Application of PRTN3 molecule and epitope thereof as tumor immunotherapy target

Abstract

The invention relates to an application of a PRTN3 molecule and an epitope thereof as a tumor immunotherapy target. Specifically, the invention relates to a polypeptide which comprises one or more CTL antigen epitopes of PRTN3, the CTL antigen epitopes of PRTN3 comprise an amino acid sequence shown in any one of SEQ ID NO: 1-21, polynucleotide for encoding the polypeptide, an expression vector, a DNA vaccine and application of the polypeptide in preparation of drugs for treating and / or preventing tumors. On the other hand, the invention further relates to application of the reagent for knocking down PRTN3 expression in preparation of drugs for treating and / or preventing tumors, and the reagent for knocking down PRTN3 expression in CAR-M or myeloid cells, especially macrophages.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. Specifically, the present invention relates to the application of the PRTN3 molecule and its epitopes as cancer stem cell antigens and tumor immunotherapy targets for macrophages to regulate the tumor microenvironment. Background Art

[0002] Although some emerging tumor immunotherapy methods, including immune checkpoint inhibitors, chimeric antigen receptor T cells (CAR-T), and tumor vaccines, have revolutionarily promoted the progress of tumor clinical treatment, there are still many problems to be solved. A major reason for the failure of solid tumor immunotherapy is tumor immune tolerance caused by the tumor immunosuppressive microenvironment, and tumor immune escape caused by cancer stem cells (CSCs) plays an important role in tumor recurrence and metastasis. Macrophages account for 30-50% of all immune cells in the solid tumor microenvironment. It can play an anti-tumor function through phagocytosis, killing, antigen presentation, and immune activation. However, due to its strong functional plasticity, it is easily "educated" to form tumor-associated macrophages (TAMs) that promote tumors and play an immunosuppressive function. The bottlenecks in the background art related to the invention mainly include the following three aspects:

[0003] (1) Targeted inhibition of CSCs can reduce tumor recurrence and metastasis in mice and improve the effect of immunotherapy. However, traditional tumor immunotherapy methods, such as PD-1 inhibitors and CAR-T, are difficult to completely eliminate CSCs, resulting in tumor recurrence and metastasis caused by CSCs immune escape.

[0004] (2) Engineered macrophages expressing antigen chimeric receptors (CAR-M) can inhibit the progression of breast cancer in mice by phagocytosing tumor cells and secreting pro-inflammatory cytokines. However, the immunosuppressive microenvironment of solid tumors can promote the polarization of CAR-M to the M2 phenotype and inhibit its anti-tumor function.

[0005] (3) Tumor immunotherapy based on immune checkpoint inhibitors, such as PD-1 inhibitors, can significantly prolong the survival time of patients with advanced lung cancer, but its treatment response rate is only 20-30%. TAMs can promote the formation of the tumor immunosuppressive microenvironment and reduce the anti-tumor effect of immune checkpoint inhibitors. Summary of the Invention

[0006] To solve the above-mentioned technical problems existing in the prior art, the present invention provides the use of a reagent targeting PRTN3 in the prevention and / or treatment of tumors.

[0007] Specifically, in the first aspect, the present invention provides a polypeptide comprising one or more CTL epitopes of PRTN3.

[0008] In a specific embodiment, the present invention provides a polypeptide comprising one or more CTL epitopes of PRTN3, and the CTL epitope of PRTN3 comprises an amino acid sequence shown in any one of SEQ ID NO: 1-21.

[0009] In a specific embodiment, the present invention provides a polypeptide comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 CTL epitopes of PRTN3, and the CTL epitope of PRTN3 comprises an amino acid sequence shown in any one of SEQ ID NO: 1-21.

[0010] In a specific embodiment, the present invention provides a polypeptide comprising 7 CTL epitopes of PRTN3, and the CTL epitope of PRTN3 comprises an amino acid sequence shown in any one of SEQ ID NO: 1-21.

[0011] In a specific embodiment, the present invention provides a polypeptide comprising the CTL epitopes of PRTN3 as shown in SEQ ID NO: 1-7.

[0012] In a specific embodiment, the present invention provides a polypeptide comprising the CTL epitopes of PRTN3 as shown in SEQ ID NO: 8-14.

[0013] In a specific embodiment, the present invention provides a polypeptide comprising the CTL epitopes of PRTN3 as shown in SEQ ID NO: 15-21.

[0014] In the second aspect, the present invention provides a polynucleotide encoding one or more polynucleotides of CTL epitopes of PRTN3, and the CTL epitope of PRTN3 comprises an amino acid sequence shown in any one of SEQ ID NO: 1-21.

[0015] In a specific embodiment, the present invention provides a polynucleotide encoding the CTL epitopes of PRTN3 as shown in SEQ ID NO: 1-7.

[0016] In a specific embodiment, the present invention provides a polynucleotide encoding a CTL epitope of PRTN3 as shown in SEQ ID NO: 8-14.

[0017] In a specific embodiment, the present invention provides a polynucleotide encoding a CTL epitope of PRTN3 as shown in SEQ ID NO: 15-21.

[0018] In a third aspect, the present invention provides an expression vector comprising a polynucleotide encoding one or more CTL epitopes of PRTN3, wherein the CTL epitopes of PRTN3 comprise an amino acid sequence selected from any one of SEQ ID NO: 1-21.

[0019] In a specific embodiment, the present invention provides an expression vector comprising a polynucleotide encoding a CTL epitope of PRTN3 as shown in SEQ ID NO: 1-7.

[0020] In a specific embodiment, the present invention provides an expression vector comprising a polynucleotide encoding a CTL epitope of PRTN3 as shown in SEQ ID NO: 8-14.

[0021] In a specific embodiment, the present invention provides an expression vector comprising a polynucleotide encoding a CTL epitope of PRTN3 as shown in SEQ ID NO: 15-21.

[0022] In a fourth aspect, the present invention provides a DNA vaccine comprising the expression vector described herein and a pharmaceutically acceptable immunoadjuvant.

[0023] In a specific embodiment, the DNA vaccine of the present invention is prepared in a formulation suitable for use by a method selected from intravenous injection, arterial injection, intramuscular injection, subcutaneous injection, organ injection, thoracic injection, or intraperitoneal injection.

[0024] In a specific embodiment, the DNA vaccine of the present invention is an aqueous solution or a lyophilized powder for reconstitution that can be administered via injection or via mucosal administration.

[0025] In a fifth aspect, the present invention provides the use of the polypeptide, polynucleotide, expression vector, or DNA vaccine according to the present invention in the preparation of a medicament for the prevention and / or treatment of tumors; in particular, the present invention provides the use of the polypeptide, polynucleotide, expression vector, or DNA vaccine according to the present invention in the preparation of a medicament for the prevention of tumors.

[0026] In a specific embodiment, the tumors are selected from: lung cancer and breast cancer.

[0027] In a sixth aspect, the present invention provides the use of an agent for knocking down the expression of PRTN3 in the preparation of a drug for treating and / or preventing tumors, wherein the agent for knocking down the expression of PRTN3 knocks down the expression of PRTN3 in CAR-M or myeloid cells, particularly macrophages.

[0028] In a specific embodiment, the agent for knocking down the expression of PRTN3 is a gene editing system, including but not limited to the Cre-loxP recombination system. More preferably, the gene editing system is Lyz2-Cre; Prtn3 loxP / loxP 。

[0029] In a specific embodiment, the agent for knocking down the expression of PRTN3 is an interfering RNA, which is selected from siRNA, shRNA, single-stranded interfering RNA, and microRNA.

[0030] In a seventh aspect, the present invention provides the use of a combination of an agent for knocking down the expression of PRTN3 and an immune checkpoint inhibitor in the preparation of a drug for treating and / or preventing tumors, wherein the agent for knocking down the expression of PRTN3 knocks down the expression of PRTN3 in CAR-M or myeloid cells, particularly macrophages.

[0031] In a specific embodiment, the immune checkpoint inhibitor is selected from anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies; specifically, the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0032] In a specific embodiment, the tumors are selected from: lung cancer and breast cancer.

[0033] Term

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] As used herein, "PRTN3 (Proteinase 3)" is a serine protease that is mainly present in the cytoplasmic granules of neutrophils. It plays an important role in inflammatory responses, immune system regulation, and certain diseases (such as vasculitis and autoimmune diseases).

[0036] The PRTN3 protein contains 256 amino acid residues. Among them, the first 25 amino acids are the signal peptide, which can guide it to cross the cell membrane and be secreted extracellularly; the 26th and 27th amino acids and the 250th to 256th amino acids at the carboxyl terminus are the leader peptide. During translation, the signal peptide in the primary structure is cleaved and hydrolyzed by signal peptidase, and then the leader peptide is cleaved during endoplasmic reticulum processing, finally forming its mature form. Correspondingly, the molecular weight of the PRTN3 precursor protein is 35 kDa, and the molecular weight of the mature form of the protein is 29 kDa. The catalytic active site of PRTN3 consists of histidine (His57), serine (Ser195), and aspartic acid (Asp102).

[0037] As used herein, the term "CTL (Cytotoxic T Lymphocyte) antigen epitope" refers to a short peptide antigen that is presented by MHC class I molecules (HLA-A / B / C in humans, H2-K / H2-D in mice) and can be recognized and activated by CD8+ T cells.

[0038] As used herein, "the CTL antigen epitope of PRTN3" is intended to represent the CTL antigen epitope derived from the PRTN3 protein.

[0039] The MHC class I molecules in mice mainly include H2-K b , H2-D b and H2-L. H2-K b and H2-D b are the two MHC class I molecules most commonly used in immunological research because they are highly expressed in C57BL / 6 mice, and C57BL / 6 is a commonly used mouse strain in immunological research.

[0040] H2-K b is a specific subtype of the MHC class I molecule of the mouse major histocompatibility complex (MHC, Major Histocompatibility Complex), belonging to the K region of the H-2 complex (mouse MHC system). In humans, the MHC class I molecule corresponds to the HLA (Human Leukocyte Antigen) system, and H2-Kb is similar to HLA-B in humans.

[0041] H2-D b is a type of the MHC class I molecule of the mouse major histocompatibility complex (MHC, Major Histocompatibility Complex), belonging to the D region of the H-2 complex. It is functionally similar to H2-K b and is responsible for presenting endogenous antigens (such as viral proteins or tumor antigens) in the immune response mediated by CD8+ cytotoxic T cells (CTLs).

[0042] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and are composed of multiple amino acids linked by peptide bonds.

[0043] As used herein, the term "expression vector" is a polynucleotide used to express a specific foreign gene in a host cell to produce a protein, polypeptide or RNA. Specifically, the expression vector includes, but is not limited to, plasmids, viral vectors or artificial chromosomes.

[0044] As used herein, the terms "macrophages expressing chimeric antigen receptors", "CAR-M cells", "CAR-M" or "CAR-macrophages" all refer to macrophages expressing CAR. The CAR-M cells of the present invention can target tumor surface antigens (such as HER2) and are used to treat tumors with high expression or positive of tumor antigens (such as HER2).

[0045] Chimeric antigen receptors (CARs) are composed of an extracellular antigen recognition region, usually a scFv (single-chain variable fragment), a transmembrane region and an intracellular co-stimulatory signal region.

[0046] As used herein, the term "immune checkpoint inhibitor" includes, but is not limited to, PD-1 (programmed death receptor 1) inhibitors such as Pembrolizumab (Keytruda), Nivolumab (Opdivo); PD-L1 (programmed death ligand 1) inhibitors such as Atezolizumab, Durvalumab and Avelumab; and CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) inhibitors such as Ipilimumab (Yervoy). BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Showing the structures of three plasmids P1, P1 and P3 constructed containing multiple epitopes of the PRTN3 antigen;

[0048] Figure 2 Showing the identification results of plasmids by PCR;

[0049] Figure 3 Showing that the P1 / P2 / P3 vaccine can activate CD8+ T cells and kill CSCs;

[0050] Figure 4 Showing that the P1 vaccine inhibits the growth and metastasis of tumors in mice;

[0051] Figure 5 Show the expression of CAR molecules on the surface of different macrophages;

[0052] Figure 6 Show that CAR-M-shPrtn3 cells have stronger antitumor effects;

[0053] Figure 7 Show the identification of Lyz2-Cre; Prtn3flox / flox mice;

[0054] Figure 8 Show that knocking out the Prtn3 gene in macrophages enhances the tumor inhibitory effect of PD-1 antibodies. Detailed implementation

[0055] The following examples are intended to illustrate the present invention in more detail, but the solutions of the present invention are not limited thereto.

[0056] For the experimental methods without specific conditions in the examples, they are usually carried out according to conventional conditions or according to the conditions recommended by the raw material or commodity manufacturers; for the reagent materials without specific sources, they are obtained by purchasing from the market.

[0057] Example 1. Construction of a DNA vaccine based on the PRTN3 antigen epitope

[0058] A total of 21 CTL antigen epitopes of PRTN3 were predicted using the online websites NetMHCpan4.0 and IEDB respectively. The screening process for us was as follows: 1. Select the top 5 epitopes predicted by the NetMHCpan4.0 website that strongly bind to H2-K b the top 5 epitopes that strongly bind to H2-D b the top 5 epitopes that strongly bind to H2-K b and 6 epitopes that bind to both H2-K b and H2-D b the top 5 epitopes that strongly bind to H2-K b the top 5 epitopes that strongly bind to H2-D b the top 5 epitopes that strongly bind to H2-K b and 4 epitopes that bind to both H2-K b and H2-D b 3. Take the union of the top 10 epitopes predicted by the NetMHCpan4.0 and IEDB websites that strongly bind to H2-K b for a total of 9 epitopes, take the union of the top 10 epitopes predicted by the NetMHCpan4.0 and IEDB websites that strongly bind to H2-D bThe union of the top 10 epitopes that bind to both consists of 4 epitopes in total (Tables 1-3). Subsequently, every 7 candidate epitopes were concatenated with the AAY linker peptide, and Kozak and MITD sequences were added to construct 3 multi-epitope plasmids, which were named P1, P1, and P3 ( Figure 1 ), and the successful construction of the plasmids was confirmed by PCR and sequencing identification ( Figure 2 ).

[0059] Table 1 Epitopes that bind strongly to H2-K b and their sequences

[0060]

[0061] Table 2 Epitopes that bind strongly to H2-D b and their sequences

[0062]

[0063] Table 3 Epitopes that bind to both H2-K b and H2-D b and their sequences

[0064]

[0065] The obtained plasmid sequences are shown in Tables 4-6 below:

[0066] Table 4 Epitope sequences contained in the P1 plasmid

[0067]

[0068]

[0069] Table 5 Epitope sequences contained in the P2 plasmid

[0070] Amino acid position Sequence SEQ ID NO: 39-48 HSRPYVASLQ 8 239-248 SMYVDWIQNV 9 227-235 ASLQFPDFF 10 172-180 LQELNVTVV 11 140-148 SLPQQDQTL 12 55-63 SHFCGGTLI 13 241-249 YVDWIQNVL 14

[0071] Table 6 Epitope sequences contained in the P3 plasmid

[0072] Amino acid position Sequence SEQ ID NO: 78-86 ISWQLVTVV 15 78-87 ISWQLVTVVL 16 170-180 RVLQELNVTVV 17 85-93 LQLNRTASL 18 124-132 VVLGAHDLL 19 221-229 FVIRECASL 20 239-249 SMYVDWIQNVL 21

[0073] Example 2. In vitro analysis of the ability of P1 / P2 / P3 vaccines to activate CD8+ T cells and kill CSCs

[0074] The P1, P2, and P3 plasmids were separately electroporated into DC2.4 cells and co-cultured with CD8 + T cells from the spleens of tumor-bearing mice. The results showed that the P1 plasmid had the strongest ability to activate CD8 + T cells, as indicated by the highest expression of Granzyme B, IFNγ, CD25, and CD69 ( Figure 3A). CD8 activated by P1, P2, and P3 respectively + Co - culture CD8+ T cells activated by P1 plasmid with lewis CSCs, and it was found that CD8+ T cells activated by P1 plasmid + had the strongest ability to kill CSCs ( Figure 3 B).

[0075] Example 3. In - vivo evaluation of the anti - tumor effect of P1 vaccine

[0076] Next, we studied the effect of P1 vaccine on tumor growth in mice. First, we injected lewis tumor stem cells subcutaneously into C57BL / 6J mice to establish a subcutaneous transplanted lung cancer model, and divided the mice into a control group (untreated) and a P1 group (immunized with P1 vaccine three times) (inject tumor stem cells first, then inject P1 vaccine). The results showed that the tumor growth in the P1 group was significantly slowed down and the survival time of the mice was prolonged ( Figure 4 A and B). In addition, inoculated mouse breast cancer cells 4T1 into the mammary fat pad of Balb / c mice and divided the mice into a control group and a P1 group. The results of small animal in - vivo imaging showed that the primary tumors in the P1 group mice were reduced and the metastasis was also decreased ( Figure 4 C and D).

[0077] Our results indicate that the DNA vaccine constructed based on the PRTN3 antigen epitope can inhibit tumor growth and metastasis in mice.

[0078] Example 4. Construction of CAR - M cells (CAR - M - shPrtn3) targeting human HER2 protein and knocking down PRTN3

[0079] First, use the pLV - H1 - EF1 - puro lentiviral vector to construct RAW264.7 - shPrtn3 cells with stable knockdown of Prtn3, and then use the pLVX - EF1 - MCS - IRES - Bsd lentiviral vector to construct CAR - M - shPrtn3 cells that stably express the CAR molecule (CD8α signal peptide + human HER2 scFv + CD8α hinge + CD8α transmembrane domain + CD3ζ intracellular domain) on the basis of the above cells. The expression level of the CAR molecule on the cell surface is as Figure 5 shown.

[0080] Example 5. In - vivo evaluation of the anti - tumor effect of CAR - M - shPrtn3 cells

[0081] Inoculate the mouse breast cancer cell line 4T1 stably expressing human HER2 protein into the fourth mammary fat pad of Balb / c mice. One week later, start the adoptive transfer treatment of CAR - M cells by intratumoral injection once a week for a total of four times. The results showed that the tumor inhibitory effect of CAR - M - shPrtn3 cells was the bestFigure 6 of A and B).

[0082] Our results showed that knockdown of the PRTN3 gene in CAR-M cells could enhance their antitumor effect.

[0083] Example 6. Transgenic mouse model with conditional knockout of Prtn3 gene in macrophages

[0084] Beijing Weishang Lide Biotechnology Co., Ltd. and Jiangsu Jicui Yakang Biotechnology Co., Ltd. were commissioned to prepare transgenic mice with myeloid cell-specific knockout of PRTN3, namely Lyz2-Cre; Prtn3flox / flox mice. The identification results are as Figure 7 shown in A and B).

[0085] Example 7. Specific knockout of Prtn3 gene in mouse macrophages can enhance the antitumor effect of PD-1 antibody

[0086] Lewis cells were subcutaneously inoculated into wild C57 mice and Lyz2-Cre; Prtn3flox / flox mice respectively. After tumor formation (about one week), treatment was started by injecting PD-1 antibody (anti-mouse CD279 antibody, company: BioLegend, catalog number: 135246) every other day. The results showed that specific knockout of PRTN3 in macrophages could significantly enhance the tumor inhibitory effect of PD-1 antibody ( Figure 8 of A and B).

[0087] Our results showed that knockout / knockdown of the Prtn3 gene in macrophages could enhance the antitumor effect of PD-1 inhibitors.

[0088] In summary, by analyzing and identifying CSCs antigens and preparing related tumor vaccines to activate T cell immune responses, the seed cells that cause tumor recurrence and metastasis can be cleared, thereby inhibiting tumor growth and metastasis; gene-modified CAR-M cells can more effectively inhibit tumor growth and metastasis; gene editing of tumor-infiltrating macrophages can enhance the antitumor effect of PD-1 inhibitors.

Claims

1. A polypeptide comprising one or more CTL antigen epitopes of PRTN3, Preferably, the CTL antigen epitope of PRTN3 comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-21; More preferably, the polypeptide comprises 7 CTL antigen epitopes of PRTN3, and the CTL antigen epitope of PRTN3 comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-21; Further preferably, the polypeptide comprises: The CTL antigen epitope of PRTN3 as shown in SEQ ID NO: 1-7; The CTL antigen epitope of PRTN3 as shown in SEQ ID NO: 8-14; or The CTL antigen epitopes of PRTN3 are shown in SEQ ID NOs: 15-21.

2. A polynucleotide encoding the polypeptide of claim 1. An expression vector comprising the polynucleotide according to claim 2.

4. A DNA vaccine comprising the expression vector of claim 3 and a pharmaceutically acceptable immune adjuvant; Preferably, the DNA vaccine is formulated for injection or administration via the mucosa; More preferably, the DNA vaccine is formulated for intravenous injection, arterial injection, intramuscular injection, subcutaneous injection, organ injection, thoracic injection or intraperitoneal injection.

5. Use of the polypeptide according to claim 1, the polynucleotide according to claim 2, the expression vector according to claim 3 or the DNA vaccine according to claim 4 in the preparation of a medicament for preventing and / or treating tumors; Preferably, the tumor is lung cancer or breast cancer.

6. Use of an agent for knocking down PRTN3 expression in the preparation of a drug for treating and / or preventing tumors, wherein the agent for knocking down PRTN3 expression knocks down the expression of PRTN3 in CAR-M or myeloid cells, especially macrophages.

7. Use of a combination of an agent for knocking down PRTN3 expression and an immune checkpoint inhibitor in the preparation of a medicament for treating and / or preventing tumors, wherein the agent for knocking down PRTN3 expression knocks down the expression of PRTN3 in CAR-M or myeloid cells, especially macrophages; Preferably, the immune checkpoint inhibitor is selected from anti-PD-1 antibody, anti-PD-L1 antibody and anti-CTLA-4 antibody; More preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody.

8. The use according to claim 6 or 7, wherein the reagent for knocking down PRTN3 expression is a gene editing system, preferably a Cre-loxP recombination system; More preferably, the gene editing system is Lyz2-Cre; Prtn3 loxP / loxP .

9. The use according to claim 6 or 7, wherein the agent for knocking down PRTN3 expression is interfering RNA, Preferably, the agent for knocking down PRTN3 expression is selected from siRNA, shRNA, single-stranded interfering RNA and microRNA.

10. The use according to any one of claims 6 to 9, wherein the tumor is lung cancer or breast cancer.

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