the Calerin 1.1/1.9 and the P3 sets of signaling pathways.
FIG. 7 is a graph of the effect of triple therapy on the survival time of TC-1 tumor-bearing mice;
FIG. 7A is a graph of the effect of vaccine binding to PD-1 inhibitory antibodies on the survival time of TC-1 mice;
FIG. 7B is a graph showing the effect of triple treatment of the calein polypeptide, vaccine and PD-1 inhibitory antibody on the survival time of TC-1 mice;
FIG. 7C is a graph of the TC-1 tumor completely cleared mice in the assay of FIG. 7B, which cleared the TC-1 tumor from the second inoculation;
FIG. 7D left panel shows HPV16E 7-specific CD8+ T cell responses in tumor size and tumor draining lymph nodes and spleen 30 days after tumor inoculation.
Detailed Description
Example 1
MTT assay for inhibition of tumor cell growth in vitro of Caerin1.1 and mutations CAerin1.1 and 1.9
5×103TC-1 cells or B16 cells were plated in 96-well cell culture plates, and P3, calein 1.1, mutant calein 1.1 polypeptide or calein 1.9 polypeptide was added to the plates to a final concentration of 5, 10, 15. mu.g/ml, respectively, in PRMI1640 medium containing 10% calf serum at 37 ℃ and 5% CO2The culture was carried out overnight. The growth of TC-1 (left) and B16 cells (right) was examined using the MTT method.
The results show that: the mutant calein 1.1 had better growth inhibitory effect on TC-1 and B16 than that of calein 1.1.
Example 2
Effect of triple treatment on Arg 1-negative tumor-infiltrating macrophages
The experimental method comprises the following steps: C57/BL6 mice were inoculated subcutaneously with 5X 105The TC-1 tumor cell of (1). 3 days after tumor inoculation, TC-1 tumor-bearing mice were divided into three groups. Each group had 3 mice. One group is a control group, and mice are immunized or locally injected with tumors or injected with abdominal cavities of the mice by using vaccine immunization, locally injecting the calein polypeptide into the tumors and injecting PBS with the same volume as the PD-1 antibody; the second and third groups were injected subcutaneously with 100. mu.l of vaccine containing 40. mu.g Ex (10. mu.g of each polypeptide)/10. mu.g MPLA/300. mu.g anti-IL-10 receptor antibody twice 3 days and 9 days after tumor inoculation, respectively, and were injected intraperitoneally with anti-300. mu.g PD-1 antibody twice on days 9 and 21 days after tumor inoculation. On days 3-9 post tumor inoculation, tumors were injected locally with 30 μ g each of Caerin1.1 and 1.9 (1: 1 weight ratio of Caerin1.1 to 1.9 in the treatment group) or 30 μ g of control peptide P3(GTELPSPPSVWFEAEFK-OH) (control group) for 7 consecutive days. One group of TC-1 tumor-bearing mice was mock-treated with PBS as a control group. On day 30 after tumor inoculation, tumors were taken for single cell RNA sequencing analysis.
The experimental results show that: six macrophage populations were detected, including M2M Φ, MHCIIhi M Φ, M Φ/DC, EarhiM Φ, M Φ/NKT, and TAM. Due to the fact thatHere, the correlation between each two mrphi was deduced based on the expression of genes with significant upregulation, which confirmed a strong correlation (0.6-0.8) or higher between these mrphi. Notably, M2M Φ and MHCIIhi M Φ were highly correlated with M Φ/NKT, respectively (fig. 2A), indicating their intimacy in the development of M Φ lineage. The number of cells of different M Φ in the three groups was compared (fig. 2B), indicating that both the treated and control groups significantly down-regulated M2M Φ by more than 70%; addition of calein 1.1/1.9 induced a greater amount of MHCIIhi and Ear than control P3hiM phi. In addition, the cairin 1.1/1.9 group significantly adjusted the ratio of several M Φ to the overall M Φ level (profile) (fig. 2C). The proportion of M2M Φ in the cairin 1.1/1.9 and P3 groups decreased from 73.2% in untreated tumors to 16.2% and 18.9%, respectively. In contrast, the proportion of MHCIIhi M Φ was increased approximately five-fold and four-fold in the Caerinn 1.1/1.9 and P3 groups compared to untreated TC-1 tumors. Furthermore, in the treated tumors, M.phi./DC and Ear were compared to the untreated tumorshiThe number of cells of M Φ increases greatly. Expression of genes associated with key lineage of M2M Φ was compared in parallel with their expression in other macrophages (fig. 2D). Several marker genes appear to be expressed only at M2M Φ, e.g., Mmp12, Arg1, Mmp13 and Slc6a8, whose abnormal elevation is positively correlated with tumor angiogenesis and invasiveness. Arg1+ macrophages had a strong immunosuppressive effect, with Arg1 being clearly upregulated in macrophages of MC38 tumors. It was also found that some marker genes for M2M Φ were highly expressed in TAMs, indicating that there may be similarities in cell function between these two macrophages.
The distribution of M2M Φ without or with the CAERIN1.1/1.9 treatment is compared in FIG. 2E, where there is a significant difference with low overlap. Some marker genes, such as Rcgg, Ndrg1 and Egln3, whose expression profiles closely matched those of M2M Φ post-treatment, observed changes indicated the likelihood that expression of these genes was stimulated by calein 1.1/1.9. Relative expression of the first 40 marker genes of M2M Φ was hierarchical clustered and compared under three conditions (fig. 2F). With the exception of Mmp13(P3) and Ndrg1 (calein 1.1/1.9), all other genes were significantly down-regulated by both treatments. To assess the importance of these variations, the expression values of the selected genes were further compared, where significant down-regulation of Arg1, Mmp13, Pf4 and Hmox1 occurred using calirin 1.1/1.9 relative to P3 treatment (fig. 2G). Enrichment of marker genes for M2M Φ after caerin1.1/1.9 treatment biological processes related to immune responses including apoptotic processes, programmed cell death, response to stimuli and organisms, cytokine production and secretion, and T cell differentiation (fig. 2H).
The triple treatment significantly increased Arg 1-negative tumor-infiltrating macrophages.
Example 3
Triple treatment for reducing the number of tumor infiltrating B cells
The experimental procedure was as in example 2.
The B cell population was the major tumor-associated cell population, accounting for 4.84% of the total number of CD45+ cells isolated from untreated TC-1 tumors. In contrast, the proportion of B cells decreased to 1.61% (87 cells) and 0.26% (15 cells) in the P3-treated and the CAERIN 1.1/1.9-treated groups, respectively. GO analysis of B cells showed that enriched biological processes included activation (corrected P value of 9.95E-15), differentiation (corrected P value of 8.27E-07) and proliferation (corrected P value of 3.49E-10) B cells, modulating B cell receptor signaling (corrected P value of 3.53E-04) (fig. 3A). KEGG enrichment analysis also revealed that the B cell receptor signaling pathway was the second most important pathway following ribosomes (P value 5.76E-06), while also enriching antigen processing and presentation (P value 0.0282) (fig. 3B). The expression of marker genes in TC-1 tumors, untreated and Caerin1.1/1.9 and P3 treated, are compared in FIG. 3C, indicating that the number of cells expressing these genes is significantly reduced with both treatments, especially the addition of Caerin 1.1/1.9. I.e., triple treatment significantly reduced the number of tumor infiltrating B cells.
Example 4
Effect of Calerin 1.1/1.9 treatment on CD8+ T cells in mice following Vaccination and PD-1 blockade
The experimental method comprises the following steps: the same as in example 2.
The results show that FIG. 4A shows the levels of all T cell types in the untreated group, P3, the calerin 1.1/1.9 polypeptide under the three conditions. It was shown that CD8+ T cells were increased more than 10-fold in the CAERIN1.1/1.9 and P3 treated groups, respectively, accounting for 40% and 37% of all T cell populations, respectively. The phenotype of these T cell populations in TC-1 tumors is elucidated in further detail. A total of four T cell populations.
Hierarchical clustering of the first 40 significantly up-regulated genes of the CD8+ T cell population is shown in fig. 4B.
FIG. 4C shows that Cd8a, Klrc1 and Lag3 are almost unique to CD8+ T cells, while Cxcr6 expression is comparable to that in CD4-CD8-T cells. It was determined that the expression of Cd8a, Cd8b1 and Lag3 was low in pDC. Ribosomes were identified as the most enriched KEGG pathway, followed by T cell receptor signaling and natural killer cell-mediated cytotoxicity (fig. 4D). To further reveal the potential functional heterogeneity of these four T cell subsets in TC-1 tumors, GO analyses were performed on CD4+ CD8+, CD8+, CD4+ CD25+ and CD4-CD8-T cells, comparing the first 25 enriched biological processes based on marker gene expression in fig. 4E.
Example 5
Triple therapy for activating tumor infiltrating NK cells
The experimental method comprises the following steps: the same as in example 2.
The treatment of calerin 1.1/1.9 resulted in a significant increase in NK cell population. There was some overlap in marker gene distribution among NK, CD4+ CD8+ and CD8+ T cells (fig. 5A). The distribution of some marker genes in the different treatment groups for different treatments is compared in FIG. 5B, where a significantly higher number of cells was detected in the calerin 1.1/1.9 treatment group than in the other two cases. Enrichment analysis of NK cell marker genes showed that it was highly correlated with immune responses, such as natural killer cell-mediated cytotoxicity and T cell receptor signaling pathways as well as differentiation of Th1, Th2 and Th17 (fig. 5C). The expression of selected marker genes (Log2 values) was compared in three samples in FIG. 5B, where significant upregulation of these genes was detected in NK cells treated with calein 1.1/1.9. Among the up-regulated genes of importance, the identification of Sell, Klrc1, GATA3 indicates the presence of CD56bright NK cells in this population; calerin 1.1/1.9 further induced the expression of other marker genes for CD56bright NK, such as Impdh1, Inpp4b and Cd 69. The presence of Pik3r1, Cd27 and Pdcd4 in this population also suggests a "transitional NK" feature, possibly related to the transition from Cd56bright to the presence of "mature NK" in this population, with CTSW, Hpox and Manf expression supporting this view of Cd56 dark NK cells. It was recently found that Klrc2 is a key marker for "adaptive NK", whereas treatment with calein 1.1/1.9 significantly upregulated Klrc2 expression. Other characteristics of "adaptive NK" are also present in this population, including Id2, Cd7, and Klrd 1. Notably, previously reported marker genes for "active", "terminal" and "inflamed" NK cells were not reliably detected here. Thus, this NK cell population represents a mixture of CD56bright, mature, transitional, and adaptive NK cells, which are the hallmarks of the least inflammatory functional NK cells. Expression of Klrc2 in NK cells under different conditions is shown in fig. 5D, which clearly shows that Klrc2 is expressed in more cells induced by calein 1.1/1.9. Furthermore, we found that the percentage of Klrc2 was higher in cells treated with calirin 1.1/1.9. Following treatment with calirin 1.1/1.9, the Reactome-rich pathway of NK cells includes apoptosis, the intrinsic pathway of apoptosis, and some CD28 stimulation-related pathways (fig. 5F), which implies an active immune response.
Example 6
Calerin 1.1/1.9 induced a stronger immune response than untreated and P3 treated.
The experimental method comprises the following steps: the same as in example 2.
Triple therapy results in significant up-regulation of proteins involved in immune response and regulatory processes, such as Gzma, Gzmc, Irf5, Tgtp1, Prg2 and Ighg 1. Figure 6A shows that the protein levels associated with immunity were significantly altered after triple treatment compared to the untreated group (these proteins were not significant in P3 group). Analysis of the significant upregulated protein-protein interactions determined strong interactions in both treatment cases, and therefore interactions supported by more evidence were filtered out using a high confidence value of 0.7, as shown in fig. 6B and 6C. In FIG. 6B, 337 interactions were identified in the 285 proteins, forming a highly connected network (PPI enriched P value < 1.0E-16); the P3-treated PPI consisted of 153 upregulated proteins and 113 interactions (fig. 6C).
Fig. 6D reveals the correlation between the proteins significantly upregulated by the triple treatment group and the cell population identified in the scRNA-seq (fig. 6D). These proteins show fold changes of greater than 2, and many are closely related to the normalized expression of genes in the monocyte, M Φ, and DC (e.g., Iigp1, Gbp2, Irf5, and Parvg) populations. In T cells and NK cells, there are several proteins that are more closely related to their gene expression, including Satb1, Spn, Dok2 and Hip1 r. The highest node on the protein interaction network of the triple treatment group was Stat1, which was upregulated 2.2-fold compared to untreated tumors and was detected as a marker gene only in monocytes, M Φ/DC and CD4+ CD25+ T cells. The marker gene Gzmc appears to be unique to NK cells and is significantly up-regulated only in the triple treatment group. Stat1 was detected as an up-regulated gene in almost all cell populations except B cells, and proteomic analysis showed a significant increase in concentration only in the triple treatment group, indicating that Stat1 was up-regulated to a large extent by calein 1.1/1.9.
Comparing the KEGG signaling pathway enrichment pathway for up-regulated proteins (P value <0.05) in FIG. 6E, treatment with calirin 1.1/1.9 activated more pathways including apoptosis, natural killer cell-mediated cytotoxicity, necrosis, nod-like receptor signaling, TNF, chemokines, NF-. kappa. B, RIG-I-like receptors and Toll-like receptors and several disease-related pathways. Among these KEGG signaling pathways, nod-like receptor signaling was identified as the most enriched pathway (P-value ═ 1.55E-10). Notably, antigen processing and presentation KEGG enrichment analysis was less significant in the calirin 1.1/1.9 (P-value 6.0E-4) compared to the P3 (P-value 1.0E-11) treatment, which is consistent with single cell sequencing results, and the B cell population of the calirin peptide treated group (promoting tumor growth) was almost eliminated.
Example 7
Experimental methods and experimental results:
FIG. 7(A) C57/BL6 mice were implanted subcutaneously at 5X 105And TC-1 tumor cells. 4 days after tumor inoculation, mice bearing TC-1 tumors were divided into five groups. Two groups are respectively spacedImmunization with Ex/MPLA/anti-IL-10 receptor antibody as described in example 2 was performed twice on day 7, with one group injected intraperitoneally with anti-PD-1 antibody on day 10 and day 30 after tumor inoculation, and the other group injected with control antibody. Third, four groups were mock immunized with PBS, followed by intraperitoneal injection of anti-PD 1 antibody or control antibody on days 10 and 30 post tumor challenge. The fifth group was mock immunized with PBS only and mock intraperitoneal injections. The survival of tumor bearing mice was monitored.
(B) C57/BL6 mice were inoculated subcutaneously with 5X 105The TC-1 tumor cell of (1). TC-1 tumor-bearing mice were divided into three groups. 6-8 mice per group. One group is a control group, and the mice are immunized or locally injected with tumors or injected in the abdominal cavity of the mice by using PBS with the same volume; the other two groups were injected subcutaneously twice 3 days and 9 days after tumor inoculation with 100. mu.l of vaccine containing 40. mu.g of Ex (10. mu.g of each polypeptide)/10. mu.g of MPLA/300. mu.g of anti-IL-10 receptor antibody, and intraperitoneally with two anti-300. mu.g of PD-1 antibody on days 9 and 21 after tumor inoculation. On days 3-9 post tumor inoculation, one group of tumors was injected locally with 30 μ g each of Caerin1.1 and/1.9, and the other group with 30 μ g of control peptide P3(GTELPSPPSVWFEAEFK-OH) for 7 consecutive days. One group of TC-1 tumor-bearing mice was simulated with PBS as a control group. At day 30 after tumor inoculation, spleens or tumor draining lymph nodes of tumor-bearing mice were taken and examined for HPV16E 7-specific CD8+ T cells using the ELISPOT method. The remaining mice were observed for survival of tumor bearing mice. Mice with tumor diameters greater than 2cm were considered dead. The survival of tumor bearing mice was monitored.
(C) The pure natural C57BL/6 mice or mice completely eradicating TC-1 tumors 100 days after tumor challenge were subcutaneously transplanted with 5X 105And TC-1 tumor cells. The survival of tumor bearing mice was monitored.
(D) At 30 days post tumor challenge, spleens and draining lymph nodes were isolated from control and vaccinated, PD-1 blocked, calrin 1.1 and calrin 1.9 or P3 treated TC-1 tumor-bearing mice, single cells were prepared, and subsequently cultured overnight in the presence of HPV16E 7CTL epitope RAHYNIVTF for ELISPOTT analysis as described in materials and methods. Left: tumor weight; the method comprises the following steps: splenic HPV16E 7-specific CD8+ T cells; and (3) right: draining lymph node HPV16E 7-specific CD8+ T cells.
Results and conclusions: the result shows that the therapeutic vaccine immunization is combined with the PD-1 antibody treatment, so that the efficiency of the therapeutic vaccine can be improved, and the survival time of the mouse can be prolonged.
Claims (10)
- A mutant of a calein 1.1 peptide having the amino acid sequence: GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2。
- 2. Use of the mutant of the calein 1.1 peptide according to claim 1 in the preparation of an antitumor drug.
- 3. The pharmaceutical composition consists of a calein 1.1 peptide and a calein 1.9 peptide, wherein the weight ratio of the calein 1.1 peptide to the calein 1.9 peptide is as follows: 1:0.1-1.
- 4. A pharmaceutical composition consisting of the calein 1.1 peptide mutant and the calein 1.9 peptide according to claim 1, wherein the weight ratio of the calein 1.1 peptide mutant to the calein 1.9 peptide is: 1:0.1-1.
- 5. The pharmaceutical composition of claim 3, wherein the weight ratio of the calein 1.1 peptide to the calein 1.9 peptide is: 1:0.5-1.
- 6. The pharmaceutical composition of claim 4, wherein the weight ratio of the mutant calein 1.1 peptide to the calein 1.9 peptide is: 1:0.5-1.
- 7. Use of a pharmaceutical composition according to any one of claims 3 to 6 for the preparation of an anti-tumor medicament.
- The application of the calirin 1.1 mutant, the calirin 1.1/1.9 polypeptide or the calirin 1.1 mutant/calirin 1.9 in the preparation of antitumor drugs by combining with vaccines and PD-1 inhibitors.
- 9. The use according to claim 8, wherein the vaccine is an HPV16E7 polypeptide vaccine consisting of 4 overlapping polypeptides EX containing the full length of HPV16E7, adjuvant MPLA, and anti-interleukin 10 receptor antibody, wherein EX is a mixture of A, B, C, D4 amino acids, wherein A, B, C, D is present in a weight ratio of 1: 1: 1: the sequence of amino acids 1, A, B, C, D is as follows:A:MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEE,B:LNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKC,C:DRAHYNIVTFCCKCDSTLRLCVQSTHVDIR,D:CVQSTHVDIRTLEDLLMGTLGIVCPICSQKP。
- 10. the use of claim 8, wherein the PD-1 inhibitor is an anti-PD-1 monoclonal antibody.
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CN117982626A (en) * | 2024-03-14 | 2024-05-07 | 中奥生物医药技术(广东)有限公司 | Application of F1/F3 polypeptide in preparation of medicines for inducing tumor cell apoptosis |
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WO2010091294A2 (en) * | 2009-02-05 | 2010-08-12 | The Regents Of The University Of California | New targeted antimicrobial moieties |
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CN111303265A (en) * | 2020-03-24 | 2020-06-19 | 中奥生物医药技术(广州)有限公司 | One kind contains131I-labeled Caerin1.1 polypeptide and application thereof |
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CN109200270A (en) * | 2018-09-30 | 2019-01-15 | 倪国颖 | A kind of combination medicine that can improve polypeptide vaccine to HPV infection oncotherapy effect and its application |
CN111303265A (en) * | 2020-03-24 | 2020-06-19 | 中奥生物医药技术(广州)有限公司 | One kind contains131I-labeled Caerin1.1 polypeptide and application thereof |
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CN117982626A (en) * | 2024-03-14 | 2024-05-07 | 中奥生物医药技术(广东)有限公司 | Application of F1/F3 polypeptide in preparation of medicines for inducing tumor cell apoptosis |
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